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Polychlorinated biphenyls (PCBs) cause a wide spectrum of toxic effects in the brain through undefined mechanisms. Exposure to the PCB mixture Aroclor-1254 (A1254) increases the repressor element-1 silencing transcription factor (REST) expression, leading to neuronal death. This study sought to understand the sequence of some molecular mechanisms to determine whether A1254 could increase REST expression and the cytoprotective effect of the phorbol ester tetradecanoylphorbol acetate (TPA) on A1254-induced toxicity in SH-SY5Y cells. As shown by Western blot analysis, A1254 (10 µg/ml) downregulates extracellular signal-related kinase 2 (ERK2) phosphorylation in a time-dependent manner, thereby triggering the binding of specificity protein 1 (Sp1) and Sp3 to the REST gene promoter as revealed by chromatin immunoprecipitation analysis. This chain of events results in an increase in REST mRNA and cell death, as assessed by quantitative real-time polymerase chain reaction and dimethylthiazolyl-2-5-diphenyltetrazolium-bromide assay, respectively. Accordingly, TPA prevented both the A1254-induced decrease in ERK2 phosphorylation and the A1254-induced increase in Sp1, Sp3, and REST protein expression. After 48 hr, TPA prevented A1254-induced cell death. ERK2 overexpression counteracted the A1254-induced increase in Sp1 and Sp3 protein expression and prevented A1254-induced Sp1 and Sp3 binding to the REST gene promoter, thus counteracting the increase in REST mRNA expression induced by the toxicant. In neuroblastoma SH-SY5Y cells, ERK2/Sp1/SP3/REST is a new pathway underlying the neurotoxic effect of PCB. The ERK2/Sp1/Sp3/REST pathway, which underlies A1254-induced neuronal death, might represent a new drug signaling cascade in PCB-induced neuronal toxicity

The initiation of microglial responses to the ischemic injury involves modifications of calcium homeostasis. Changes in [Ca2+]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, [Ca2+]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.

Mitochondria are now recognized as one of the main intracellular calcium-storing organelles which play a key role in the intracellular calcium signalling. Indeed, besides performing oxidative phosphorylation, mitochondria are able to sense and shape calcium (Ca(2+)) transients, thus controlling cytosolic Ca(2+) signals and Ca(2+)-dependent protein activity. It has been well established for many years that mitochondria have a huge capacity to accumulate calcium. While the physiological significance of this pathway was hotly debated until relatively recently, it is now clear that the ability of mitochondria in calcium handling is a ubiquitous phenomenon described in every cell system in which the issue has been addressed.In this chapter, we will review the molecular mechanisms involved in the regulation of mitochondrial calcium cycling in physiological conditions with particular regard to the role played by the mitochondrial Na(+)/Ca(2+) exchanger

Ischemic preconditioning is a neuroprotective mechanism in which a brief non-injurious episode of ischemia protects the brain from a subsequent lethal insult. Recently, it has been reported that modified reperfusion subsequent to a prolonged ischemic episode may also confer neuroprotection, a phenomenon termed postconditioning. Mitogen-activated protein kinases (MAPK) play a key role in these two neuroprotective mechanisms. The aim of this study was to evaluate whether Na(+)/Ca(2+) exchangers (NCXs), a family of ionic transporters that contribute to the maintenance of intracellular ionic homeostasis, contribute to the neuroprotection elicited by ischemic preconditioning and postconditioning.Results of this study indicated that (1) NCX1 and NCX3 are upregulated in those brain regions protected by preconditioning, while (2) postconditioning treatment induces an upregulation only in NCX3 expression. (3) NCX1 upregulation and NCX3 upregulation are mediated by p-AKT since its inhibition reverted the neuroprotective effect of preconditioning and postconditioning and prevented NCXs overexpression. (4) The involvement of NCX in preconditioning and postconditioning neuroprotection is further supported by the results of experiments showing that a partial reversion of the protective effect induced by preconditioning was obtained by silencing NCX1 or NCX3, while the silencing of NCX3 was able to mitigate the protection induced by ischemic postconditioning.Altogether, the data presented here suggest that NCX1 and NCX3 -represent two promising druggable targets for setting on new strategies in stroke therapy

The ubiquitous sodium-calcium exchanger isoform 1 (NCX1) is a -bidirectional transporter that plays a relevant role under physiological and pathophysiological conditions including brain ischemia by regulating intraneuronal Ca(2+) and Na(+) homeostasis. Although changes in ncx1 protein and transcript expression have been detected during stroke, its transcriptional regulation is still largely unexplored. Here, we reviewed our recent findings on several transcription factors including cAMP response element-binding protein (CREB), nuclear factor kappa B (NF-κB), and hypoxia-inducible factor-1 (HIF-1) in the control of the ncx1 gene expression in neuronal cells.

