301 results on '"Pannaccione, Anna"'
Search Results
152. ANESTETICI LOCALI
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A Pannaccione, Annunziato L, Di Renzo G, and Pannaccione, Anna
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- 2015
153. FARMACI NEL TRATTAMENTO DEL DETERIORAMENTO COGNITIVO
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F. Moroni, A. Pannaccione, Annunziato L, Di Renzo G, Moroni, F., and Pannaccione, Anna
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- 2015
154. Nitric oxide induces [Ca2+]ioscillations in pituitary GH3cells: involvement ofIDRand ERG K+currents
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Rossana Sirabella, Agnese Secondo, Lucio Annunziato, Luigi Formisano, Anna Pannaccione, Mauro Cataldi, Gianfranco Di Renzo, Secondo, Agnese, Pannaccione, Anna, Cataldi, Mauro, Sirabella, Rossana, Formisano, Luigi, DI RENZO, GIANFRANCO MARIA LUIGI, and Annunziato, Lucio
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Nitroprusside ,medicine.medical_specialty ,Pituitary gland ,Arginine ,Physiology ,S-Nitroso-N-Acetylpenicillamine ,Nitric Oxide ,Cell Line ,Nitric oxide ,chemistry.chemical_compound ,Software Design ,Internal medicine ,medicine ,Animals ,Nitric Oxide Donors ,Calcium Signaling ,Enzyme Inhibitors ,voltage-gated potassium channel ,biology ,fast-inactivating outward currents ,Cell Biology ,Voltage-gated potassium channel ,Ether-A-Go-Go Potassium Channels ,Rats ,Nitric oxide synthase ,NG-Nitroarginine Methyl Ester ,Endocrinology ,medicine.anatomical_structure ,chemistry ,Pituitary Gland ,Potassium ,Biophysics ,biology.protein ,ether-à-go-go-related gene potassium channel ,slow-inactivating outward current ,Nitric Oxide Synthase ,S-Nitroso-N-acetylpenicillamine ,Erg ,Algorithms ,Intracellular ,Nitroso Compounds - Abstract
The role of nitric oxide (NO) in the occurrence of intracellular Ca2+concentration ([Ca2+]i) oscillations in pituitary GH3cells was evaluated by studying the effect of increasing or decreasing endogenous NO synthesis with l-arginine and nitro-l-arginine methyl ester (l-NAME), respectively. When NO synthesis was blocked with l-NAME (1 mM) [Ca2+]i, oscillations disappeared in 68% of spontaneously active cells, whereas 41% of the quiescent cells showed [Ca2+]ioscillations in response to the NO synthase (NOS) substrate l-arginine (10 mM). This effect was reproduced by the NO donors NOC-18 and S-nitroso- N-acetylpenicillamine (SNAP). NOC-18 was ineffective in the presence of the L-type voltage-dependent Ca2+channels (VDCC) blocker nimodipine (1 μM) or in Ca2+-free medium. Conversely, its effect was preserved when Ca2+release from intracellular Ca2+stores was inhibited either with the ryanodine-receptor blocker ryanodine (500 μM) or with the inositol 1,4,5-trisphosphate receptor blocker xestospongin C (3 μM). These results suggest that NO induces the appearance of [Ca2+]ioscillations by determining Ca2+influx. Patch-clamp experiments excluded that NO acted directly on VDCC but suggested that NO determined membrane depolarization because of the inhibition of voltage-gated K+channels. NOC-18 and SNAP caused a decrease in the amplitude of slow-inactivating ( IDR) and ether-à-go-go-related gene ( ERG) hyperpolarization-evoked, deactivating K+currents. Similar results were obtained when GH3cells were treated with l-arginine. The present study suggests that in GH3cells, endogenous NO plays a permissive role for the occurrence of spontaneous [Ca2+]ioscillations through an inhibitory effect on IDRand on IERG.
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- 2006
155. First- and second-generation H1 antihistamines: from the molecular basis of their interaction with HERG K+ channels to physiological and pathophysiological implication
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Agnese Secondo, Pasqualina Castaldo, Anna Pannaccione, Lucio Annunziato, Mauro Cataldi, Maurizio Taglialatela, Francesca Boscia, Taglialatela, Maurizio, Castaldo, P., Pannaccione, Anna, Secondo, Agnese, Cataldi, Mauro, Boscia, Francesca, and Annunziato, Lucio
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biology ,HERG CHANNELS ,Chemistry ,ARRHYTHMIA ,Immunology ,hERG ,Cardiac action potential ,Pharmacology ,Potassium channel ,biology.protein ,Immunology and Allergy ,Neuroscience ,ANTIHISTAMINES ,K channels - Abstract
Summary The present article reports on the recent findings addressing the molecular basis for some rare but serious cardiovascular side-effects exerted by some non-sedating H1-blocking antihistamines. These latest developments, which have allowed fundamental insights into the role played by a specific class of potassium channels, the so-called human ether-a-gogo-related gene (HERG) channels, in the regulation of cardiac action potential duration and rhythm regulation, have also opened new areas of investigation into its participation in neuronal and endocrine (adenohypophyseal, chromaffin, and pancreatic) cells functioning.
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- 2004
156. Histidines 578 and 587 in the S5-S6Linker of the Human Ether-a-gogo Related Gene-1K+ Channels Confer Sensitivity to Reactive Oxygen Species
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Maurizio Taglialatela, Eckhard Ficker, Anna Pannaccione, Pasqualina Castaldo, Lucio Annunziato, Pannaccione, Anna, Castaldo, Pasqualina, Ficker, E, Annunziato, Lucio, and Taglialatela, Maurizio
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ERG1 Potassium Channel ,Potassium Channels ,Stereochemistry ,Molecular Sequence Data ,Xenopus ,Ether ,Deferoxamine ,medicine.disease_cause ,Biochemistry ,Histidines 578 and 587 ,Structure-Activity Relationship ,Xenopus laevis ,chemistry.chemical_compound ,medicine ,Animals ,Humans ,Histidine ,Amino Acid Sequence ,Molecular Biology ,Gene ,chemistry.chemical_classification ,Mutation ,Reactive oxygen species ,biology ,Chemistry ,Cell Biology ,biology.organism_classification ,Ether-A-Go-Go Potassium Channels ,Enzyme ,Potassium Channels, Voltage-Gated ,Catalase ,biology.protein ,Biophysics ,Reactive Oxygen specie ,K+ channels ,Reactive Oxygen Species ,Linker ,Phenanthrolines - Abstract
The K(+) channels encoded by the human Ether-a-gogo Related Gene-1 (hERG1) are crucially involved in controlling heart and brain excitability and are selectively influenced by reactive oxygen species (ROS). To localize the molecular regions involved in ROS-induced modulation of hERG1, segmental exchanges between the ROS-sensitive hERG1 and the ROS-insensitive bovine ether-a-gogo gene (bEAG) K(+) channels were generated, and the sensitivity of these chimeric channels to ROS was studied with the two-microelectrode voltage-clamp technique upon their expression in Xenopus oocytes. Substitution of the S(5)-S(6) linker of hERG1 with the corresponding bEAG region removed channel sensitivity to ROS, whereas the reverse chimeric exchange introduced ROS sensitivity into bEAG. Mutation of each of the two hERG1 histidines at positions 578 and 587 within the S(5)-S(6) linker generated K(+) channels insensitive to modulation by ROS. In addition, the two iron chelators desferrioxamine (1 mm) and o-phenanthroline (0.2 mm) significantly inhibited hERG1 outward K(+) currents and prevented hERG1 inhibition induced by the ROS-scavenging enzyme catalase (1000 units/ml). Finally, the hERG1-inhibitory effect exerted by the iron chelators was prevented by the hERG1 H578D/H587Y double mutation. Collectively, the results obtained suggest that histidines at positions 578 and 587 in the S(5)-S(6) linker region of hERG1 K(+) channels are crucial players in ROS-induced modulation of hERG1 K(+) channels.
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- 2002
157. NCX1 is a new rest target gene: role in cerebral ischemia
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Agnese Secondo, Lucio Annunziato, Rossana Sirabella, Antonella Casamassa, Giuseppe Pignataro, Anna Pannaccione, Lorella M.T. Canzoniero, Gianfranco Di Renzo, Maria Josè Sisalli, Luigi Formisano, Francesca Boscia, Pasquale Molinaro, Valeria Valsecchi, Natascia Guida, Antonio Vinciguerra, Formisano, L, Guida, N, Valsecchi, V, Pignataro, Giuseppe, Vinciguerra, A, Pannaccione, Anna, Secondo, Agnese, Boscia, Francesca, Molinaro, Pasquale, Sisalli, Mj, Sirabella, Rossana, Casamassa, A, Canzoniero, Lm, DI RENZO, GIANFRANCO MARIA LUIGI, and Annunziato, Lucio
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Chromatin Immunoprecipitation ,Blotting, Western ,Molecular Sequence Data ,Ischemia ,Fluorescent Antibody Technique ,Electrophoretic Mobility Shift Assay ,RE1-silencing transcription factor (REST) ,Hippocampal formation ,Biology ,Real-Time Polymerase Chain Reaction ,Transfection ,Hippocampus ,Neuroprotection ,Sodium-Calcium Exchanger ,SH-SY5Y cells ,Brain Ischemia ,lcsh:RC321-571 ,Rats, Sprague-Dawley ,Brain ischemia ,Organ Culture Techniques ,Gene expression ,medicine ,Animals ,Humans ,Gene silencing ,Rats, Wistar ,lcsh:Neurosciences. Biological psychiatry. Neuropsychiatry ,Transcription factor ,Rest (music) ,Neurons ,NCX1 ,Microscopy, Confocal ,Base Sequence ,REST ,Epigenetic ,Acetylation ,medicine.disease ,Rats ,Cell biology ,Repressor Proteins ,Stroke ,Gene Expression Regulation ,Neurology ,Na+–Ca2 + exchanger 1 (NCX1) ,Mutagenesis, Site-Directed ,cardiovascular system ,Neuroscience - Abstract
The Na + –Ca 2 + exchanger 1 (NCX1), a bidirectional transporter that mediates the electrogenic exchange of one calcium ion for three sodium ions across the plasmamembrane, is known to be involved in brain ischemia. Since the RE1-silencing transcription factor (REST) is a key modulator of neuronal gene expression in several neurological conditions, we studied the possible involvement of REST in regulating NCX1 gene expression and activity in stroke. We found that: (1) REST binds in a sequence specific manner and represses through H4 deacetylation, ncx1 gene in neuronal cells by recruting CoREST, but not mSin3A. (2)In neurons and in SH-SY5Y cells REST silencing by siRNA and site-direct mutagenesis of REST consensus sequence on NCX1 brain promoter determined an increase in NCX1 promoter activity. (3)By contrast, REST overexpression caused a reduction in NCX1 protein expression and activity. (4)Interestingly, in rats subjected to transient middle cerebral artery occlusion (tMCAO) and in organotypic hippocampal slices or SH-SY5Y cells exposed to oxygen and glucose deprivation (OGD) plus reoxygenation (RX), the increase in REST was associated with a decrease in NCX1. However, this reduction was reverted by REST silencing. (5)REST knocking down, along with the deriving NCX1overexpression in the deep V and VIb cortical layers caused a marked reduction in infarct volume after tMCAO. Double silencing of REST and NCX1 completely abolished neuroprotection induced by siREST administration. Collectively, these results demonstrate that REST, by regulating NCX1 expression, may represent a potential druggable target for the treatment of brain ischemia.
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- 2013
158. Neurounina-1, a Novel Compound that Increases Na+/Ca2+ Exchanger Activity, Effectively Protects Against Stroke Damage
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Maria Cantile, Mauro Robello, Gianfranco Di Renzo, Agnese Secondo, Giambattista Bonanno, Ferdinando Fiorino, Elena Gatta, Anna Pannaccione, Giuseppe Pignataro, Maria Josè Sisalli, Beatrice Severino, Antonella Scorziello, Marco Milanese, Giuseppe Caliendo, Pasquale Molinaro, Paolo Ambrosino, Ornella Cuomo, Vincenzo Santagada, Lucio Annunziato, Molinaro, Pasquale, Cantile, M, Cuomo, Ornella, Secondo, Agnese, Pannaccione, Anna, Ambrosino, P, Pignataro, Giuseppe, Fiorino, Ferdinando, Severino, Beatrice, Gatta, E, Sisalli, Mj, Milanese, M, Scorziello, Antonella, Bonanno, G, Robello, M, Santagada, Vincenzo, Caliendo, Giuseppe, DI RENZO, GIANFRANCO MARIA LUIGI, and Annunziato, Lucio
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Flumazenil ,Male ,Programmed cell death ,Patch-Clamp Techniques ,Pyrrolidines ,Glutamic Acid ,Pharmacology ,Receptors, N-Methyl-D-Aspartate ,Neuroprotection ,Sodium-Calcium Exchanger ,Mice ,Dogs ,In vivo ,Cricetinae ,Animals ,GABA-A Receptor Antagonists ,Patch clamp ,Receptor ,Cells, Cultured ,gamma-Aminobutyric Acid ,Neurons ,Benzodiazepinones ,Cell Death ,Chemistry ,Glutamate receptor ,Infarction, Middle Cerebral Artery ,Microfluorimetry ,Receptors, GABA-A ,In vitro ,Mitochondria ,Rats ,Mice, Inbred C57BL ,Stroke ,Ca2+ homeostasi ,Neuroprotective Agents ,Biochemistry ,stroke therapy ,Mutation ,Molecular Medicine ,Calcium ,NCX ,Synaptosomes - Abstract
Previous studies have demonstrated that the knockdown or knockout of the three Na(+)/Ca(2+) exchanger (NCX) isoforms, NCX1, NCX2, and NCX3, worsens ischemic brain damage. This suggests that the activation of these antiporters exerts a neuroprotective action against stroke damage. However, drugs able to increase the activity of NCXs are not yet available. We have here succeeded in synthesizing a new compound, named neurounina-1 (7-nitro-5-phenyl-1-(pyrrolidin-1-ylmethyl)-1H-benzo[e][1,4]diazepin-2(3H)-one), provided with an high lipophilicity index and able to increase NCX activity. Ca(2+) radiotracer, Fura-2 microfluorimetry, and patch-clamp techniques revealed that neurounina-1 stimulated NCX1 and NCX2 activities with an EC(50) in the picomolar to low nanomolar range, whereas it did not affect NCX3 activity. Furthermore, by using chimera strategy and site-directed mutagenesis, three specific molecular determinants of NCX1 responsible for neurounina-1 activity were identified in the α-repeats. Interestingly, NCX3 became responsive to neurounina-1 when both α-repeats were replaced with the corresponding regions of NCX1. In vitro studies showed that 10 nM neurounina-1 reduced cell death of primary cortical neurons exposed to oxygen-glucose deprivation followed by reoxygenation. Moreover, in vitro, neurounina-1 also reduced γ-aminobutyric acid (GABA) release, enhanced GABA(A) currents, and inhibited both glutamate release and N-methyl-d-aspartate receptors. More important, neurounina-1 proved to have a wide therapeutic window in vivo. Indeed, when administered at doses of 0.003 to 30 μg/kg i.p., it was able to reduce the infarct volume of mice subjected to transient middle cerebral artery occlusion even up to 3 to 5 hours after stroke onset. Collectively, the present study shows that neurounina-1 exerts a remarkable neuroprotective effect during stroke and increases NCX1 and NCX2 activities.