The present invention describes compounds of formula (I), and in particular the 7- nitro-5-phenyl-1 -(pyrrolidin-1 - ylmethyl)-1 H-benzo[e][1,4]diazepin-2(3H)-one compound and their pharmaceuticall acceptable salts. The compounds of the invention are useful for medical use, particularly as active ingredient potentially useful for the treatment of cerebral ischemia and other disease characterized by deregulation of sodium and calcium homeostasis

The silencing in PC-12 cells or the knocking-out in mice of the ncx3 gene, that encodes for the Na+/Ca2+ exchanger type 3, prevented the enhancement of both I(NCX) and Ca(2+) content in ER stores induced by beta -amyloid 1-42, suggesting that NCX3 was involved in the increase of ER Ca(2+) content stimulated by this peptide. By contrast, in the late phase (72 h), when the NCX3 proteolytic cleavage abruptly decreased, the occurrence of a parallel reduction in ER Ca(2+) content triggered ER stress, as revealed by caspase-12 activation. Concomitantly, the late increase in [Ca(2+)](i) coincided with neuronal death. Interestingly, NCX3 silencing caused an earlier activation of Aβ(1-42)-induced caspase-12. Indeed, in NCX3-silenced neurons, Aβ(1-42) exposure hastened caspase-dependent apoptosis, thus reinforcing neuronal cell death. These results suggest that Aβ(1-42), through Ca(2+)-dependent calpain activation, generates a hyperfunctional form of the Na+/Ca2+ exchanger NCX3 that, by increasing Ca(2+) content into ER, delays caspase-12 activation and thusER-stress and neuronal death.

We evaluated whether changes in expression and activity of the three sodium/calcium exchanger isoforms, NCX1, NCX2 and NCX3 occurred in PC12 cells when the ERK1/2, JNK, and p38 MAPKs were silenced, pharmacologically blocked, or activated with NGF. Several findings suggesting that MAPKs control NCX emerged: (1) A decrease in NCX1 and NCX3 basal expression occurred when JNK or MEK1, the ERK1/2 upstream activator, were pharmacologically blocked, respectively; (2) NGF increased CREB1 and Sp1 binding to ncx1 promoter and in CREB1 binding to two different sequences close to ncx2 transcription start site on genomic DNA; (3) An up-regulation of NCX1 and NCX3, abrogated upon either MEK1 or p38 blockade, and a down-regulation of NCX2, abolished upon p38 blockade, occurred upon NGF-induced MAPK activation. NCX1 up-regulation was abolished upon either CREB1 or Sp1 silencing, whereas NCX2 down-regulation was abrogated only by CREB1 silencing. NCX3 up-regulation was unaffected by CREB1 or Sp1 silencing and abolished upon proteasomal inhibition; (4) Whole-cell Na(+) /Ca(2+) exchange decreased when MEK1 and JNK were blocked and increased when MAPKs were activated by NGF. Collectively, these results demonstrate a MAPK-dependent regulation of NCX expression and activity which could be relevant in mediating some of the effects of MAPKs in neurons.

Background and Purpose— The sodium–calcium exchanger-1 (NCX1) represents a key mediator for maintaining [Na + ] i and [Ca 2+ ] i homeostasis. Although changes in NCX1 protein and transcript expression have been detected during stroke, its transcriptional regulation is still unknown. Thus far, however, there is evidence that hypoxia-inducible factor-1 (HIF-1) is a nuclear factor required for transcriptional activation of several genes implicated in stroke. The main objective of this study was to investigate whether NCX1 gene might be a novel target of HIF-1 in the brain. Methods and Results— Here we report that: (1) in neuronal cells, NCX1 increased expression after oxygen and glucose deprivation or cobalt-induced HIF-1 activation was prevented by silencing HIF-1; (2) the brain NCX1 promoter cloned upstream of the firefly-luciferase gene contained 2 regions of HIF-1 target genes called hypoxia-responsive elements that are sensitive to oxygen and glucose deprivation or cobalt chloride; (3) HIF-1 specifically bound hypoxia-responsive elements on brain NCX1, as demonstrated by band-shift and chromatin immunoprecipitation assays; (4) HIF-1α silencing prevented NCX1 upregulation and neuroprotection induced by ischemic preconditioning; and (5) NCX1 silencing partially reverted the preconditioning-induced neuroprotection in rats. Conclusions— NCX1 gene is a novel HIF-1 target, and HIF-1 exerts its prosurvival role through NCX1 upregulation during brain preconditioning.

Ischemic brain damage represents a major source of morbidity and mortality in westernized society and poses a significant financial burden on the health care system. To date, few effective therapies have been realized to treat stroke and promising avenues have not proven clinically useful. Recent evidence, however, suggests that channels, pumps, and ionic exchangers are involved in CNS ischemia and ischemic stroke, but the potential contribution of these channels for curing stroke is far less understood than for many other normal and pathological conditions. New Strategies in Stroke Intervention: Ionic Channels, Pumps, and Transporters analyzes the roles played by targets in stroke development and the potential action of drugs modulating these proteins. This book provides a groundbreaking review of these ionic channels, pumps, and transporters as regulators of neuronal ionic homeostasis, providing a better understanding of ischemic brain disorders and the new pharmacological avenues for a cure. It will be a useful tool for researchers working in this field, and any student interested in the physiological, pathophysiological, and pharmacological features of stroke damage.

Full color throughout Ischemic brain damage represents a major source of morbidity and mortality in westernized society and poses a significant financial burden on the health care system. To date, few effective therapies have been realized to treat stroke and promising avenues have not proven clinically useful. Recent evidence, however, suggests that channels, pumps, and ionic exchangers are involved in CNS ischemia and ischemic stroke, but the potential contribution of these channels for curing stroke is far less understood than for many other normal and pathological conditions. New Strategies in Stroke Intervention: Ionic Channels, Pumps, and Transporters analyzes the roles played by targets in stroke development and the potential action of drugs modulating these proteins. This book provides a groundbreaking review of these ionic channels, pumps, and transporters as regulators of neuronal ionic homeostasis, providing a better understanding of ischemic brain disorders and the new pharmacological avenues for a cure. It will be a useful tool for researchers working in this field, and any student interested in the physiological, pathophysiological, and pharmacological features of stroke damage.

Il brevetto riguarda composti di formula (I), ed in particolare 7-nitro-5-fenil-1(pirrolidin-1-ilmetil)-1H-benzo[e][1,4] diazepin-2(3H)-one, e loro sali farmaceuticamente accettabili, per l’uso medico ed in particolare come principio farmacologicamente attivo per il trattamento dell’ischemia cerebrale e di ogni altra patologia caratterizzata dalla perdita del controllo dell’omeostasi degli ioni sodio e calcio.

Lo scopo principale degli autori del presente trattato è stato quello di correlare le conoscenze sempre più approfondite sull’azione molecolare dei farmaci con gli effetti farmacologici esercitati nell’uomo e che costituiscono i presupposti razionali della terapia medica. Uno spazio adeguato è stato inoltre riservato alla trattazione degli aspetti farmacocinetici clinici, delle reazioni avverse e delle interazioni tra i farmaci, che rappresentano un aspetto in continuo divenire e che sono spesso alla base di eventi avversi clinicamente rilevanti.[...] [...] L’ intento del testo è stato quello di fornire un’informazione la più completa possibile con l’inserimento nella trattazione di argomenti che hanno assunto negli ultimi anni particolare rilevanza nel campo delle possibili prospettive terapeutiche più moderne. Nel trattato sono presenti infatti capitoli innovativi quali quello sui Farmaci Biotecnologici a bersaglio definito, sulla Farmacogenetica e sulla Terapia Genica. Completano la trattazione argomenti inerenti gli aspetti socio-sanitari , economici e normativi della Farmacologia contenuti nei capitoli sulla Sperimentazione Clinica dei Farmaci, sulla Farmacoeconomia e sulla Farmacologia del Doping. Infine, in considerazione del largo uso che si fa dei prodotti di origine vegetale , si è ritenuto utile affrontare tale problematica nel capitolo Principi di fitoterapia nella pratica clinica

Because clinical trials of pharmacological neuroprotective strategies in stroke have been disappointing, attention has turned to the brain's own endogenous strategies for neuroprotection. Two endogenous mechanisms have been characterized so far, namely ischemic preconditioning and ischemic postconditioning. The neuroprotective concept of preconditioning is based on the observation that a brief, noninjurious episode of ischemia is able to protect the brain from a subsequent longer ischemic insult. Recently, a hypothesis has been offered that modified reperfusion subsequent to a prolonged ischemic episode may also confer ischemic neuroprotection, a phenomenon termed postconditioning. Many pathways have been proposed as plausible mechanisms to explain the neuroprotection offered by preconditioning and postconditioning. Unfortunately, so far, none of them has clearly identified the mechanism involved in preconditioning and postconditioning. The present article will review the main mechanisms reported to date to explain the neuroprotective effect of both ischemic preconditioning and postconditioning.