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- 2013
159. Genetically Modified Mice as a Strategy to Unravel the Role Played by the Na+/Ca2+ Exchanger in Brain Ischemia and in Spatial Learning and Memory Deficits
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Giuseppe Pignataro, Ornella Cuomo, Francesca Boscia, Agnese Secondo, Mauro Cataldi, André Herchuelz, Rossana Sirabella, Antonella Scorziello, Anna Pannaccione, Lucio Annunziato, Sophie Sokolow, Pasquale Molinaro, Davide Viggiano, Gianfranco Di Renzo, Molinaro, Pasquale, Cataldi, Mauro, Cuomo, Ornella, Viggiano, D, Pignataro, Giuseppe, Sirabella, Rossana, Secondo, Agnese, Boscia, Francesca, Pannaccione, Anna, Scorziello, Antonella, Sokolow, S, Herchuelz, A, DI RENZO, GIANFRANCO MARIA LUIGI, and Annunziato, Lucio
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NCX1 ,Pathology ,medicine.medical_specialty ,Sodium-calcium exchanger ,Antiporter ,Ischemia ,Biology ,medicine.disease ,NCX2 ,NCX3 ,Learning and memory ,Brain ischemia ,Conditional gene knockout ,Knockout mouse ,cardiovascular system ,medicine ,Memory consolidation ,Neuroscience ,Homeostasis ,Knockout mice - Abstract
Because no isoform-specific blocker of NCX has ever been synthesized, a more selective strategy to identify the role of each antiporter isoform in the brain was represented by the generation of knockout and knockin mice for the different isoforms of the antiporter.Experiments performed in NCX2 and NCX3 knockout mice provided evidence that these two isoforms participate in spatial learning and memory consolidation, although in an opposite manner. These new data from ncx2-/- and ncx3-/- mice may open new experimental avenues for the development of effective therapeutic compounds that, by selectively inhibiting or activating these molecular targets, could treat patients affected by cognitive impairment including Alzheimer's, Parkinson's, Huntington's diseases, and infarct dementia.More importantly, knockout and knockin mice also provided new relevant information on the role played by NCX in maintaining the intracellular Na(+) and Ca(2+) homeostasis and in protecting neurons during brain ischemia. In particular, both ncx2-/- and ncx3-/- mice showed an increased neuronal vulnerability after the ischemic insult induced by transient middle cerebral artery occlusion.As the ubiquitous deletion of NCX1 brings about to an early death of embryos because of a lack of heartbeat, this strategy could not be successfully pursued. However, information on the role of NCX1 in normal and ischemic brain could be obtained by developing conditional knockout mice lacking NCX1 in the brain. Preliminarily results obtained in these conditional mice suggest that also NCX1 protects neurons from ischemic cell death.Overall, the use of genetic-modified mice for NCX1, NCX2, and NCX3 represents a fruitful strategy to characterize the physiological role exerted by NCX in CNS and to identify the isoforms of the antiporter as potential molecular targets for therapeutic intervention in cerebral ischemia.
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- 2012
160. Silencing or knocking out the Na+/Ca2+ exchanger-3 (NCX3) impairs oligodendrocyte differentiation
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Anna Pannaccione, Antonella Casamassa, Agnese Secondo, Lucio Annunziato, Luigi Formisano, Natascia Guida, André Herchuelz, Francesca Boscia, Carla D’Avanzo, Sophie Sokolow, Boscia, Francesca, D'Avanzo, C, Pannaccione, Anna, Secondo, Agnese, Casamassa, A, Formisano, L, Guida, N, Sokolow, S, Herchuelz, A, and Annunziato, Lucio
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Programmed cell death ,Cellular differentiation ,Nerve Tissue Proteins ,Sodium-Calcium Exchanger ,Cell Line ,Mice ,Myelin ,Neural Stem Cells ,Downregulation and upregulation ,medicine ,Animals ,Humans ,Protein Isoforms ,Gene silencing ,Calcium Signaling ,Gene Silencing ,RNA, Messenger ,Molecular Biology ,Myelin Sheath ,Calcium signaling ,Mice, Knockout ,Original Paper ,biology ,Sodium-calcium exchanger ,Oligodendrocyte differentiation ,Cell Differentiation ,Cell Biology ,Oligodendrocyte ,Rats ,Myelin basic protein ,Cell biology ,Oligodendroglia ,medicine.anatomical_structure ,Gene Expression Regulation ,nervous system ,Biochemistry ,biology.protein ,Corrigendum - Abstract
Changes in intracellular [Ca(2+)](i) levels have been shown to influence developmental processes that accompany the transition of human oligodendrocyte precursor cells (OPCs) into mature myelinating oligodendrocytes and are required for the initiation of the myelination and re-myelination processes. In the present study, we explored whether calcium signals mediated by the selective sodium calcium exchanger (NCX) family members NCX1, NCX2, and NCX3, play a role in oligodendrocyte maturation. Functional studies, as well as mRNA and protein expression analyses, revealed that NCX1 and NCX3, but not NCX2, were divergently modulated during OPC differentiation into oligodendrocyte phenotype. In fact, whereas NCX1 was downregulated, NCX3 was strongly upregulated during oligodendrocyte development. The importance of calcium signaling mediated by NCX3 during oligodendrocyte maturation was supported by several findings. Indeed, whereas knocking down the NCX3 isoform in OPCs prevented the upregulation of the myelin protein markers 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) and myelin basic protein (MBP), its overexpression induced an upregulation of CNPase and MBP. Furthermore, NCX3-knockout mice showed not only a reduced size of spinal cord but also marked hypo-myelination, as revealed by decrease in MBP expression and by an accompanying increase in OPC number. Collectively, our findings indicate that calcium signaling mediated by NCX3 has a crucial role in oligodendrocyte maturation and myelin formation.
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- 2012
161. Nitric oxide stimulates NCX1 and NCX2 but inhibits NCX3 isoform by three distinct molecular determinants
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Lucio Annunziato, Maria Cantile, Alba Esposito, Pasquale Molinaro, Pellegrino Lippiello, Gianfranco Di Renzo, Rossana Sirabella, Agnese Secondo, Anna Pannaccione, Takahiro Iwamoto, Secondo, Agnese, Molinaro, Pasquale, Pannaccione, Anna, Esposito, A, Cantile, M, Lippiello, P, Sirabella, Rossana, Iwamoto, T, DI RENZO, GIANFRANCO MARIA LUIGI, and Annunziato, Lucio
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Gene isoform ,Patch-Clamp Techniques ,Blotting, Western ,Biology ,S-Nitroso-N-Acetylpenicillamine ,Kidney ,Nitric Oxide ,Sodium-Calcium Exchanger ,Nitric oxide ,Cell Line ,Membrane Potentials ,chemistry.chemical_compound ,Cricetinae ,Baby hamster kidney cell ,Animals ,Protein Isoforms ,Patch clamp ,Binding site ,Peptide sequence ,Pharmacology ,NCX1 ,Binding Sites ,Snap ,Transfection ,Flow Cytometry ,Molecular biology ,NCX2 ,chemistry ,Mutagenesis, Site-Directed ,Nitric Oxide (NO) ,Molecular Medicine ,Calcium - Abstract
In this study, the role of nitric oxide (NO) in the modulation of the activity of NCX1, NCX2, and NCX3 exchangers was investigated in baby hamster kidney cells singly transfected with each of these isoforms by single-cell Fura-2-microfluorometry and patch clamp. Furthermore, the molecular determinants of NO on each isoform were identified by deletion, site-directed mutagenesis, and chimera strategies. Our data revealed four main findings. First, the NO-donor S-nitroso-N-acetylpenicillamine (SNAP; 10 nM) and the NO-precursor L-arginine (10 mM) were both able to increase NCX1 activity in a cGMP-independent way. Moreover, within the amino acid sequence 723 to 734 of the f-loop, Cys730 resulted as the target of NO on NCX1. Second, SNAP and L-arginine were able to increase NCX2 activity, but this effect was prevented by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). In addition, the membrane-permeable 8-bromoguanosine-cGMP alone was able to mimic the stimulatory effect of the gaseous mediator, suggesting the involvement of a cGMP-dependent mechanism. Within the amino acid sequence 699 to 744 of the f-loop, Ser713 was the NO molecular determinant on the NCX2 protein; Third, NCX3 activity was instead down-regulated by NO in a cGMP-independent manner. This NO-inhibitory action was exerted at the level of Cys156 in the α1-region outside the f-loop. Finally, the activity of the two NCX3 chimeras-obtained by the replacement of the NO-insensitive NCX3 region with the homologous NO-sensitive segments of NCX1 or NCX2-was potentiated by SNAP. Together, the present data demonstrate that NO differently regulates the activity of the three gene products NCX1, NCX2, and NCX3 by modulating specific molecular determinants.
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- 2011
162. Na+-Ca2+ Exchanger (NCX3) Knock-Out Mice Display an Impairment in Hippocampal Long-Term Potentiation and Spatial Learning and Memory
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Robert Nisticò, Gianfranco F. Di Renzo, Anna Pannaccione, Davide Viggiano, Agnese Secondo, André Herchuelz, Pasquale Molinaro, Rossana Sirabella, Lucio Annunziato, Sophie Sokolow, Antonella Scorziello, Francesca Boscia, Bisan Mehdawy, Molinaro, Pasquale, Viggiano, Davide, Nisticò, R, Sirabella, Rossana, Secondo, Agnese, Boscia, Francesca, Pannaccione, Anna, Scorziello, Antonella, Mehdawy, B, Sokolow, S, Herchuelz, A, DI RENZO, GIANFRANCO MARIA LUIGI, and Annunziato, Lucio
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Male ,Animals ,Cells, Cultured ,Gene Silencing ,Hippocampus ,Long-Term Potentiation ,Maze Learning ,Memory ,Mice ,Mice, Inbred C57BL ,Mice, Knockout ,Sodium-Calcium Exchanger ,Spatial Behavior ,Synaptic Transmission ,Cells ,Knockout ,Hippocampal formation ,Biology ,Inbred C57BL ,memory ,Fear conditioning ,Cultured ,learning ,General Neuroscience ,Settore BIO/14 ,Na+-Ca2+ Exchanger (NCX3) ,Long-term potentiation ,Depolarization ,Articles ,Barnes maze ,Cell biology ,Animals, Cells ,Cultured, Gene Silencing, Hippocampus ,metabolism/physiopathology, Long-Term Potentiation ,genetics, Male, Maze Learning ,physiology, Memory ,physiology, Mice, Mice ,Inbred C57BL, Mice ,Knockout, Sodium-Calcium Exchanger ,genetics/metabolism, Spatial Behavior ,physiology, Synaptic Transmission ,genetics ,nervous system ,Knockout mouse ,Neuroscience ,Homeostasis - Abstract
Long-term potentiation (LTP) depends on the coordinated regulation of an ensemble of proteins related to Ca2+homeostasis, including Ca2+transporters. One of the major players in the regulation of intracellular Ca2+([Ca2+]i) homeostasis in neurons is the sodium/calcium exchanger (NCX), which represents the principal mechanism of Ca2+clearance in the synaptic sites of hippocampal neurons. Because NCX3, one of the three brain isoforms of the NCX family, is highly expressed in the hippocampal subfields involved in LTP, we hypothesized that it might represent a potential candidate for LTP modulation. To test this hypothesis, we first examined the effect ofncx3gene ablation on NCX currents (INCX) and Ca2+homeostasis in hippocampal neurons.ncx3−/−neurons displayed a reducedINCX, a higher basal level of [Ca2+]i, and a significantly delayed clearance of [Ca2+]ifollowing depolarization. Furthermore, measurement of field EPSPs, recorded from the CA1 area, revealed thatncx3−/−mice had an impaired basal synaptic transmission. Moreover, hippocampal slices fromncx3−/−mice exhibited a worsening in LTP compared with congenicncx3+/+. Consistently, immunohistochemical and immunoblot analysis indicated that in the hippocampus ofncx3−/−mice both Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) expression and the phosphoCaMKIIα/CaMKIIα ratio were significantly reduced compared withncx3+/+. Interestingly,ncx3−/−mice displayed a reduced spatial learning and memory performance, as revealed by the novel object recognition, Barnes maze, and context-dependent fear conditioning assays. Collectively, our findings demonstrate that the deletion of thencx3gene in mice has detrimental consequences on basal synaptic transmission, LTP regulation, spatial learning, and memory performance.
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- 2011
163. NCX1 expression and functional activity increase in microglia invading the infarct core
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Agnese Secondo, Gianfranco Di Renzo, Francesca Boscia, Anna Pannaccione, Lucio Annunziato, Rosaria Gala, Antonella Scorziello, Boscia, Francesca, Gala, Rosaria, Pannaccione, Anna, Secondo, Agnese, Scorziello, Antonella, DI RENZO, GIANFRANCO MARIA LUIGI, and Annunziato, Lucio
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Male ,Pathology ,medicine.medical_specialty ,cerebral ischemia ,Sodium-Calcium Exchanger ,Brain ischemia ,Rats, Sprague-Dawley ,Downregulation and upregulation ,Cell Movement ,Medicine ,Animals ,Cells, Cultured ,Advanced and Specialized Nursing ,Na+/Ca2+ exchanger ,NCX1 ,Microglia ,Sodium-calcium exchanger ,business.industry ,Cerebral infarction ,Cerebral Infarction ,medicine.disease ,Rats ,Up-Regulation ,Blot ,medicine.anatomical_structure ,Gene Expression Regulation ,IB4 ,cardiovascular system ,pMCAO ,Immunohistochemistry ,Neurology (clinical) ,Cardiology and Cardiovascular Medicine ,business ,Ex vivo - Abstract
Background and Purpose— The sodium–calcium exchanger NCX1 represents a key mediator for maintaining [Na + ] i and [Ca 2+ ] i in anoxic conditions. To date, no information is available on NCX1 protein expression and activity in microglial cells under ischemic conditions. Methods— By means of Western blotting, patch-clamp electrophysiology, single-cell Fura-2 acetoxymethyl-ester microfluorometry, immunohistochemistry, and confocal microscopy, we investigated the regional and temporal changes of NCX1 protein in microglial cells of the peri-infarct and core regions after permanent middle cerebral artery occlusion. The exchanger expression and activity were measured in primary microglia isolated ex vivo from the core region of adult rat brains 7 days after permanent middle cerebral artery occlusion and in cultured microglia under in vitro hypoxia. Results— One day after permanent middle cerebral artery occlusion, NCX1 protein expression was detected in some microglial cells adjacent to the soma of neurons in the infarct core. More interestingly, 3 and 7 days after permanent middle cerebral artery occlusion, NCX1 signal strongly increased in the round-shaped microglia invading the infarct core. Cultured microglial cells obtained from the core also displayed increased NCX1 expression as compared with contralateral cells and showed enhanced NCX activity in the reverse mode of operation. Similarly, NCX activity and NCX1 protein expression were significantly enhanced in BV2 microglia exposed to oxygen and glucose deprivation, whereas NCX2 and NCX3 were downregulated. Interestingly, in NCX1-silenced cells, [Ca 2+ ] i increase induced by hypoxia was completely prevented. Conclusion– The upregulation of NCX1 expression and activity observed in microglia after permanent middle cerebral artery occlusion suggests a relevant role of NCX1 in modulating microglia functions in the postischemic brain.
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- 2009
164. Molecular pharmacology of the amiloride analog 3-amino-6-chloro-5-[(4-chloro-benzyl)amino]-n-[[(2,4-dimethylbenzyl)-amino]iminomethyl]-pyrazinecarboxamide (CB-DMB) as a pan inhibitor of the Na+-Ca2+ exchanger isoforms NCX1, NCX2, and NCX3 in stably transfected cells
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Agnese, Secondo, Anna, Pannaccione, Pasquale, Molinaro, Paolo, Ambrosino, Pellegrino, Lippiello, Alba, Esposito, Maria, Cantile, Priti R, Khatri, Daniela, Melisi, Gianfranco, Di Renzo, Lucio, Annunziato, Secondo, Agnese, Pannaccione, Anna, Molinaro, Pasquale, Ambrosino, P, Lippiello, Pellegrino, Esposito, A, Cantile, Maria, Khatri, Pr, Melisi, Daniela, DI RENZO, GIANFRANCO MARIA LUIGI, and Annunziato, Lucio
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NCX1 ,Transfection ,Sodium-Calcium Exchanger ,NCX2 ,Cell Line ,Rats ,Amiloride ,Dogs ,Cricetinae ,Animals ,Humans ,Protein Isoforms ,Na+-Ca2+ exchanger isoform ,Cells, Cultured ,and NCX3 - Abstract
With the help of single-cell microflorimetry, (45)Ca(2+) radiotracer fluxes, and patch-clamp in whole-cell configuration, we examined the effect of the amiloride derivative 3-amino-6-chloro-5-[(4-chloro-benzyl)amino]-N-[[(2,4-dimethylbenzyl)amino]iminomethyl]-pyrazinecarboxamide (CB-DMB) on the activity of the three isoforms of the Na(+)/Ca(2+) exchanger (NCX) and on several other membrane currents including voltage- and pH-sensitive ones. This amiloride analog suppressed the bidirectional activity of all NCX isoforms in a concentration-dependent manner. The IC(50) values of CB-DMB were in the nanomolar range for the outward and the inward components of the bidirectional NCX1, NCX2, and NCX3 activity. Deletion mutagenesis showed that CB-DMB inhibited NCX activity mainly at level of the f-loop but not through the interaction with Gly833 located at the level of the alpha(2) repeat. On the other hand, CB-DMB suppressed in the micromolar range the other plasma membrane currents encoded by voltage-dependent Ca(2+) channels, tetrodotoxin-sensitive Na(+) channels, and pH-sensitive ASIC1a. Collectively, the data of the present study showed that CB-DMB, when used in the nanomolar range, is one of the most potent compounds that can block the activity of the three NCX isoforms when they work both in the forward and in the reverse modes of operation without interfering with other ionic channels.
- Published
- 2009
165. Targeted disruption of Na+/Ca2+ exchanger 3 (NCX3) gene leads to a worsening of ischemic brain damage
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Francesca Boscia, Rosaria Gala, Annagrazia Adornetto, Sophie Sokolow, Gianfranco Di Renzo, Rossana Sirabella, André Herchuelz, Agnese Secondo, Antonella Scorziello, Lucio Annunziato, Davide Viggiano, Anna Pannaccione, Ornella Cuomo, Giuseppe Pignataro, Stéphane Schurmans, Pasquale Molinaro, Molinaro, Pasquale, Cuomo, Ornella, Pignataro, Giuseppe, Boscia, Francesca, Sirabella, Rossana, Pannaccione, Anna, Secondo, Agnese, Scorziello, Antonella, Adornetto, A, Gala, Rosaria, Viggiano, D, Sokolow, S, Herchuelz, A, Schurmans, S, DI RENZO, GIANFRANCO MARIA LUIGI, and Annunziato, Lucio
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Gene isoform ,medicine.medical_specialty ,Cell Survival ,Ischemia ,Brain damage ,Hippocampal formation ,Biology ,Hippocampus ,cerebral ischemia ,Sodium-Calcium Exchanger ,Brain Ischemia ,Mice ,Mice, Congenic ,Organ Culture Techniques ,Internal medicine ,medicine ,Animals ,Homeostasis ,Hypoxia, Brain ,Mice, Knockout ,Neurons ,Sodium-calcium exchanger ,Cell Death ,General Neuroscience ,OGD ,Membrane Transport Proteins ,Articles ,Hypoxia (medical) ,medicine.disease ,sodium calcium exchanger ,Mice, Inbred C57BL ,Endocrinology ,organotypic hippocampal cultures ,Gene Targeting ,Disease Progression ,medicine.symptom ,MCAO ,Neuroscience ,Intracellular ,NCX ,Signal Transduction - Abstract
Na+/Ca2+exchanger 3 (NCX3), one of the three isoforms of the NCX family, is highly expressed in the brain and is involved in the maintenance of intracellular Na+and Ca2+homeostasis. Interestingly, whereas the function of NCX3 under physiological conditions has been determined, its role under anoxia is still unknown. To assess NCX3 role in cerebral ischemia, we exposed ncx3−/− mice to transient middle cerebral artery occlusion followed by reperfusion. In addition, to evaluate the effect of ncx3 ablation on neuronal survival, organotypic hippocampal cultures and primary cortical neurons from ncx3−/− mice were subjected to oxygen glucose deprivation (OGD) plus reoxygenation. Here we report that ncx3 gene suppression leads to a worsening of brain damage after focal ischemia and to a massive neuronal death in all the hippocampal fields of organotypic cultures as well as in cortical neurons from ncx3−/− mice exposed to OGD plus reoxygenation. In addition, in ncx3−/− cortical neurons exposed to hypoxia, NCX currents, recorded in the reverse mode of operation, were significantly lower than those detected in ncx3+/+. From these results, NCX3 protein emerges as a new molecular target that may have a potential therapeutic value in modulating cerebral ischemia.
- Published
- 2008
166. Up-regulation and increased activity of KV3.4 channels and their accessory subunit MinK-related peptide 2 induced by amyloid peptide are involved in apoptotic neuronal death
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Maurizio Taglialatela, G.F. Di Renzo, Anna Pannaccione, Pasqualina Castaldo, Lucio Annunziato, Francesca Boscia, Rossana Sirabella, Annagrazia Adornetto, Antonella Scorziello, Pannaccione, Anna, Boscia, Francesca, Scorziello, Antonella, Adornetto, Annagrazia, Castaldo, P, Sirabella, Rossana, Taglialatela, Maurizio, DI RENZO, GIANFRANCO MARIA LUIGI, and Annunziato, Lucio
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Programmed cell death ,Patch-Clamp Techniques ,Protein subunit ,Peptide ,Apoptosis ,Biology ,Hippocampus ,PC12 Cells ,Cnidarian Venoms ,medicine ,Animals ,Patch clamp ,RNA, Messenger ,Rats, Wistar ,Cells, Cultured ,Pharmacology ,chemistry.chemical_classification ,Neurons ,Amyloid beta-Peptides ,Cell Death ,Neurodegeneration ,P3 peptide ,NF-kappa B ,NFKB1 ,medicine.disease ,Molecular biology ,Peptide Fragments ,Rats ,Up-Regulation ,Electrophysiology ,Nerve growth factor ,Sea Anemones ,chemistry ,Shaw Potassium Channels ,Molecular Medicine ,Peptides - Abstract
The aim of the present study was to investigate whether K(V)3.4 channel subunits are involved in neuronal death induced by neurotoxic beta-amyloid peptides (Abeta). In particular, to test this hypothesis, three main questions were addressed: 1) whether the Abeta peptide can up-regulate both the transcription/translation and activity of K(V)3.4 channel subunit and its accessory subunit, MinK-related peptide 2 (MIRP2); 2) whether the increase in K(V)3.4 expression and activity can be mediated by the nuclear factor-kappaB (NF-kappaB) family of transcriptional factors; and 3) whether the specific inhibition of K(V)3.4 channel subunit reverts the Abeta peptide-induced neurodegeneration in hippocampal neurons and nerve growth factor (NGF)-differentiated PC-12 cells. We found that Abeta(1-42) treatment induced an increase in K(V)3.4 and MIRP2 transcripts and proteins, detected by reverse transcription-polymerase chain reaction and Western blot analysis, respectively, in NGF-differentiated PC-12 cells and hippocampal neurons. Patch-clamp experiments performed in whole-cell configuration revealed that the Abeta peptide caused an increase in I(A) current amplitude carried by K(V)3.4 channel subunits, as revealed by their specific blockade with blood depressing substance-I (BDS-I) in both hippocampal neurons and NGF-differentiated PC-12 cells. The inhibition of NF-kappaB nuclear translocation with the cell membrane-permeable peptide SN-50 prevented the increase in K(V)3.4 protein and transcript expression. In addition, the SN-50 peptide was able to block Abeta(1-42)-induced increase in K(V)3.4 K(+) currents and to prevent cell death caused by Abeta(1-42) exposure. Finally, BDS-I produced a similar neuroprotective effect by inhibiting the increase in K(V)3.4 expression. As a whole, our data indicate that K(V)3.4 channels could be a novel target for Alzheimer's disease pharmacological therapy.
- Published
- 2007
167. Nuclear factor-kappa B activation by reactive oxygen species mediates voltage-gated K+ current enhancement by neurotoxic beta-amyloid peptides in nerve growth factor-differentiated PC-12 cells and hippocampal neurones
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Pannaccione, A, Secondo, A, Scorziello, A, Cali, G, Taglialatela, Maurizio, Annunziato, L., Pannaccione, Anna, Secondo, Agnese, Scorziello, Antonella, Calì, G, Taglialatela, Maurizio, and Annunziato, Lucio
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Patch-Clamp Techniques ,Time Factors ,Charybdotoxin ,Blotting, Western ,nuclear factor-κB ,Hippocampus ,PC12 Cells ,Membrane Potentials ,Cnidarian Venoms ,GAP-43 Protein ,mental disorders ,Nerve Growth Factor ,Potassium Channel Blockers ,intracellular Ca2+ concentration ,Animals ,Drug Interactions ,4-Aminopyridine ,Enzyme Inhibitors ,Vitamin A ,Egtazic Acid ,Cells, Cultured ,Chelating Agents ,Neurons ,Protein Synthesis Inhibitors ,reactive oxygen specie ,Amyloid beta-Peptides ,Dose-Response Relationship, Drug ,neuronal cell death ,NF-kappa B ,Tetraethylammonium ,Cell Differentiation ,Alzheimer's disease ,voltage-gated K+ channels ,Embryo, Mammalian ,Electric Stimulation ,Peptide Fragments ,Rats ,Enzyme Activation ,Actin Cytoskeleton ,Potassium Channels, Voltage-Gated ,Dactinomycin ,Calcium ,Peptides ,Reactive Oxygen Species - Abstract
Increased activity of plasma membrane K+ channels, leading to decreased cytoplasmic K+ concentrations, occurs during neuronal cell death. In the present study, we showed that the neurotoxic beta-amyloid peptide Abeta(25-35) caused a dose-dependent (0.1-10 microm) and time-dependent (> 12 h) enhancement of both inactivating and non-inactivating components of voltage-dependent K+ (VGK) currents in nerve growth factor-differentiated rat phaeochromocytoma (PC-12) cells and primary rat hippocampal neurones. Similar effects were exerted by Abeta(1-42), but not by the non-neurotoxic Abeta(35-25) peptide. Abeta(25-35) and Abeta(1-42) caused an early (15-20 min) increase in intracellular Ca(2+) concentration. This led to an increased production of reactive oxygen species (ROS), which peaked at 3 h and lasted for 24 h; ROS production seemed to trigger the VGK current increase as vitamin E (50 microm) blocked both the Abeta(25-35)- and Abeta(1-42)-induced ROS increase and VGK current enhancement. Inhibition of protein synthesis (cycloheximide, 1 microg/mL) and transcription (actinomycin D, 50 ng/mL) blocked Abeta(25-35)-induced VGK current enhancement, suggesting that this potentiation is mediated by transcriptional activation induced by ROS. Interestingly, the specific nuclear factor-kappaB inhibitor SN-50 (5 microm), but not its inactive analogue SN-50M (5 microm), fully counteracted Abeta(1-42)- or Abeta(25-35)-induced enhancement of VGK currents, providing evidence for a role of this family of transcription factors in regulating neuronal K+ channel function during exposure to Abeta.
- Published
- 2005
168. GABA and glutamate receptors are involved in modulating pacemaker activity in hydra
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G Kass-Simon, P Pierobon, A Pannaccione, KASS SIMON, G, Pannaccione, Anna, and Pierobon, P.
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Agonist ,medicine.medical_specialty ,Physiology ,medicine.drug_class ,Hydra ,Glutamic Acid ,glutamate ,Kainate receptor ,AMPA receptor ,Biology ,Biochemistry ,GABA Antagonists ,GABA ,chemistry.chemical_compound ,Receptors, GABA ,Biological Clocks ,Internal medicine ,Culture Techniques ,Quinoxalines ,medicine ,Excitatory Amino Acid Agonists ,Animals ,GABA-A Receptor Agonists ,GABA-A Receptor Antagonists ,Molecular Biology ,GABA Agonists ,alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid ,gamma-Aminobutyric Acid ,Kainic Acid ,Glutamate receptor ,LGIC ,Bicuculline ,GABA receptor antagonist ,Endocrinology ,nervous system ,chemistry ,Receptors, Glutamate ,pacemaker systems ,embryonic structures ,NBQX ,Excitatory Amino Acid Antagonists ,medicine.drug ,Ionotropic effect - Abstract
The effects of gamma-amino butyric acid (GABA) and glutamate, their ionotropic agonists and antagonists on hydra’s ectodermal and endodermal pacemaker systems were studied. GABA decreased ectodermal body contraction bursts (CBs) and the number of pulses in a burst (PyCB) and endodermal rhythmic potentials (RPs); tentacle pulses (TPs) were not affected. The GABA agonist, muscimol, and the benzodiazepine receptor agonist, diazepam, mimicked the A effects of GABA on the endodermal system. The GABA antagonist bicuculline counteracted GABA’s effects. Low A concentrations of glutamate increased CBs and RPs. Higher concentrations required concanavalin A (Con A) to produce the same effect on CBs and PyCB. TPs were increased by high concentrations of glutamate and kainate. The ionotropic glutamate agonist, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) also required Con A to increase CBs and RPs. The effects of AMPA were antagonized by 6-nitro-7-sulfamoylbenzowfxquinoxaline-2,3-dione (NBQX), which, per se, decreased CBs. The results indicate that GABA and glutamate, acting on their ionotropic receptors, modify the impulses of hydra’s pacemaker systems. On the whole GABA decreased the outputs of both ectodermal and endodermal impulse generating systems, while glutamate increased them. 2003 Elsevier Science Inc. All rights reserved.