Background: National drugs reports demonstrate increasing consumption of Angiotensin II Receptor antagonist (+ 17%); Trend of cost: +8,3% for Losartan; +42% for Valsartan; +11,3% for Irbesartan. Methods: We evaluated almost 200 studies, by searching Pub-Med, OVID, Embase. First, 103 studies were excluded because of lack of randomization and control; then 28 studies were excluded because of combination therapy. We included 25 studies in a systematic review. Results: We identify as clinical effectiveness outcome the decrease in mean systolic and diastolic pressure after 8 weeks of therapy. We found 4 comparable studies to this outcome; lack of standard error of mean or CI in two studies made impossible to generate pooled estimates of outcome. After funding review, we retrieved 4 studies with no explicit Drug’s Companies support: one of them favours valsartan versus olmesartan in 115 randomized patients; one other favours valsartan versus amlodipine in elderly patients,one other reports equivalence between irbesartan and valsartan, the last one favours olmesartan versus losartan and valsartan, without SE of means. Either in all studies, either in the independent ones outcome was substantial equivalence in the antihypertensive effects of the Angiotensin II Receptor antagonist. Conclusions: Angiotensin II Receptor antagonist are emerging therapeutic alternatives; however to generate consistent evidence is crucial to implement best practice and effective therapeutic choices. To design an independent big trial is a difficult challenge in the actual research environment, a smart observational study could constitute an effective alternative.

La presente invenzione si riferisce al campo delle formulazioni farmaceutiche in particolare a formulazioni farmaceutiche utili per il trattamento dei danni cerebrali secondari all’insulto ischemico o ad altre malattie del sistema nervoso centrale e periferico. Stato dell’arte La trombina, una serina-proteasi generata dal taglio proteolitico della protrombina, oltre al suo noto ruolo nella cascata della coagulazione, esercita effetti extravascolari mediati da una famiglia di recettori attivati dalle proteasi (PARs). I PARs sono recettori accoppiati a proteina G attivati dal taglio proteolitico della loro estremità NH2-terminale, mediato dalla trombina. Finora sono state descritte quattro isoforme di tali recettori, delle quali PAR1, PAR3 e PAR4 sono attivate dalla trombina, mentre PAR2 è attivato dalla tripsina ed è stato dimostrato che PAR1 è espresso nel cervello sia a livello neuronale che a livello gliale cosi come il precursore del suo ligando endogeno, la protrombina. In particolare, PAR1 è stato ritrovato in quelle regioni cerebrali sensibili al danno ischemico e studi in vitro, su fettine ippocampali, hanno dimostrato che la sua espressione risulta aumentata dopo anossia. Inoltre, l’attivazione di PAR 1 sembra essere responsabile della genesi di molteplici eventi patologici quali per esempio, la morte cellulare in colture ippocampali e in motoneuroni, la retrazione dei neuriti e il potenziamento della tossicità da NMDA. Parallelamente all’attivazione di PAR1 anche i livelli di trombina aumentano nel core ischemico, o perché la barriera ematoencefalica diventa più permeabile a proteine ad alto peso molecolare, permettendo così l’ingresso della protrombina o perchè il cervello stesso può essere una fonte di protrombina. A supporto di questa ultima ipotesi vi è l’osservazione che nel Sistema nervoso centrale (SNC) i livelli di RNAm per la protrombina risultano aumentati sia dopo ischemia cerebrale che dopo danno a carico del midollo spinale Infine, la dimostrazione della presenza del fattore X a livello cerebrale supporta l’ipotesi che la trombina possa essere prodotta direttamente in questa sede. Recenti evidenze hanno dimostrato che l’infusione intracerebrale dell’inibitore della trombina, irudina, effettuata subito dopo l’induzione del danno ischemico mediante occlusione transitoria dell’arteria cerebrale media (tMCAO), e prima dell’inizio della riperfusione, riduce il volume ischemico e migliora i deficit neurologici. Inoltre, è stato anche dimostrato che i livelli di ATIII, una glicoproteina appartenente alla famiglia delle serpine in grado di bloccare la formazione e l’attività della trombina, si riducono in pazienti in cui si è manifestato un episodio di ischemia cerebrale.