- Published
- 2003
169. Apoptosis induced in neuronal cells by oxidative stress: role played by caspases and intracellular calcium ions
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Giuseppe Pignataro, Lucio Annunziato, Luigi Sibaud, Rossana Sirabella, Mauro Cataldi, Angela D'Alessio, Salvatore Amoroso, G.F. Di Renzo, Agnese Secondo, Anna Pannaccione, Annunziato, L, Amoroso, S, Pannaccione, Anna, Cataldi, Mauro, Pignataro, Giuseppe, D'Alessio, A, Sirabella, Rossana, Secondo, Agnese, Sibaud, L, DI RENZO, GIANFRANCO MARIA LUIGI, Annunziato, L., Amoroso, S., D'Alessio, A., Sibaud, L., Annunziato, Lucio, Amoroso, Salvatore, and D'Alessio, Angela
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Programmed cell death ,Free Radicals ,Caspase 2 ,Caspase 3 ,Apoptosis ,Biology ,Toxicology ,medicine.disease_cause ,Calcium in biology ,medicine ,Animals ,Humans ,Calcium ions ,chemistry.chemical_classification ,Neurons ,Reactive oxygen species ,General Medicine ,Caspase ,Cell biology ,Neuronal apoptosi ,Enzyme Activation ,Oxidative Stress ,chemistry ,Caspases ,biology.protein ,Oxidative stre ,Calcium ,Oxidative stress ,Intracellular - Abstract
Reactive oxygen species (ROS) have been implicated in the pathophysiology of many neurologic disorders and brain dysfunction. In the same pathological settings evidence has been provided in favour of a participation of intracellular Ca(2+) concentration altered homeostasis in the chain of events leading to neuronal apoptosis. In the present review literature reports and experimental data on the relationship between caspase activation and alteration of intracellular calcium concentrations in the mechanisms triggering neuronal apoptosis are discussed. The data gathered support the conclusion that during oxidative stress in neuronal cells the production of ROS triggers a mechanism that, through the release of cytochrome c from mitochondria and caspase-3 activation, leads to apoptosis; the concomitant ROS-mediated elevation of intracellular Ca(2+) concentration triggers caspase-2 activation but both events do not seem to be involved in cell death.
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- 2003
170. Modulation of ion channels by reactive oxygen and nitrogen species: a pathophysiological role in brain aging?
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Maurizio Taglialatela, Mauro Cataldi, Pasqualina Castaldo, Gianfranco Di Renzo, Anna Pannaccione, Agnese Secondo, Lucio Annunziato, Annunziato, Lucio, Pannaccione, Anna, Cataldi, Mauro, Secondo, Agnese, Castaldo, Pasqualina, DI RENZO, GIANFRANCO MARIA LUIGI, Taglialatela, Maurizio, Annunziato, L, Castaldo, P, and Taglialatela, M.
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Voltage-gated calcium channel ,Aging ,Neuronal death ,Ion Channels ,chemistry.chemical_compound ,Voltage-gated sodium channel ,Humans ,Voltage-gated potassium channel ,Ion channel ,Reactive nitrogen species ,Human ether-a-gogo related gene-1 (hERG1 or KCNH2) ,Aged ,Voltage-dependent calcium channel ,Chemistry ,General Neuroscience ,Sodium channel ,Reactive oxygen species (ROS) ,Apoptosi ,Brain ,Reactive Nitrogen Species ,Electrophysiology ,Ion homeostasis ,Biochemistry ,Reactive nitrogen species (RNS) ,Biophysics ,Neurology (clinical) ,Geriatrics and Gerontology ,Reactive Oxygen Species ,Intracellular ,Developmental Biology - Abstract
An ever increasing number of reports shows the involvement of free radicals in the functional and structural changes occurring in the brain as a part of the "normal" aging process. Given that plasma membrane and intracellular ion channels play a critical role in maintaining intracellular ion homeostasis, which is crucial for neuronal cell survival, in the present review we have attempted to elaborate on the idea that functional changes in ion channel activity induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS) might occur during the aging process. To this aim, we have reviewed the available literature and the data obtained in our laboratory on the ability of ROS and RNS to modify the activity of several plasma membrane and intracellular ion channels and transporters, in an attempt to correlate such changes with those occurring with the aging process. Particular emphasis is given to voltage-gated Na(+), Ca(2+), and K(+) channels, although second messenger-activated channels like Ca(2+)- and ATP-dependent K(+) channels, and intracellular channels controlling intracellular Ca(2+) storage and release will also be discussed. On the basis of the available data it is not yet possible to establish a strict correlation between the changes in neuronal electrophysiological properties induced by oxidative modification at the level of ion channels and the neurodegenerative process accompanying brain aging. However, an increasing amount of information suggests that the modulatory effects exerted by ROS and RNS on ion channel proteins might have a relevant role for neuronal cell survival or death. Obviously, more work is needed to establish the possible involvement of ion channels and of their modulation by ROS and RNS as important mechanisms for the aging process. Only when a more complete molecular picture of the aging process will be available, it will be possible to test the fascinating hypothesis that aging might be pharmacologically delayed by modulating ROS and RNS action on ion channels or the biochemical pathways involved in their modulation.
- Published
- 2002
171. 'Janus face' of nitric oxide action on plasma membrane and intracellular ionic channels
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Agnese Secondo, Anna Pannaccione, G.F. Di Renzo, Maurizio Taglialatela, Pasqualina Castaldo, Mauro Cataldi, S. Iossa, L. Annunziato, Taglialatela, Maurizio, Pannaccione, Anna, Cataldi, Mauro, Castaldo, P, Secondo, Agnese, Iossa, S, DI RENZO, GIANFRANCO MARIA LUIGI, Annunziato, L., Taglialatela, M, M., Taglialatela, P., Castaldo, S., Iossa, and Annunziato, Lucio
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Aging ,chemistry.chemical_compound ,Health (social science) ,Membrane ,Chemistry ,Biophysics ,Plasma ,Janus ,Geriatrics and Gerontology ,Gerontology ,Ionic Channels ,Intracellular ,Nitric oxide - Published
- 2001
172. Inhibition of depolarization-induced [3H]noradrenaline release from SH-SY5Y human neuroblastoma cells by some second-generation H(1) receptor antagonists through blockade of store-operated Ca(2+) channels (SOCs)
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Taglialatela, Maurizio, Secondo, A, Fresi, A, Rosati, B, Pannaccione, A, Castaldo, P, Giorgio, G, Wanke, E, Annunziato, L., Taglialatela, Maurizio, Secondo, Agnese, Fresi, A, Rosati, B, Pannaccione, Anna, Castaldo, P, Giorgio, G, Wanke, E, Annunziato, L., Taglialatela, M, and Annunziato, Lucio
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ERG1 Potassium Channel ,Potassium Channels ,Depolarization-induced norepinephrine release ,Tritium ,Neuroblastoma ,Norepinephrine ,Transcriptional Regulator ERG ,Tumor Cells, Cultured ,Humans ,Receptors, Histamine H1 ,Cation Transport Proteins ,Second-generation antihistamine ,Ca2+ channels activated by Cai2+ store depletion ,hERG K+ channel ,Astemizole ,Loratadine ,Calcium Channel Blockers ,Cetirizine ,Ether-A-Go-Go Potassium Channels ,DNA-Binding Proteins ,Potassium Channels, Voltage-Gated ,Hydroxyzine ,Histamine H1 Antagonists ,Trans-Activators ,SH-SY5Y human neuroblastoma cell ,Calcium ,Calcium Channels ,Terfenadine ,Long QT syndrome - Abstract
In the present study, the effect of the blockade of membrane calcium channels activated by intracellular Ca(2+) store depletion on basal and depolarization-induced [3H]norepinephrine ([3H]NE) release from SH-SY5Y human neuroblastoma cells was examined. The second-generation H(1) receptor blockers astemizole, terfenadine, and loratadine, as well as the first-generation compound hydroxyzine, inhibited [3H]NE release induced by high extracellular K(+) concentration ([K(+)](e)) depolarization in a concentration-dependent manner (the IC(50)s were 2.3, 1.7, 4.8, and 9.4 microM, respectively). In contrast, the more hydrophilic second-generation H(1) receptor blocker cetirizine was completely ineffective (0.1-30 microM). The inhibition of high [K(+)](e)-induced [3H]NE release by H(1) receptor blockers seems to be related to their ability to inhibit Ca(2+) channels activated by Ca(i)(2+) store depletion (SOCs). In fact, astemizole, terfenadine, loratadine, and hydroxyzine, but not cetirizine, displayed a dose-dependent inhibitory action on the increase in intracellular Ca(2+) concentrations ([Ca(2+)](i)) obtained with extracellular Ca(2+) reintroduction after Ca(i)(2+) store depletion with thapsigargin (1 microM), an inhibitor of the sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA) pump. The rank order of potency for SOC inhibition by these compounds closely correlated with their inhibitory properties on depolarization-induced [3H]NE release from SH-SY5Y human neuroblastoma cells. Nimodipine (1 microM) plus omega-conotoxin (100 nM) did not interfere with the present model for SOC activation. In addition, the inhibition of depolarization-induced [3H]NE release does not seem to be attributable to the blockade of the K(+) currents carried by the K(+) channels encoded by the human Ether-a-Gogo Related Gene (I(HERG)) by these antihistamines. In fact, whole-cell voltage-clamp experiments revealed that the IC(50) for astemizole-induced hERG blockade is about 300-fold lower than that for the inhibition of high K(+)-induced [3H]NE release. Furthermore, current-clamp experiments in SH-SY5Y cells showed that concentrations of astemizole (3 microM) which were effective in preventing depolarization-induced [3H]NE release were unable to interfere with the cell membrane potential under depolarizing conditions (100 mM [K(+)](e)), suggesting that hERG K(+) channels do not contribute to membrane potential control during exposure to elevated [K(+)](e). Collectively, the results of the present study suggest that, in SH-SY5Y human neuroblastoma cells, the inhibition of SOCs by some second-generation antihistamines can prevent depolarization-induced neurotransmitter release.
- Published
- 2001
173. Inhibition of HERG1 K+ channels by the novel second-generation antihistamine mizolastine
- Author
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Taglialatela, M., Anna PANNACCIONE, Castaldo, P., Giorgio, G., Annunziato, L., Taglialatela, Maurizio, Pannaccione, Anna, Castaldo, P, Giorgio, G, Annunziato, Lucio, and Annunziato, L.
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drug cardiotoxicity ,ERG1 Potassium Channel ,Potassium Channels ,Xenopus ,Astemizole ,antihistamine ,Ether-A-Go-Go Potassium Channels ,Cell Line ,DNA-Binding Proteins ,Transcriptional Regulator ERG ,Potassium Channels, Voltage-Gated ,Papers ,Histamine H1 Antagonists ,Trans-Activators ,mizolastine ,Animals ,Humans ,Benzimidazoles ,Female ,Potassium channel ,Cation Transport Proteins ,arrhythmias - Abstract
1. Ventricular arrhythmias are rare but life-threatening side effects of therapy with the second-generation H(1) receptor antagonists terfenadine and astemizole. Blockade of the K(+) channels encoded by the Human Ether-à-go-go-Related Gene 1 (HERG1) K(+) channels, which is the molecular basis of the cardiac repolarizing current I(Kr), by prolonging cardiac repolarization, has been recognized as the mechanism underlying the cardiac toxicity of these compounds. 2. In the present study, the potential blocking ability of the novel second-generation H(1) receptor antagonist mizolastine of the HERG1 K(+) channels heterologously expressed in XENOPUS: oocytes and in HEK 293 cells or constitutively present in SH-SY5Y human neuroblastoma cells has been examined and compared to that of astemizole. 3. Mizolastine blocked HERG1 K(+) channels expressed in XENOPUS: oocytes with an estimated IC(50) of 3.4 microM. Mizolastine blockade was characterized by a fast dissociation rate when compared to that of astemizole; when fitted to a monoexponential function, the time constants for drug dissociation from the K(+) channel were 72.4+/-11.9 s for 3 microM mizolastine, and 1361+/-306 s for 1 microM astemizole. 4. In human embryonic kidney 293 cells (HEK 293 cells) stably transfected with HERG1 cDNA, extracellular application of mizolastine exerted a dose-related inhibitory action on I(HERG1), with an IC(50) of 350+/-76 nM. Furthermore, mizolastine dose-dependently inhibited HERG1 K(+) channels constitutively expressed in SH-SY5Y human neuroblastoma clonal cells. 5. The results of the present study suggest that the novel second-generation H(1) receptor antagonist mizolastine, in concentrations higher than those achieved in vivo during standard therapy, is able to block in some degree both constitutively and heterologously expressed HERG1 K(+) channels, and confirm the heterogeneity of molecules belonging to this therapeutical class with respect to their HERG1-inhibitory action.
- Published
- 2000
174. Electrophysiological and molecular basis for the adverse cardiovascular effects of histamine H1 receptor antagonists
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Anna Pannaccione, L And Annunziato, Giovanna Giorgio, Maurizio Taglialatela, Pasqualina Castaldo, M. Taglialatela, A. Pannaccione, P. Castaldo, G. Giorgio, L. Annunziato, Taglialatela, Maurizio, Pannaccione, Anna, P., Castaldo, G., Giorgio, and Annunziato, Lucio
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Histamine H1 Receptor Antagonists ,Electrophysiology ,medicine.medical_specialty ,Endocrinology ,business.industry ,Internal medicine ,medicine ,Pharmacology ,business ,antihistamine ,H1 receptor - Abstract
Electrophysiological and molecular biology techniques have widely expanded our knowledge of the diverse functions where K+ channels are implicated as potential and proven pharmacological targets. The aim of the present commentary is to review the recent progress in the understanding of the functional role of the K+ channels encoded by the human ether-a-gogo related gene (HERG), with particular emphasis on their direct pharmacological modulation by drugs, or on their regulation by pharmacologically relevant phenomena. About 3 years have passed since the cloning, expression, and description of the pathophysiological role of HERG K+ channels in human cardiac repolarization. Despite this short lapse of time, these K+ channels have already gained considerable attention as pharmacological targets. In fact, interference with HERG K+ channels seems to be the main mechanism explaining both the therapeutic actions of the class III antiarrhythmics and the potential cardiotoxicity of second-generation H1 receptor antagonists such as terfenadine and astemizole, as well as of psychotropic drugs such as some antidepressants and neuroleptics. It seems possible to anticipate that the main tasks for future investigation will be, on the one side, the better understanding of the intimate mechanism of action of HERG K+ channel-blocking drugs in order to elucidate the conditions regulating the delicate balance between antiarrhythmic and proarrhythmic potential and, on the other, to unravel the pathophysiological role of this K+ channel in the function of the brain and of other excitable tissues.