The present study explores the hypothesis that the new anti-epileptic drug levetiracetam (LEV) could interfere with the inositol 1,4,5-trisphosphate (IP(3))-dependent release of intracellular Ca(2+) initiated by G(q)-coupled receptor activation, a process that plays a role in triggering and maintaining seizures. We assessed the effect of LEV on the amplitude of [Ca(2+)](i) response to bradykinin (BK) and ATP in single Fura-2/acetoxymethyl ester-loaded PC12 rat pheochromocytoma cells, which express very high levels of LEV binding sites. LEV dose-dependently reduced the [Ca(2+)](i) increase, elicited either by 1 microM BK or by 100 microM ATP (IC(50), 0.39 +/- 0.01 microM for BK and 0.20 +/- 0.01 microM for ATP; Hill coefficients, 1.33 +/- 0.04 for BK and 1.38 +/- 0.06 for ATP). Interestingly, although the discharge of ryanodine stores by a process of calcium-induced calcium release also took place as part of the [Ca(2+)](i) response to BK, LEV inhibitory effect was mainly exerted on the IP(3)-dependent stores. In fact, the drug was still effective after the pharmacological blockade of ryanodine receptors. Furthermore, LEV did not affect Ca(2+) stored in the intracellular deposits since it did not reduce the amplitude of [Ca(2+)](i) response either to thapsigargin or to ionomycin. In conclusion, LEV inhibits Ca(2+) release from the IP(3)-sensitive stores without reducing Ca(2+) storage into these deposits. Because of the relevant implications of IP(3)-dependent Ca(2+) release in neuron excitability and epileptogenesis, this novel effect of LEV could provide a useful insight into the mechanisms underlying its antiepileptic properties

The 2-phenylaminopyrimidine derivative imatinib-mesylate, a powerful protein tyrosine kinase (PTK) inhibitor that targets abl, c-kit, and the platelet-derived growth factor receptors, is rapidly gaining a relevant role in the treatment of several types of neoplasms. Because first generation PTK inhibitors affect the activity of a large number of voltage-dependent ion channels, the present study explored the possibility that imatinib-mesylate could interfere with the activity of T-type channels, a class of voltage-dependent Ca2+ channels that take part in the chain of events elicited by PTK activation. The effect of the drug on T-type channel activity was examined using the whole-cell patch-clamp technique with Ba2+ (10 mM) as the permeant ion in human embryonic kidney-293 cells, stably expressing the rat Ca(V)3.3 channels. Imatinib-mesylate concentrations, ranging from 30 to 300 microM, reversibly decreased Ca(V)3.3 current amplitude with an IC(50) value of 56.9 microM. By contrast, when imatinib-mesylate (500 microM) was intracellularly dialyzed with the pipette solution, no reduction in Ba2+ current density was observed. The 2-phenylaminopyrimidine derivative modified neither the voltage dependence of activation nor the steady-state inactivation of Ca(V)3.3 channels. The decrease in extracellular Ba2+ concentration from 10 to 2 mM and the substitution of Ca2+ for Ba2+ increased the extent of 30 microM imatinib-mesylate-induced percentage of channel blockade from 25.9 +/- 2.4 to 36.3 +/- 0.9% in 2 mM Ba2+ and 44.2 +/- 2.3% in 2 mM Ca2+. In conclusion, imatinib-mesylate blocked the cloned Ca(V)3.3 channels by a PTK-independent mechanism. Specifically, the drug did not affect the activation or the inactivation of the channel but interfered with the ion permeation process

1. In C6 glioma cells exposed to chemical hypoxia a massive release of lactate dehydrogenase (LDH) occurred at 3 and 6 h, coupled with an increased number of propidium-iodide positive dead cells. 2. Extracellular Na+ removal, which activates the Na(+)-Ca2+ exchanger as a Na+ efflux pathway and prevents Na+ entrance, significantly reduced LDH release and the number of propidium iodide positive C6 cells. 3. During chemical hypoxia, in the presence of extracellular Na+ ions, a progressive increase of [Ca2+]i occurred; in the absence of extracellular Na+ ions [Ca2+]i was enhanced to a greater extent. 4. The blockade of the Na(+)-Ca2+ exchanger by the amiloride derivative 5-(N-4-chlorobenzyl)-2',4'-dimethylbenzamil (CB-DMB), lanthanum (La3+) and the Ca2+ chelator EGTA, completely reverted the protective effect exerted by the removal of Na+ ions on C6 glioma cells exposed to chemical hypoxia. 5. The inhibition of the Na(+)-Ca2+ antiporter enhanced chemical hypoxia-induced LDH release when C6 glioma cells were incubated in the presence of physiological concentrations of extracellular Na+ ions (145 mM), suggesting that the blockade of the Na(+)-Ca2+ antiporter during chemical hypoxia can lead to increased cell damage. 6. Collectively, these results suggest that activation of the Na(+)-Ca2+ exchanger protects C6 glioma cells exposed to chemical hypoxia, whereas its pharmacological blockade can exacerbate cellular injury.