- Published
- 2000
175. Modulation of the K+ channels encoded by the human ethera-gogo-related gene-1 (hERG1) by nitric oxide
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Taglialatela, M., Pannaccione, A., Iossa, S., Castaldo, P., LUCIO ANNUNZIATO, Taglialatela, M, Pannaccione, Anna, Iossa, S, Castaldo, P, Annunziato, Lucio, and Taglialatela, Maurizio
- Subjects
K(+) channels. human ether-a-gogo-related gene-1 (hERG1) ,nitric oxide - Abstract
The inhibition of nitric oxide synthase by N-nitro-L-arginine methyl ester (0.03-3 mM) dose-dependently reduced nitric oxide (NO(*)) levels and enhanced the outward currents carried by human ether-a-gogo-related gene-1 (hERG1) K(+) channels expressed in Xenopus laevis oocytes, whereas the increase in NO(*) levels achieved by exposure to L-arginine (0.03-10 mM) inhibited these currents. Furthermore, four NO(*) donors belonging to such different chemical classes as sodium nitroprusside (1-1000 microM), 3-morpholino-sydnonimine (100-1000 microM), (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1, 2-diolate (NOC-18; 1-300 microM), and S-nitroso N-acetylpenicillamine (1-300 microM) dose-dependently inhibited hERG1 outward K(+) currents. By contrast, the NO(*) donor NOC-18 (0.3 mM) did not affect other cloned K(+) channels such as rat neuroblastoma-glioma K(+) channel 2, rat delayed rectifier K(+) channel 1, bovine ether-a-gogo gene, rat ether-a-gogo-related gene-2, and rat ether-a-gogo-related gene-3. The inhibitory effect of NO(*) donors on hERG1 K(+) channels was prevented by the NO(*) scavengers 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide and hemoglobin. The membrane permeable analog of cGMP, 8-bromo-cGMP (1 mM), failed to reproduce the inhibitory action of NO(*) donors on hERG1 outward currents; furthermore, the specific inhibitor of the NO(*)-dependent guanylyl cyclase, 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxalin-1-one (50 microM), neither interfered with outward hERG1 K(+) currents nor prevented their inhibition by 0.3 mM NOC-18. Both L-arginine (10 mM) and NOC-18 (0.3 mM) counteracted the stimulatory effect on hERG1 outward currents induced by the radical oxygen species-generating system FeSO(4) (25 microM)/ascorbic acid (50 microM; Fe/Asc). Finally, L-arginine (10 mM) and NOC-18 (0.3 mM) inhibited both basal and Fe/Asc (0.1 mM/0.2 mM)-stimulated lipid peroxidation in X. laevis oocytes. Collectively, the present results suggest that NO(*), both endogenously produced and pharmacologically delivered, may exert in a cGMP-independent way an inhibitory effect on hERG1 outward K(+) currents via an interaction with radical oxygen species either generated under resting conditions or triggered by Fe/Asc.
- Published
- 1999
176. Cardiac ion channels and antihistamines: possible mechanisms of cardiotoxicity
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Taglialatela, M., Castaldo, P., Anna PANNACCIONE, Giorgio, G., Genovese, A., Marone, G., Annunziato, L., Taglialatela, Maurizio, Castaldo, Pasqualina, Pannaccione, Anna, Giorgio, G, Genovese, Arturo, Marone, G, and Annunziato, Lucio
- Subjects
ERG1 Potassium Channel ,Potassium Channels ,Histamine Antagonists ,cardiotoxicity ,ion channels ,Heart ,antihistamine ,Ether-A-Go-Go Potassium Channels ,DNA-Binding Proteins ,Electrocardiography ,Structure-Activity Relationship ,Transcriptional Regulator ERG ,Potassium Channels, Voltage-Gated ,Potassium Channel Blockers ,Trans-Activators ,Humans ,Cation Transport Proteins - Abstract
Despite the enormous success of second generation antihistamines, in the mid-1980s, about 10 years after their introduction in the market, several reports appeared in the literature indicating the rare occurrence of a form of polymorphic ventricular dysrhythmia, the 'torsade de pointes', after the administration of astemizole or terfenadine. This cardiac side-effect has been interpreted as a consequence of the interference of these drugs with cardiac K+ channels involved in action potential repolarization, and in particular with the IKr component of the cardiac repolarizing current. As the K+ channels encoded by the human ether-a-gogo-related gene (HERG) seem to represent the molecular basis of IKr, this cardiac K+ channel was soon recognized as a primary target for second generation antihistamine-induced proarrhythmic effects. In fact, both terfenadine and astemizole have been shown to block HERG K+ channels in a concentration range similar to that found in the plasma of subjects with cardiotoxic manifestations. However, no correlation can be found between the ability to prolong the cardiac action potential duration and the H1-antagonistic activity by several antihistamines, suggesting that HERG blockade and cardiotoxic potential are not class properties of second generation antihistamines. In fact, other molecules such as cetirizine, loratadine, acrivastine, and fexofenadine seem to lack both cardiotoxic potential and HERG-blocking ability at therapeutically relevant concentrations. The marked heterogeneity displayed by second generation antihistamines in their ability to prolong the cardiac action potential duration and to block HERG K+ channels might be of considerable therapeutical significance for those patients at risk of developing cardiac dysrhythmias and in need of therapy with H1-receptor blockers; it also emphasizes the importance of an evaluation of the possible blockade of HERG K+ channels during the early developmental phases of novel compounds belonging to this therapeutical class.
- Published
- 1999
177. Molecular basis for the lack of HERG K+ channel block-related cardiotoxicity by the H1 receptor blocker cetirizine compared with other second-generation antihistamines
- Author
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Gianni Marone, Zhengfeng Zhou, Giovanna Giorgio, Craig T. January, Maurizio Taglialatela, Arturo Genovese, Pasqualina Castaldo, Lucio Annunziato, Anna Pannaccione, Taglialatela, Maurizio, Pannaccione, Anna, Castaldo, Pasqualina, Giorgio, G, Zhou, Z, January, Ct, Genovese, Arturo, Marone, G, Annunziato, Lucio, Taglialatela, M, Castaldo, P, Annunziato, L., and Marone, Gianni
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ERG1 Potassium Channel ,Potassium Channels ,Tertiary amine ,hERG ,Histamine H1 receptor ,Pharmacology ,Loratadine ,Transfection ,Xenopus laevis ,Transcriptional Regulator ERG ,Tumor Cells, Cultured ,medicine ,Animals ,Humans ,Terfenadine ,Receptors, Histamine H1 ,cardiovascular diseases ,HERG K+ channel ,cetirizine ,Cation Transport Proteins ,H1 receptor ,Voltage-gated ion channel ,biology ,Chemistry ,Heart ,Astemizole ,Ether-A-Go-Go Potassium Channels ,Potassium channel ,DNA-Binding Proteins ,Electrophysiology ,Potassium Channels, Voltage-Gated ,Histamine H1 Antagonists ,Oocytes ,Trans-Activators ,biology.protein ,Molecular Medicine ,Female ,medicine.drug - Abstract
In the current study, the potential blocking ability of K+ channels encoded by the human ether-a-go-go related gene (HERG) by the piperazine H1 receptor antagonist cetirizine has been examined and compared with that of other second-generation antihistamines (astemizole, terfenadine, and loratadine). Cetirizine was completely devoid of any inhibitory action on HERG K+ channels heterologously expressed in Xenopus laevis oocytes in concentrations up to 30 microM. On the other hand, terfenadine and astemizole effectively blocked HERG K+ channels with nanomolar affinities (the estimated IC50 values were 330 and 480 nM, respectively), whereas loratadine was approximately 300-fold less potent (IC50 approximately 100 microM). In addition, in contrast to terfenadine, cetirizine did not show use-dependent blockade. In SH-SY5Y cells, a human neuroblastoma clone that constitutively expresses K+ currents carried by HERG channels (IHERG), as well as in human embryonic kidney 293 cells stably transfected with HERG cDNA, extracellular perfusion with 3 microM cetirizine did not exert any inhibitory action on IHERG. Astemizole (3 microM), on the other hand, was highly effective. Terfenadine (3 microM) caused a marked (approximately 80%) inhibition of IHERG in SH-SY5Y cells, whereas loratadine, at the same concentration, caused a 40% blockade. Furthermore, the application of cetirizine (3 microM) on the intracellular side of the membrane of HERG-transfected human embryonic kidney 293 cells did not affect IHERG, whereas the same intracellular concentration of astemizole caused a complete block. The results of the current study suggest that second-generation antihistamines display marked differences in their ability to block HERG K+ channels. Cetirizine in particular, which possesses more polar and smaller substituent groups attached to the tertiary amine compared with other antihistamines, lacks HERG-blocking properties, possibly explaining the absence of torsade de pointes ventricular arrhythmias associated with its therapeutical use.
- Published
- 1998
178. Regulation of the human ether-a-gogo related gene (HERG) K+ channels by reactive oxygen species
- Author
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Lucio Annunziato, Anna Pannaccione, S. Iossa, Eckhard Ficker, Angela Fresi, Maurizio Taglialatela, Pasqualina Castaldo, Taglialatela, Maurizio, Castaldo, Pasqualina, Iossa, S, Pannaccione, Anna, Fresi, A, Ficker, E, Annunziato, Lucio, Taglialatela, M, Castaldo, P, and Annunziato, L.
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ERG1 Potassium Channel ,Potassium Channels ,Xenopus ,hERG ,Ascorbic Acid ,reactive oxygen species (ROS) ,Ferric Compounds ,Membrane Potentials ,Superoxide dismutase ,Transcriptional Regulator ERG ,Malondialdehyde ,Animals ,Humans ,Cloning, Molecular ,Cation Transport Proteins ,chemistry.chemical_classification ,Membrane potential ,Reactive oxygen species ,Multidisciplinary ,biology ,Voltage-gated ion channel ,Free Radical Scavengers ,Biological Sciences ,Ascorbic acid ,Ether-A-Go-Go Potassium Channels ,Recombinant Proteins ,Potassium channel ,DNA-Binding Proteins ,Kinetics ,Biochemistry ,chemistry ,Potassium Channels, Voltage-Gated ,Human ether-a-gogo related gene (HERG) ,Oocytes ,Trans-Activators ,biology.protein ,Biophysics ,Female ,K+ channels ,Reactive Oxygen Species - Abstract
Human ether-a-gogo related gene (HERG) K+channels are key elements in the control of cell excitability in both the cardiovascular and the central nervous systems. For this reason, the possible modulation by reactive oxygen species (ROS) of HERG and other cloned K+channels expressed inXenopusoocytes has been explored in the present study. Exposure ofXenopusoocytes to an extracellular solution containing FeSO4(25–100 μM) and ascorbic acid (50–200 μM) (Fe/Asc) increased both malondialdehyde content and 2′,7′-dichlorofluorescin fluorescence, two indexes of ROS production. Oocyte perfusion with Fe/Asc caused a 50% increase of the outward K+currents carried by HERG channels, whereas inward currents were not modified. This ROS-induced increase in HERG outward K+currents was due to a depolarizing shift of the voltage-dependence of channel inactivation, with no change in channel activation. No effect of Fe/Asc was observed on the expressed K+currents carried by other K+channels such as bEAG, rDRK1, and mIRK1. Fe/Asc-induced stimulation of HERG outward currents was completely prevented by perfusion of the oocytes with a ROS scavenger mixture (containing 1,000 units/ml catalase, 200 ng/ml superoxide dismutase, and 2 mM mannitol). Furthermore, the scavenger mixture also was able to reduce HERG outward currents in resting conditions by 30%, an effect mimicked by catalase alone. In conclusion, the present results seem to suggest that changes in ROS production can specifically influence K+currents carried by the HERG channels.
- Published
- 1997
179. Na + /Ca 2+ exchanger isoform 1 (NCX1) and canonical transient receptor potential channel 6 (TRPC6) are recruited by STIM1 to mediate Store-Operated Calcium Entry in primary cortical neurons.
- Author
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Tedeschi V, Sisalli MJ, Pannaccione A, Piccialli I, Molinaro P, Annunziato L, and Secondo A
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- Animals, Calcium Signaling, Membrane Proteins metabolism, Mice, ORAI1 Protein genetics, Protein Isoforms genetics, Rats, Calcium metabolism, Neurons metabolism, Sodium-Calcium Exchanger genetics, Stromal Interaction Molecule 1 genetics, TRPC6 Cation Channel
- Abstract
Excessive calcium (Ca
2+ ) release from the endoplasmic reticulum (ER) represents an important hallmark of several neurodegenerative diseases. ER is recharged from Ca2+ through the so-called Store-Operated Calcium Entry (SOCE) thus providing Ca2+ signals to regulate critical cell functions. Single transmembrane-spanning domain protein stromal interacting molecule 1 (STIM1), mainly residing in the ER, and plasmalemmal channel Orai1 represent the SOCE key components at neuronal level. However, many other proteins participate to ER Ca2+ refilling including the Na+ /Ca2+ exchanger isoform 1 (NCX1), whose regulation by ER remains unknown. In this study, we tested the possibility that neuronal NCX1 may take part to SOCE through the interaction with STIM1. In rat primary cortical neurons and in nerve growth factor (NGF)-differentiated PC12 cells NCX1 knocking down by siRNA strategy significantly prevented SOCE as well as SOCE pharmacological inhibition by SKF-96365 and 2-APB. A significant reduction of SOCE was recorded also in synaptosomes from ncx1- /- mice brain compared with ncx1+ /+ mice. Double labeling confocal experiments showed a large co-localization between NCX1 and STIM1 in rat primary cortical neurons. Accordingly, NCX1 and STIM1 co-immunoprecipitated and functionally interacted each other during ischemic preconditioning, a phenomenon inducing ischemic tolerance. However, STIM1 knocking down reduced NCX1 activity recorded by either patch-clamp electrophysiology or Fura-2 single-cell microfluorimetry. Furthermore, canonical transient receptor potential channel 6 (TRPC6) was identified as the mechanism mediating local increase of sodium (Na+ ) useful to drive NCX1 reverse mode and, therefore, NCX1-mediated Ca2+ refilling. In fact, TRPC6 not only interacted with STIM1, as shown by the co-localization and co-immunoprecipitation with the ER Ca2+ sensor, but it also mediated 1,3-Benzenedicarboxylic acid, 4,4'-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,12-benzofurandiyl)]bis-, tetrakis[(acetyloxy)methyl] ester (SBFI)-monitored Na+ increase elicited by thapsigargin in primary cortical neurons. Accordingly, efficient TRPC6 knockdown prevented thapsigargin-induced intracellular Na+ elevation and SOCE. Collectively, we identify NCX1 as a new partner of STIM1 in mediating SOCE, whose activation in the reverse mode may be facilitated by the local increase of Na+ concentration due to the interaction between STIM1 and TRPC6 in primary cortical neurons., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)- Published
- 2022
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180. New Insights into the Structure-Activity Relationship and Neuroprotective Profile of Benzodiazepinone Derivatives of Neurounina-1 as Modulators of the Na + /Ca 2+ Exchanger Isoforms.
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Magli E, Fattorusso C, Persico M, Corvino A, Esposito G, Fiorino F, Luciano P, Perissutti E, Santagada V, Severino B, Tedeschi V, Pannaccione A, Pignataro G, Caliendo G, Annunziato L, Secondo A, and Frecentese F
- Subjects
- Animals, Benzodiazepinones chemistry, Drug Design, Protein Isoforms metabolism, Sodium-Calcium Exchanger metabolism, Structure-Activity Relationship, Benzodiazepinones pharmacology, Neuroprotective Agents pharmacology, Protein Isoforms antagonists & inhibitors, Pyrrolidines chemistry, Sodium-Calcium Exchanger antagonists & inhibitors
- Abstract
Due to the neuroprotective role of the Na
+ /Ca2+ exchanger (NCX) isoforms NCX1 and NCX3, we synthesized novel benzodiazepinone derivatives of the unique NCX activator Neurounina-1 , named compounds 1-19 . The derivatives are characterized by a benzodiazepinonic nucleus linked to five- or six-membered cyclic amines via a methylene, ethylene, or acetyl spacer. The compounds have been screened on NCX1/NCX3 isoform activities by a high-throughput screening approach, and the most promising were characterized by patch-clamp electrophysiology and Fura-2AM video imaging. We identified two novel modulators of NCX: compound 4 , inhibiting NCX1 reverse mode, and compound 14 , enhancing NCX1 and NCX3 activity. Compound 1 displayed neuroprotection in two preclinical models of brain ischemia. The analysis of the conformational and steric features led to the identification of the molecular volume required for selective NCX1 activation for mixed NCX1/NCX3 activation or for NCX1 inhibition, providing the first prototypal model for the design of optimized isoform modulators.- Published
- 2021
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181. The Na + /Ca 2+ Exchanger 3 Is Functionally Coupled With the Na V 1.6 Voltage-Gated Channel and Promotes an Endoplasmic Reticulum Ca 2+ Refilling in a Transgenic Model of Alzheimer's Disease.
- Author
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Piccialli I, Ciccone R, Secondo A, Boscia F, Tedeschi V, de Rosa V, Cepparulo P, Annunziato L, and Pannaccione A
- Abstract
The remodelling of neuronal ionic homeostasis by altered channels and transporters is a critical feature of the Alzheimer's disease (AD) pathogenesis. Different reports converge on the concept that the Na
+ /Ca2+ exchanger (NCX), as one of the main regulators of Na+ and Ca2+ concentrations and signalling, could exert a neuroprotective role in AD. The activity of NCX has been found to be increased in AD brains, where it seemed to correlate with an increased neuronal survival. Moreover, the enhancement of the NCX3 currents (INCX ) in primary neurons treated with the neurotoxic amyloid β 1-42 (Aβ1-42 ) oligomers prevented the endoplasmic reticulum (ER) stress and neuronal death. The present study has been designed to investigate any possible modulation of the INCX , the functional interaction between NCX and the NaV 1.6 channel, and their impact on the Ca2+ homeostasis in a transgenic in vitro model of AD, the primary hippocampal neurons from the Tg2576 mouse, which overproduce the Aβ1-42 peptide. Electrophysiological studies, carried in the presence of siRNA and the isoform-selective NCX inhibitor KB-R7943, showed that the activity of a specific NCX isoform, NCX3, was upregulated in its reverse, Ca2+ influx mode of operation in the Tg2576 neurons. The enhanced NCX activity contributed, in turn, to increase the ER Ca2+ content, without affecting the cytosolic Ca2+ concentrations of the Tg2576 neurons. Interestingly, our experiments have also uncovered a functional coupling between NCX3 and the voltage-gated NaV 1.6 channels. In particular, the increased NaV 1.6 currents appeared to be responsible for the upregulation of the reverse mode of NCX3, since both TTX and the Streptomyces griseolus antibiotic anisomycin, by reducing the NaV 1.6 currents, counteracted the increase of the INCX in the Tg2576 neurons. In agreement, our immunofluorescence analyses revealed that the NCX3/NaV 1.6 co-expression was increased in the Tg2576 hippocampal neurons in comparison with the WT neurons. Collectively, these findings indicate that NCX3 might intervene in the Ca2+ remodelling occurring in the Tg2576 primary neurons thus emerging as a molecular target with a neuroprotective potential, and provide a new outcome of the NaV 1.6 upregulation related to the modulation of the intracellular Ca2+ concentrations in AD neurons., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Piccialli, Ciccone, Secondo, Boscia, Tedeschi, de Rosa, Cepparulo, Annunziato and Pannaccione.)- Published
- 2021
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182. The Anemonia sulcata Toxin BDS-I Protects Astrocytes Exposed to Aβ 1-42 Oligomers by Restoring [Ca 2+ ] i Transients and ER Ca 2+ Signaling.
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Piccialli I, Tedeschi V, Boscia F, Ciccone R, Casamassa A, de Rosa V, Grieco P, Secondo A, and Pannaccione A
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- Animals, Cells, Cultured, Rats, Amyloid beta-Peptides toxicity, Astrocytes drug effects, Calcium metabolism, Calcium Signaling drug effects, Cnidarian Venoms pharmacology, Endoplasmic Reticulum metabolism, Peptide Fragments toxicity
- Abstract
Intracellular calcium concentration ([Ca
2+ ]i ) transients in astrocytes represent a highly plastic signaling pathway underlying the communication between neurons and glial cells. However, how this important phenomenon may be compromised in Alzheimer's disease (AD) remains unexplored. Moreover, the involvement of several K+ channels, including KV 3.4 underlying the fast-inactivating currents, has been demonstrated in several AD models. Here, the effect of KV 3.4 modulation by the marine toxin blood depressing substance-I (BDS-I) extracted from Anemonia sulcata has been studied on [Ca2+ ]i transients in rat primary cortical astrocytes exposed to Aβ1-42 oligomers. We showed that: (1) primary cortical astrocytes expressing KV 3.4 channels displayed [Ca2+ ]i transients depending on the occurrence of membrane potential spikes, (2) BDS-I restored, in a dose-dependent way, [Ca2+ ]i transients in astrocytes exposed to Aβ1-42 oligomers (5 µM/48 h) by inhibiting hyperfunctional KV 3.4 channels, (3) BDS-I counteracted Ca2+ overload into the endoplasmic reticulum (ER) induced by Aβ1-42 oligomers, (4) BDS-I prevented the expression of the ER stress markers including active caspase 12 and GRP78/BiP in astrocytes treated with Aβ1-42 oligomers, and (5) BDS-I prevented Aβ1-42 -induced reactive oxygen species (ROS) production and cell suffering measured as mitochondrial activity and lactate dehydrogenase (LDH) release. Collectively, we proposed that the marine toxin BDS-I, by inhibiting the hyperfunctional KV 3.4 channels and restoring [Ca2+ ]i oscillation frequency, prevented Aβ1-42 -induced ER stress and cell suffering in astrocytes.- Published
- 2020
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183. The new K V 3.4 inhibitor BDS-I[1-8] as a potential pharmacological opportunity in Alzheimer's disease therapy.
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Piccialli I, Ciccone R, and Pannaccione A
- Abstract
Competing Interests: None
- Published
- 2020
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184. The Na + /Ca 2+ exchanger in Alzheimer's disease.
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Pannaccione A, Piccialli I, Secondo A, Ciccone R, Molinaro P, Boscia F, and Annunziato L
- Subjects
- Animals, Homeostasis, Humans, Mitochondria metabolism, Models, Biological, Neuroprotection, Alzheimer Disease metabolism, Sodium-Calcium Exchanger metabolism
- Abstract
As a pivotal player in regulating sodium (Na
+ ) and calcium (Ca2+ ) homeostasis and signalling in excitable cells, the Na+ /Ca2+ exchanger (NCX) is involved in many neurodegenerative disorders in which an imbalance of intracellular Ca2+ and/or Na+ concentrations occurs, including Alzheimer's disease (AD). Although NCX has been mainly implicated in neuroprotective mechanisms counteracting Ca2+ dysregulation, several studies highlighted its role in the neuronal responses to intracellular Na+ elevation occurring in several pathophysiological conditions. Since the alteration of Na+ and Ca2+ homeostasis significantly contributes to synaptic dysfunction and neuronal loss in AD, it is of crucial importance to analyze the contribution of NCX isoforms in the homeostatic responses at neuronal and synaptic levels. Some studies found that an increase of NCX activity in brains of AD patients was correlated with neuronal survival, while other research groups found that protein levels of two NCX subtypes, NCX2 and NCX3, were modulated in parietal cortex of late stage AD brains. In particular, NCX2 positive synaptic terminals were increased in AD cohort while the number of NCX3 positive terminals were reduced. In addition, NCX1, NCX2 and NCX3 isoforms were up-regulated in those synaptic terminals accumulating amyloid-beta (Aβ), the neurotoxic peptide responsible for AD neurodegeneration. More recently, the hyperfunction of a specific NCX subtype, NCX3, has been shown to delay endoplasmic reticulum stress and apoptotic neuronal death in hippocampal neurons exposed to Aβ insult. Despite some issues about the functional role of NCX in synaptic failure and neuronal loss require further studies, these findings highlight the putative neuroprotective role of NCX in AD and open new strategies to develop new druggable targets for AD therapy., Competing Interests: Declaration of Competing Interest The authors declare no competing interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)- Published
- 2020
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185. Nuclear localization of NCX: Role in Ca 2+ handling and pathophysiological implications.
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Secondo A, Petrozziello T, Tedeschi V, Boscia F, Pannaccione A, Molinaro P, and Annunziato L
- Subjects
- Animals, Humans, Models, Biological, Calcium metabolism, Calcium Signaling, Cell Nucleus metabolism, Disease, Sodium-Calcium Exchanger metabolism
- Abstract
Numerous lines of evidence indicate that nuclear calcium concentration ([Ca
2+ ]n ) may be controlled independently from cytosolic events by a local machinery. In particular, the perinuclear space between the inner nuclear membrane (INM) and the outer nuclear membrane (ONM) of the nuclear envelope (NE) likely serves as an intracellular store for Ca2+ ions. Since ONM is contiguous with the endoplasmic reticulum (ER), the perinuclear space is adjacent to the lumen of ER thus allowing a direct exchange of ions and factors between the two organelles. Moreover, INM and ONM are fused at the nuclear pore complex (NPC), which provides the only direct passageway between the nucleoplasm and cytoplasm. However, due to the presence of ion channels, exchangers and transporters, it has been generally accepted that nuclear ion fluxes may occur across ONM and INM. Within the INM, the Na+ /Ca2+ exchanger (NCX) isoform 1 seems to play an important role in handling Ca2+ through the different nuclear compartments. Particularly, nuclear NCX preferentially allows local Ca2+ flowing from nucleoplasm into NE lumen thanks to the Na+ gradient created by the juxtaposed Na+ /K+ -ATPase. Such transfer reduces abnormal elevation of [Ca2+ ]n within the nucleoplasm thus modulating specific transductional pathways and providing a protective mechanism against cell death. Despite very few studies on this issue, here we discuss those making major contribution to the field, also addressing the pathophysiological implication of nuclear NCX malfunction., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2019 Elsevier Ltd. All rights reserved.)- Published
- 2020
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186. The Na + /Ca 2+ exchangers in demyelinating diseases.
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Boscia F, de Rosa V, Cammarota M, Secondo A, Pannaccione A, and Annunziato L
- Subjects
- Animals, Axons metabolism, Axons pathology, Humans, Models, Biological, Nerve Degeneration metabolism, Nerve Degeneration pathology, Oligodendroglia metabolism, Demyelinating Diseases metabolism, Sodium-Calcium Exchanger metabolism
- Abstract
Intracellular [Na
+ ]i and [Ca2+ ]i imbalance significantly contribute to neuro-axonal dysfunctions and maladaptive myelin repair or remyelination failure in chronic inflammatory demyelinating diseases such as multiple sclerosis. Progress in recent years has led to significant advances in understanding how [Ca2+ ]i signaling network drive degeneration or remyelination of demyelinated axons. The Na+ /Ca2+ exchangers (NCXs), a transmembrane protein family including three members encoded by ncx1, ncx2, and ncx3 genes, are emerging important regulators of [Na+ ]i and [Ca2+ ]i both in neurons and glial cells. Here we review recent advance highlighting the role of NCX exchangers in axons and myelin-forming cells, i.e. oligodendrocytes, which represent the major targets of the aberrant inflammatory attack in multiple sclerosis. The contribution of NCX subtypes to axonal pathology and myelin synthesis will be discussed. Although a definitive understanding of mechanisms regulating axonal pathology and remyelination failure in chronic demyelinating diseases is still lacking and requires further investigation, current knowledge suggest that NCX activity plays a crucial role in these processes. Defining the relative contributions of each NCX transporter in axon pathology and myelinating glia will constitute not only a major advance in understanding in detail the intricate mechanism of neurodegeneration and remyelination failure in demyelinating diseases but also will help to identify neuroprotective or remyelinating strategies targeting selective NCX exchangers as a means of treating MS., (Copyright © 2019 Elsevier Ltd. All rights reserved.)- Published
- 2020
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187. Synthesis and Pharmacological Evaluation of a Novel Peptide Based on Anemonia sulcata BDS-I Toxin as a New K V 3.4 Inhibitor Exerting a Neuroprotective Effect Against Amyloid-β Peptide.
- Author
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Ciccone R, Piccialli I, Grieco P, Merlino F, Annunziato L, and Pannaccione A
- Abstract
There is increasing evidence that the fast-inactivating potassium current I
A , encoded by KV 3. 4 channels, plays an important role in Alzheimer's Disease (AD), since the neurotoxic β-amyloid peptide1-42 (Aβ1-42 ) increases the IA current triggering apoptotic processes. The specific inhibition of KV 3.4 by the marine toxin extracted from Anemonia sulcata , named blood depressing substance-I (BDS-I), reverts the Aβ peptide-induced cell death. The aim of the present study was to identify the smallest fragments of BDS-I, obtained by peptide synthesis, able to inhibit KV 3.4 currents. For this purpose, whole-cell patch clamp technique was used to evaluate the effects of BDS-I fragments on KV 3.4 currents in CHO cells heterologously expressing KV 3.4. We found that BDS-I[1-8] fragment, containing the N-terminal octapeptide sequence of full length BDS-I, was able to inhibit KV 3.4 currents in a concentration dependent manner, whereas the scrambled sequence of BDS-I[1-8] and all the other fragments obtained from BDS-I full length were ineffective. As we demonstrated in a previous study, BDS-I full length is able to counteract Aβ1-42 -induced enhancement of KV 3.4 activity, preventing Aβ1-42 -induced caspase-3 activation and the abnormal nuclear morphology in NGF-differentiated PC-12 cells. Similarly to BDS-I, we found that BDS-I[1-8] blocking KV 3.4 currents prevented Aβ1-42 -induced caspase-3 activation and apoptotic processes. Collectively, these results suggest that BDS-I[1-8] could represent a lead compound to be developed as a new drug targeting KV 3.4 channels.- Published
- 2019
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188. Erratum: Author Correction: Na + /Ca 2+ exchanger 1 on nuclear envelope controls PTEN/Akt pathway via nucleoplasmic Ca 2+ regulation during neuronal differentiation.
- Author
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Secondo A, Esposito A, Petrozziello T, Boscia F, Molinaro P, Tedeschi V, Pannaccione A, Ciccone R, Guida N, Di Renzo G, and Annunziato L
- Abstract
[This corrects the article DOI: 10.1038/s41420-017-0018-1.].
- Published
- 2019
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189. D-Aspartate treatment attenuates myelin damage and stimulates myelin repair.
- Author
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de Rosa V, Secondo A, Pannaccione A, Ciccone R, Formisano L, Guida N, Crispino R, Fico A, Polishchuk R, D'Aniello A, Annunziato L, and Boscia F
- Subjects
- Animals, Cell Line, Female, Humans, Male, Mice, Inbred C57BL, Oligodendrocyte Precursor Cells drug effects, Oligodendrocyte Precursor Cells physiology, Rats, Wistar, Receptors, AMPA metabolism, Receptors, N-Methyl-D-Aspartate metabolism, Sodium-Calcium Exchanger metabolism, Treatment Outcome, D-Aspartic Acid administration & dosage, Demyelinating Diseases drug therapy, Myelin Sheath metabolism, Neuroprotective Agents administration & dosage
- Abstract
Glutamate signaling may orchestrate oligodendrocyte precursor cell (OPC) development and myelin regeneration through the activation of glutamate receptors at OPC-neuron synapses. D-Aspartate is a D-amino acid exerting modulatory actions at glutamatergic synapses. Chronic administration of D-Aspartate has been proposed as therapeutic treatment in diseases related to myelin dysfunction and NMDA receptors hypofunction, including schizophrenia and cognitive deficits. Here, we show, by using an in vivo remyelination model, that administration of D-Aspartate during remyelination improved motor coordination, accelerated myelin recovery, and significantly increased the number of small-diameter myelinated axons. Chronically administered during demyelination, D-Aspartate also attenuated myelin loss and inflammation. Interestingly, D-Aspartate exposure stimulated OPC maturation and accelerated developmental myelination in organotypic cerebellar slices. D-Aspartate promoting effects on OPC maturation involved the activation of glutamate transporters, AMPA and NMDA receptors, and the Na
+ /Ca2+ exchanger NCX3. While blocking NMDA or NCX3 significantly prevented D-Aspartate-induced [Ca2+ ]i oscillations, blocking AMPA and glutamate transporters prevented both the initial and oscillatory [Ca2+ ]i response as well as D-Aspartate-induced inward currents in OPC Our findings reveal that D-Aspartate treatment may represent a novel strategy for promoting myelin recovery., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)- Published
- 2019
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190. Synthesis and Biological Evaluation of a New Structural Simplified Analogue of cADPR, a Calcium-Mobilizing Secondary Messenger Firstly Isolated from Sea Urchin Eggs.
- Author
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D'Errico S, Borbone N, Catalanotti B, Secondo A, Petrozziello T, Piccialli I, Pannaccione A, Costantino V, Mayol L, Piccialli G, and Oliviero G
- Subjects
- Animals, Cell Line, Tumor, Neurons metabolism, PC12 Cells, Rats, Signal Transduction physiology, Structure-Activity Relationship, Calcium metabolism, Cyclic ADP-Ribose chemistry, Cyclic ADP-Ribose metabolism, Ovum metabolism, Sea Urchins metabolism
- Abstract
Herein, we reported on the synthesis of cpIPP, which is a new structurally-reduced analogue of cyclic ADP-ribose (cADPR), a potent Ca
2+ -releasing secondary messenger that was firstly isolated from sea urchin eggs extracts. To obtain cpIPP the "northern" ribose of cADPR was replaced by a pentyl chain and the pyrophosphate moiety by a phophono-phosphate anhydride. The effect of the presence of the new phosphono-phosphate bridge on the intracellular Ca2+ release induced by cpIPP was assessed in PC12 neuronal cells in comparison with the effect of the pyrophosphate bridge of the structurally related cyclic N1-butylinosine diphosphate analogue (cbIDP), which was previously synthesized in our laboratories, and with that of the linear precursor of cpIPP, which, unexpectedly, revealed to be the only one provided with Ca2+ release properties., Competing Interests: The authors declare no conflict of interest.- Published
- 2018
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191. Na + /Ca 2+ exchanger 1 on nuclear envelope controls PTEN/Akt pathway via nucleoplasmic Ca 2+ regulation during neuronal differentiation.
- Author
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Secondo A, Esposito A, Petrozziello T, Boscia F, Molinaro P, Tedeschi V, Pannaccione A, Ciccone R, Guida N, Di Renzo G, and Annunziato L
- Abstract
Nuclear envelope (NE) is a Ca
2+ -storing organelle controlling neuronal differentiation through nuclear Ca2+ concentrations ([Ca2+ ]n ). However, how [Ca2+ ]n regulates this important function remains unknown. Here, we investigated the role of the nuclear form of the Na+ /Ca2+ exchanger 1(nuNCX1) during the different stages of neuronal differentiation and the involvement of PTEN/PI3'K/Akt pathway. In neuronal cells, nuNCX1 was detected on the inner membrane of the NE where protein expression and activity of the exchanger increased during NGF-induced differentiation. nuNCX1 activation by Na+ -free perfusion induced a time-dependent activation of nuclear-resident PI3K/Akt pathway in isolated nuclei. To discriminate the contribution of nuNCX1 from those of plasma membrane NCX, we generated a chimeric protein composed of the fluorophore EYFP, the exchanger inhibitory peptide, and the nuclear localization signal, named XIP-NLS. Fura-2 measurements on single nuclei and patch-clamp experiments in whole-cell configuration showed that XIP-NLS selectively inhibited nuNCX1. Once it reached the nuclear compartment, XIP-NLS increased the nucleoplasmic Ca2+ peak elicited by ATP and reduced Akt phosphorylation, GAP-43 and MAP-2 expression through nuclear-resident PTEN induction. Furthermore, in accordance with the prevention of the neuronal phenotype, XIP-NLS significantly reduced TTX-sensitive Na+ currents and membrane potential during neuronal differentiation. The selective inhibition of nuNCX1 by XIP-NLS increased the percentage of β III tubulin-positive immature neurons in mature cultures of MAP-2-positive cortical neurons, thus unraveling a new function for nuNCX1 in regulating neuronal differentiation through [Ca2+ ]n -dependent PTEN/PI3K/Akt pathway., Competing Interests: The authors declare that they have no competing interests.- Published
- 2018
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192. The expression and activity of K V 3.4 channel subunits are precociously upregulated in astrocytes exposed to Aβ oligomers and in astrocytes of Alzheimer's disease Tg2576 mice.
- Author
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Boscia F, Pannaccione A, Ciccone R, Casamassa A, Franco C, Piccialli I, de Rosa V, Vinciguerra A, Di Renzo G, and Annunziato L
- Subjects
- Amyloid beta-Peptides metabolism, Animals, Brain metabolism, Cells, Cultured, Disease Models, Animal, Glial Fibrillary Acidic Protein metabolism, Male, Mice, Mice, Inbred C57BL, Mice, Transgenic, Peptide Fragments adverse effects, Peptide Fragments metabolism, Rats, Wistar, Shaw Potassium Channels physiology, Alzheimer Disease genetics, Alzheimer Disease metabolism, Amyloid beta-Peptides adverse effects, Astrocytes drug effects, Astrocytes metabolism, Gene Expression, Shaw Potassium Channels genetics, Shaw Potassium Channels metabolism, Up-Regulation drug effects
- Abstract
Astrocyte dysfunction emerges early in Alzheimer's disease (AD) and may contribute to its pathology and progression. Recently, the voltage gated potassium channel K
V 3.4 subunit, which underlies the fast-inactivating K+ currents, has been recognized to be relevant for AD pathogenesis and is emerging as a new target candidate for AD. In the present study, we investigated both in in vitro and in vivo models of AD the expression and functional activity of KV 3.4 potassium channel subunits in astrocytes. In primary astrocytes our biochemical, immunohistochemical, and electrophysiological studies demonstrated a time-dependent upregulation of KV 3.4 expression and functional activity after exposure to amyloid-β (Aβ) oligomers. Consistently, astrocytic KV 3.4 expression was upregulated in the cerebral cortex, hippocampus, and cerebellum of 6-month-old Tg2576 mice. Further, confocal triple labeling studies revealed that in 6-month-old Tg2576 mice, KV 3.4 was intensely coexpressed with Aβ in nonplaque associated astrocytes. Interestingly, in the cortical and hippocampal regions of 12-month-old Tg2576 mice, plaque-associated astrocytes much more intensely expressed KV 3.4 subunits, but not Aβ. More important, we evidenced that the selective knockdown of KV 3.4 expression significantly downregulated both glial fibrillary acidic protein levels and Aβ trimers in the brain of 6-month-old Tg2576 mice. Collectively, our results demonstrate that the expression and function of KV 3.4 channel subunits are precociously upregulated in cultured astrocytes exposed to Aβ oligomers and in reactive astrocytes of AD Tg2576 mice., (Copyright © 2017 Elsevier Inc. All rights reserved.)- Published
- 2017
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193. NCX1 Exchanger Cooperates with Calretinin to Confer Preconditioning-Induced Tolerance Against Cerebral Ischemia in the Striatum.
- Author
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Boscia F, Casamassa A, Secondo A, Esposito A, Pannaccione A, Sirabella R, Pignataro G, Cuomo O, Vinciguerra A, de Rosa V, and Annunziato L
- Subjects
- Animals, Brain Ischemia pathology, Cell Line, Tumor, Gene Silencing, Humans, Immunoprecipitation, Interneurons metabolism, Male, Neostriatum pathology, Neurons metabolism, Neuroprotection, Phosphorylation, Proto-Oncogene Proteins c-akt metabolism, Rats, Sprague-Dawley, Up-Regulation, Brain Ischemia metabolism, Calbindin 2 metabolism, Ischemic Preconditioning, Neostriatum metabolism, Sodium-Calcium Exchanger metabolism
- Abstract
Recently, the Na(+)/Ca(+2) exchanger NCX1 and the calcium binding protein calretinin have emerged as new molecular effectors of delayed preconditioning in the brain. In the present study, we investigated whether NCX1 and calretinin cooperate within the preconditioned striatum to confer neurons greater resistance to degeneration. Confocal microscopy analysis revealed that NCX1 expression was upregulated in calretinin-positive interneurons in the rat striatum after tolerance induction. Consistently, coimmunoprecipitation assays performed on human SHSY-5Y cells, a neuronal cell line which constitutively expresses calretinin, revealed a binding between NCX1 and calretinin. Finally, silencing of calretinin expression, both in vitro and in vivo, significantly prevented preconditioning-induced neuroprotection. Interestingly, our biochemical and functional studies showed that the selective silencing of calretinin in brain cells significantly prevented not only the preconditioning-induced upregulation of NCX1 expression and activity but also the activation of the prosurvival protein kinase Akt, which is involved in calretinin and NCX1 protective actions. Collectively, our results indicate that the Na(+)/Ca(+2) exchanger NCX1 and the calcium binding protein calretinin cooperate within the striatum to confer tolerance against cerebral ischemia.
- Published
- 2016
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194. Pharmacological characterization of the newly synthesized 5-amino-N-butyl-2-(4-ethoxyphenoxy)-benzamide hydrochloride (BED) as a potent NCX3 inhibitor that worsens anoxic injury in cortical neurons, organotypic hippocampal cultures, and ischemic brain.
- Author
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Secondo A, Pignataro G, Ambrosino P, Pannaccione A, Molinaro P, Boscia F, Cantile M, Cuomo O, Esposito A, Sisalli MJ, Scorziello A, Guida N, Anzilotti S, Fiorino F, Severino B, Santagada V, Caliendo G, Di Renzo G, and Annunziato L
- Subjects
- Animals, Brain pathology, Brain physiopathology, Brain Ischemia pathology, Calcium metabolism, Cell Death drug effects, Cell Death physiology, Cell Hypoxia physiology, Cell Line, Cricetinae, Disease Models, Animal, Dogs, Dose-Response Relationship, Drug, Drug Evaluation, Preclinical, Glucose deficiency, Infarction, Middle Cerebral Artery, Mice, Inbred C57BL, Mutation, Neurons drug effects, Neurons pathology, Neurons physiology, Protein Isoforms, Rats, Sodium-Calcium Exchanger genetics, Sodium-Calcium Exchanger metabolism, Tissue Culture Techniques, Benzamides pharmacology, Brain drug effects, Brain Ischemia physiopathology, Cell Hypoxia drug effects, Central Nervous System Agents pharmacology, Sodium-Calcium Exchanger antagonists & inhibitors
- Abstract
The Na(+)/Ca(2+) exchanger (NCX), a 10-transmembrane domain protein mainly involved in the regulation of intracellular Ca(2+) homeostasis, plays a crucial role in cerebral ischemia. In the present paper, we characterized the effect of the newly synthesized compound 5-amino-N-butyl-2-(4-ethoxyphenoxy)-benzamide hydrochloride (BED) on the activity of the three NCX isoforms and on the evolution of cerebral ischemia. BED inhibited NCX isoform 3 (NCX3) activity (IC50 = 1.9 nM) recorded with the help of single-cell microflorimetry, (45)Ca(2+) radiotracer fluxes, and patch-clamp in whole-cell configuration. Furthermore, this drug displayed negligible effect on NCX2, the other isoform expressed within the CNS, and it failed to modulate the ubiquitously expressed NCX1 isoform. Concerning the molecular site of action, the use of chimera strategy and deletion mutagenesis showed that α1 and α2 repeats of NCX3 represented relevant molecular determinants for BED inhibitory action, whereas the intracellular regulatory f-loop was not involved. At 10 nM, BED worsened the damage induced by oxygen/glucose deprivation (OGD) followed by reoxygenation in cortical neurons through a dysregulation of [Ca(2+)]i. Furthermore, at the same concentration, BED significantly enhanced cell death in CA3 subregion of hippocampal organotypic slices exposed to OGD and aggravated infarct injury after transient middle cerebral artery occlusion in mice. These results showed that the newly synthesized 5-amino-N-butyl-2-(4-ethoxyphenoxy)-benzamide hydrochloride is one of the most potent inhibitor of NCX3 so far identified, representing an useful tool to dissect the role played by NCX3 in the control of Ca(2+) homeostasis under physiological and pathological conditions.
- Published
- 2015
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195. A new cell-penetrating peptide that blocks the autoinhibitory XIP domain of NCX1 and enhances antiporter activity.
- Author
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Molinaro P, Pannaccione A, Sisalli MJ, Secondo A, Cuomo O, Sirabella R, Cantile M, Ciccone R, Scorziello A, di Renzo G, and Annunziato L
- Subjects
- Amino Acid Sequence, Animals, Brain metabolism, Calcium metabolism, Cell Line, Cell Membrane drug effects, Cell Membrane metabolism, Cell-Penetrating Peptides chemistry, Cell-Penetrating Peptides metabolism, Cricetinae, Gene Expression, Ion Transport, Molecular Sequence Data, Mutagenesis, Site-Directed, Myocardium metabolism, Patch-Clamp Techniques, Peptides chemistry, Peptides metabolism, Protein Structure, Tertiary, Rats, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Sodium metabolism, Sodium-Calcium Exchanger agonists, Sodium-Calcium Exchanger antagonists & inhibitors, Sodium-Calcium Exchanger chemistry, Sodium-Calcium Exchanger genetics, Cell-Penetrating Peptides pharmacology, Peptides pharmacology, Sodium-Calcium Exchanger metabolism
- Abstract
The plasma membrane Na(+)/Ca(2+) exchanger (NCX) is a high-capacity ionic transporter that exchanges 3Na(+) ions for 1Ca(2+) ion. The first 20 amino acids of the f-loop, named exchanger inhibitory peptide (XIP(NCX1)), represent an autoinhibitory region involved in the Na(+)-dependent inactivation of the exchanger. Previous research has shown that an exogenous peptide having the same amino acid sequence as the XIP(NCX1) region exerts an inhibitory effect on NCX activity. In this study, we identified another regulatory peptide, named P1, which corresponds to the 562-688aa region of the exchanger. Patch-clamp analysis revealed that P1 increased the activity of the exchanger, whereas the XIP inhibited it. Furthermore, P1 colocalized with NCX1 thus suggesting a direct binding interaction. In addition, site-directed mutagenesis experiments revealed that the binding and the stimulatory effect of P1 requires a functional XIP(NCX1) domain on NCX1 thereby suggesting that P1 increases the exchanger activity by counteracting the action of this autoinhibitory sequence. Taken together, these results open a new strategy for developing peptidomimetic compounds that, by mimicking the functional pharmacophore of P1, might increase NCX1 activity and thus exert a therapeutic action in those diseases in which an increase in NCX1 activity might be helpful.
- Published
- 2015
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196. Involvement of the Na+/Ca2+ exchanger isoform 1 (NCX1) in neuronal growth factor (NGF)-induced neuronal differentiation through Ca2+-dependent Akt phosphorylation.
- Author
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Secondo A, Esposito A, Sirabella R, Boscia F, Pannaccione A, Molinaro P, Cantile M, Ciccone R, Sisalli MJ, Scorziello A, Di Renzo G, and Annunziato L
- Subjects
- Animals, Cell Differentiation, Endoplasmic Reticulum metabolism, Enzyme Activation, Homeostasis, Mutation, Neurites metabolism, PC12 Cells, Patch-Clamp Techniques, Phosphatidylinositol 3-Kinases metabolism, Phosphorylation, RNA, Small Interfering metabolism, Rats, Rats, Wistar, Signal Transduction, Sodium metabolism, Brain embryology, Calcium metabolism, Nerve Growth Factor pharmacology, Neurons cytology, Neurons metabolism, Sodium-Calcium Exchanger metabolism
- Abstract
NGF induces neuronal differentiation by modulating [Ca(2+)]i. However, the role of the three isoforms of the main Ca(2+)-extruding system, the Na(+)/Ca(2+) exchanger (NCX), in NGF-induced differentiation remains unexplored. We investigated whether NCX1, NCX2, and NCX3 isoforms could play a relevant role in neuronal differentiation through the modulation of [Ca(2+)]i and the Akt pathway. NGF caused progressive neurite elongation; a significant increase of the well known marker of growth cones, GAP-43; and an enhancement of endoplasmic reticulum (ER) Ca(2+) content and of Akt phosphorylation through an early activation of ERK1/2. Interestingly, during NGF-induced differentiation, the NCX1 protein level increased, NCX3 decreased, and NCX2 remained unaffected. At the same time, NCX total activity increased. Moreover, NCX1 colocalized and coimmunoprecipitated with GAP-43, and NCX1 silencing prevented NGF-induced effects on GAP-43 expression, Akt phosphorylation, and neurite outgrowth. On the other hand, the overexpression of its neuronal splicing isoform, NCX1.4, even in the absence of NGF, induced an increase in Akt phosphorylation and GAP-43 protein expression. Interestingly, tetrodotoxin-sensitive Na(+) currents and 1,3-benzenedicarboxylic acid, 4,4'-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,12-benzofurandiyl)]bis-, tetrakis[(acetyloxy)methyl] ester-detected [Na(+)]i significantly increased in cells overexpressing NCX1.4 as well as ER Ca(2+) content. This latter effect was prevented by tetrodotoxin. Furthermore, either the [Ca(2+)]i chelator(1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) (BAPTA-AM) or the PI3K inhibitor LY 294002 prevented Akt phosphorylation and GAP-43 protein expression rise in NCX1.4 overexpressing cells. Moreover, in primary cortical neurons, NCX1 silencing prevented Akt phosphorylation, GAP-43 and MAP2 overexpression, and neurite elongation. Collectively, these data show that NCX1 participates in neuronal differentiation through the modulation of ER Ca(2+) content and PI3K signaling., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2015
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197. NCX1 is a new rest target gene: role in cerebral ischemia.
- Author
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Formisano L, Guida N, Valsecchi V, Pignataro G, Vinciguerra A, Pannaccione A, Secondo A, Boscia F, Molinaro P, Sisalli MJ, Sirabella R, Casamassa A, Canzoniero LM, Di Renzo G, and Annunziato L
- Subjects
- Animals, Base Sequence, Blotting, Western, Brain Ischemia metabolism, Chromatin Immunoprecipitation, Electrophoretic Mobility Shift Assay, Fluorescent Antibody Technique, Hippocampus metabolism, Humans, Microscopy, Confocal, Molecular Sequence Data, Mutagenesis, Site-Directed, Organ Culture Techniques, Rats, Rats, Sprague-Dawley, Rats, Wistar, Real-Time Polymerase Chain Reaction, Repressor Proteins metabolism, Sodium-Calcium Exchanger metabolism, Transfection, Brain Ischemia genetics, Gene Expression Regulation genetics, Neurons metabolism, Repressor Proteins genetics, Sodium-Calcium Exchanger genetics
- Abstract
The Na(+)-Ca(2+) exchanger 1 (NCX1), a bidirectional transporter that mediates the electrogenic exchange of one calcium ion for three sodium ions across the plasma membrane, is known to be involved in brain ischemia. Since the RE1-silencing transcription factor (REST) is a key modulator of neuronal gene expression in several neurological conditions, we studied the possible involvement of REST in regulating NCX1 gene expression and activity in stroke. We found that: (1) REST binds in a sequence specific manner and represses through H4 deacetylation, ncx1 gene in neuronal cells by recruting CoREST, but not mSin3A. (2) In neurons and in SH-SY5Y cells REST silencing by siRNA and site-direct mutagenesis of REST consensus sequence on NCX1 brain promoter determined an increase in NCX1 promoter activity. (3) By contrast, REST overexpression caused a reduction in NCX1 protein expression and activity. (4) Interestingly, in rats subjected to transient middle cerebral artery occlusion (tMCAO) and in organotypic hippocampal slices or SH-SY5Y cells exposed to oxygen and glucose deprivation (OGD) plus reoxygenation (RX), the increase in REST was associated with a decrease in NCX1. However, this reduction was reverted by REST silencing. (5) REST knocking down, along with the deriving NCX1 overexpression in the deep V and VIb cortical layers caused a marked reduction in infarct volume after tMCAO. Double silencing of REST and NCX1 completely abolished neuroprotection induced by siREST administration. Collectively, these results demonstrate that REST, by regulating NCX1 expression, may represent a potential druggable target for the treatment of brain ischemia., (Copyright © 2012 Elsevier Inc. All rights reserved.)
- Published
- 2013
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198. New roles of NCX in glial cells: activation of microglia in ischemia and differentiation of oligodendrocytes.
- Author
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Boscia F, D'Avanzo C, Pannaccione A, Secondo A, Casamassa A, Formisano L, Guida N, Scorziello A, Di Renzo G, and Annunziato L
- Subjects
- Animals, Brain Ischemia genetics, Brain Ischemia pathology, Gene Expression Regulation genetics, Humans, Mice, Mice, Knockout, Microglia pathology, Myelin Basic Protein biosynthesis, Myelin Basic Protein genetics, Myelin Sheath genetics, Myelin Sheath pathology, Neural Stem Cells metabolism, Neural Stem Cells pathology, Brain Ischemia metabolism, Calcium Signaling, Cell Differentiation, Microglia metabolism, Myelin Sheath metabolism, Sodium-Calcium Exchanger metabolism
- Abstract
The initiation of microglial responses to the ischemic injury involves modifications of calcium homeostasis. Changes in [Ca(2+)](i) levels have also been shown to influence the developmental processes that accompany the transition of human oligodendrocyte precursor cells (OPCs) into mature myelinating oligodendrocytes and are required for the initiation of myelination and remyelination processes.We investigated the regional and temporal changes of NCX1 protein in microglial cells of the peri-infarct and core regions after permanent middle cerebral artery occlusion (pMCAO). Interestingly, 3 and 7 days after pMCAO, NCX1 signal strongly increased in the round-shaped microglia invading the infarct core. Cultured microglial cells from the core displayed increased NCX1 expression as compared with contralateral cells and showed enhanced NCX activity in the reverse mode of operation. Similarly, NCX activity and NCX1 protein expression were significantly enhanced in BV2 microglia exposed to oxygen and glucose deprivation, whereas NCX2 and NCX3 were downregulated. Interestingly, in NCX1-silenced cells, [Ca(2+)](i) increase induced by hypoxia was completely prevented. The upregulation of NCX1 expression and activity observed in microglia after pMCAO suggests a relevant role of NCX1 in modulating microglia functions in the postischemic brain.Next, we explored whether calcium signals mediated by NCX1, NCX2, or NCX3 play a role in oligodendrocyte maturation. Functional studies, as well as mRNA and protein expression analyses, revealed that NCX1 and NCX3, but not NCX2, were divergently modulated during OPC differentiation into oligodendrocyte. In fact, while NCX1 was downregulated, NCX3 was strongly upregulated during the oligodendrocyte development. Whereas the knocking down of the NCX3 isoform in OPCs prevented the upregulation of the myelin protein markers CNPase and MBP, its overexpression induced their upregulation. Furthermore, NCX3 knockout mice exhibited not only a reduced size of spinal cord but also a marked hypomyelination, as revealed by the decrease in MBP expression and by the accompanying increase in OPCs number. Our findings indicate that calcium signaling mediated by NCX3 plays a crucial role in oligodendrocyte maturation and myelin formation.
- Published
- 2013
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199. Neurounina-1, a novel compound that increases Na+/Ca2+ exchanger activity, effectively protects against stroke damage.
- Author
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Molinaro P, Cantile M, Cuomo O, Secondo A, Pannaccione A, Ambrosino P, Pignataro G, Fiorino F, Severino B, Gatta E, Sisalli MJ, Milanese M, Scorziello A, Bonanno G, Robello M, Santagada V, Caliendo G, Di Renzo G, and Annunziato L
- Subjects
- Animals, Calcium metabolism, Cell Death drug effects, Cells, Cultured, Cricetinae, Dogs, Flumazenil pharmacology, GABA-A Receptor Antagonists pharmacology, Glutamic Acid metabolism, Infarction, Middle Cerebral Artery complications, Male, Mice, Mice, Inbred C57BL, Mitochondria drug effects, Mitochondria metabolism, Mutation, Neurons drug effects, Neurons pathology, Patch-Clamp Techniques, Rats, Receptors, GABA-A physiology, Receptors, N-Methyl-D-Aspartate agonists, Sodium-Calcium Exchanger genetics, Stroke etiology, Stroke pathology, Synaptosomes drug effects, Synaptosomes metabolism, gamma-Aminobutyric Acid metabolism, Benzodiazepinones pharmacology, Neuroprotective Agents pharmacology, Pyrrolidines pharmacology, Sodium-Calcium Exchanger metabolism, Stroke prevention & control
- Abstract
Previous studies have demonstrated that the knockdown or knockout of the three Na(+)/Ca(2+) exchanger (NCX) isoforms, NCX1, NCX2, and NCX3, worsens ischemic brain damage. This suggests that the activation of these antiporters exerts a neuroprotective action against stroke damage. However, drugs able to increase the activity of NCXs are not yet available. We have here succeeded in synthesizing a new compound, named neurounina-1 (7-nitro-5-phenyl-1-(pyrrolidin-1-ylmethyl)-1H-benzo[e][1,4]diazepin-2(3H)-one), provided with an high lipophilicity index and able to increase NCX activity. Ca(2+) radiotracer, Fura-2 microfluorimetry, and patch-clamp techniques revealed that neurounina-1 stimulated NCX1 and NCX2 activities with an EC(50) in the picomolar to low nanomolar range, whereas it did not affect NCX3 activity. Furthermore, by using chimera strategy and site-directed mutagenesis, three specific molecular determinants of NCX1 responsible for neurounina-1 activity were identified in the α-repeats. Interestingly, NCX3 became responsive to neurounina-1 when both α-repeats were replaced with the corresponding regions of NCX1. In vitro studies showed that 10 nM neurounina-1 reduced cell death of primary cortical neurons exposed to oxygen-glucose deprivation followed by reoxygenation. Moreover, in vitro, neurounina-1 also reduced γ-aminobutyric acid (GABA) release, enhanced GABA(A) currents, and inhibited both glutamate release and N-methyl-d-aspartate receptors. More important, neurounina-1 proved to have a wide therapeutic window in vivo. Indeed, when administered at doses of 0.003 to 30 μg/kg i.p., it was able to reduce the infarct volume of mice subjected to transient middle cerebral artery occlusion even up to 3 to 5 hours after stroke onset. Collectively, the present study shows that neurounina-1 exerts a remarkable neuroprotective effect during stroke and increases NCX1 and NCX2 activities.
- Published
- 2013
- Full Text
- View/download PDF
200. Genetically modified mice as a strategy to unravel the role played by the Na(+)/Ca (2+) exchanger in brain ischemia and in spatial learning and memory deficits.
- Author
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Molinaro P, Cataldi M, Cuomo O, Viggiano D, Pignataro G, Sirabella R, Secondo A, Boscia F, Pannaccione A, Scorziello A, Sokolow S, Herchuelz A, Di Renzo G, and Annunziato L
- Subjects
- Animals, Brain metabolism, Brain pathology, Brain Ischemia genetics, Brain Ischemia pathology, Humans, Learning Disabilities genetics, Learning Disabilities pathology, Memory Disorders genetics, Memory Disorders pathology, Mice, Mice, Knockout, Nerve Tissue Proteins genetics, Neurodegenerative Diseases genetics, Neurodegenerative Diseases metabolism, Neurodegenerative Diseases pathology, Sodium-Calcium Exchanger genetics, Brain Ischemia metabolism, Learning Disabilities metabolism, Memory Disorders metabolism, Nerve Tissue Proteins metabolism, Sodium-Calcium Exchanger metabolism
- Abstract
Because no isoform-specific blocker of NCX has ever been synthesized, a more selective strategy to identify the role of each antiporter isoform in the brain was represented by the generation of knockout and knockin mice for the different isoforms of the antiporter.Experiments performed in NCX2 and NCX3 knockout mice provided evidence that these two isoforms participate in spatial learning and memory consolidation, although in an opposite manner. These new data from ncx2-/- and ncx3-/- mice may open new experimental avenues for the development of effective therapeutic compounds that, by selectively inhibiting or activating these molecular targets, could treat patients affected by cognitive impairment including Alzheimer's, Parkinson's, Huntington's diseases, and infarct dementia.More importantly, knockout and knockin mice also provided new relevant information on the role played by NCX in maintaining the intracellular Na(+) and Ca(2+) homeostasis and in protecting neurons during brain ischemia. In particular, both ncx2-/- and ncx3-/- mice showed an increased neuronal vulnerability after the ischemic insult induced by transient middle cerebral artery occlusion.As the ubiquitous deletion of NCX1 brings about to an early death of embryos because of a lack of heartbeat, this strategy could not be successfully pursued. However, information on the role of NCX1 in normal and ischemic brain could be obtained by developing conditional knockout mice lacking NCX1 in the brain. Preliminarily results obtained in these conditional mice suggest that also NCX1 protects neurons from ischemic cell death.Overall, the use of genetic-modified mice for NCX1, NCX2, and NCX3 represents a fruitful strategy to characterize the physiological role exerted by NCX in CNS and to identify the isoforms of the antiporter as potential molecular targets for therapeutic intervention in cerebral ischemia.
- Published
- 2013
- Full Text
- View/download PDF
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