Sustained release of prostaglandin E2 in fibroblasts expressing ectopically cyclooxygenase 2 impairs P2Y-dependent Ca2+-mobilization

The nucleotide uridine trisphosphate (UTP) released to the extracellular milieu acts as a signaling molecule via activation of specific pyrimidine receptors (P2Y). P2Y receptors are G protein-coupled receptors expressed in many cell types. These receptors mediate several cell responses and they are involved in intracellular calcium mobilization. We investigated the role of the prostanoid PGE2in P2Y signaling in mouse embryonic fibroblasts (MEFs), since these cells are involved in different ontogenic and physiopathological processes, among them is tissue repair following proinflammatory activation. Interestingly, Ca2+-mobilization induced by UTP-dependent P2Y activation was reduced by PGE2when this prostanoid was produced by MEFs transfected with COX-2 or when PGE2was added exogenously to the culture medium. This Ca2+-mobilization was important for the activation of different metabolic pathways in fibroblasts. Moreover, inhibition of COX-2 with selective coxibs prevented UTP-dependent P2Y activation in these cells. The inhibition of P2Y responses by PGE2involves the activation of PKCs and PKD, a response that can be suppressed after pharmacological inhibition of these protein kinases. In addition to this, PGE2reduces the fibroblast migration induced by P2Y-agonists such as UTP. Taken together, these data demonstrate that PGE2is involved in the regulation of P2Y signaling in these cells.This work was supported by Grants BFU2011-24760 and BFU2011-24743 from MINECO, S2010/BMD-2378 from Comunidad de Madrid, Red de Investigación Cardiovascular, RIC, RD12/0042/0019, and Fundación Marcelino Botín (to María Teresa Miras-Portugal). RIC and Ciberehd are funded by the Instituto de Salud Carlos III.Peer Reviewe

Nucleotides in neuroregeneration and neuroprotection

AbstractBrain injury generates the release of a multitude of factors including extracellular nucleotides, which exhibit bi-functional properties and contribute to both detrimental actions in the acute phase and also protective and reparative actions in the later recovery phase to allow neuroregeneration. A promising strategy toward restoration of neuronal function is based on activation of endogenous adult neural stem/progenitor cells. The implication of purinergic signaling in stem cell biology, including regulation of proliferation, differentiation, and cell death has become evident in the last decade. In this regard, current strategies of acute transplantation of ependymal stem/progenitor cells after spinal cord injury restore altered expression of P2X4 and P2X7 receptors and improve functional locomotor recovery. The expression of both receptors is transcriptionally regulated by Sp1 factor, which plays a key role in the startup of the transcription machinery to induce regeneration-associated genes expression. Finally, general signaling pathways triggered by nucleotide receptors in neuronal populations converge on several intracellular kinases, such as PI3K/Akt, GSK3 and ERK1,2, as well as the Nrf-2/heme oxigenase-1 axis, which specifically link them to neuroprotection. In this regard, regulation of dual specificity protein phosphatases can become novel mechanism of actions for nucleotide receptors that associate them to cell homeostasis regulation.This article is part of the Special Issue entitled ‘Purines in Neurodegeneration and Neuroregeneration’

Cerebellar astrocytes co-express several ADP receptors. Presence of functional P2Y13-like receptors

Astrocytes exhibit a form of excitability based on variations of intracellular Ca2+ concentration in response to various stimuli, including ADP, ATP, UTP and dinucleotides. Here, we investigate the presence of the recently cloned ADP-sensitive receptors, P2Y12 and P2Y13 subtypes, which are negatively coupled to adenylate cyclase, in cerebellar astrocytes. We checked the effect of specific agonists, 2-methylthioadenosine diphosphate (2MeSADP) and ADP, on adenylate cyclase stimulation induced by isoproterenol. Both agonists significantly reduced the cAMP accumulation induced by isoproterenol. The inhibitory effect was concentration-dependent with IC50 values of 46 ± 13 and 23 ± 14 nM for 2MeSADP and ADP, respectively. The experiments were carried out in the presence of MRS-2179, a specific antagonist of P2Y1 receptor, to avoid any contribution of this receptor. Using fura-2 microfluorimetry we also proved that astrocytes responded to 2MeSADP stimulations with calcium responses in the absence and also in the presence of MRS-2179. Both effects, inhibition of adenylate cyclase and intracellular calcium mobilization, were not modified by 2MeSAMP, an antagonist of P2Y12 receptor, suggesting that were mediated by P2Y13-like receptors

Variation in antiosteoporotic drug prescribing and spending across Spain. A population-based ecological cross-sectional study

Introduction: Evidence has shown that utilization of antiosteoporotic medications does not correspond with risk, and studies on other therapies have shown that adequacy of pharmaceutical prescribing might vary between regions. Nevertheless, very few studies have addressed the variability in osteoporotic drug consumption. We aimed to describe variations in pharmaceutical utilization and spending on osteoporotic drugs between Health Areas (HA) in Spain. Methods: Population-based cross-sectional ecological study of expenditure and utilization of the five therapeutic groups marketed for osteoporosis treatment in Spain in 2009. Small area variation analysis (SAVA) methods were used. The units of analysis were the 168 HA of 13 Spanish regions, including 7.2 million women aged 50 years and older. The main outcomes were the defined daily dose (DDD) per 1000 inhabitants and day (DDD/1000/Day) dispensed according to the pharmaceutical claims reimbursed, and the expenditure on antiosteoporotics at retail price per woman =50 years old and per year. Results: The average osteoporosis drug consumption was 116.8 DDD/1000W/Day, ranging from 78.5 to 158.7 DDD/1000W/Day between the HAs in the 5th and 95th percentiles. Seventy-five percent of the antiosteoporotics consumed was bisphosphonates, followed by raloxifene, strontium ranelate, calcitonins, and parathyroid hormones including teriparatide. Regarding variability by therapeutic groups, biphosphonates showed the lowest variation, while calcitonins and parathyroid hormones showed the highest variation. The annual expenditure on antiosteoporotics was €426.5 million, translating into an expenditure of €59.2 for each woman =50 years old and varying between €38.1 and €83.3 between HAs in the 5th and 95th percentiles. Biphosphonates, despite accounting for 79% of utilization, only represented 63% of total expenditure, while parathyroid hormones with only 1.6% of utilization accounted for 15% of the pharmaceutical spending. Conclusion: This study highlights a marked geographical variation in the prescription of antiosteoporotics, being more pronounced in the case of costly drugs such as parathyroid hormones. The differences in rates of prescribing explained almost all of the variance in drug spending, suggesting that the difference in prescription volume between territories, and not the price of the drugs, is the main source of variation in this setting. Data on geographical variation of prescription can help guide policy proposals for targeting areas with inadequate antiosteoporotic drug use

Echoes of time. The mobility of Brazilian researchers and students in Portugal

A investigação que apresentamos, de caráter exploratório, recaiu sobre histórias biográficas de brasileiros que escolhem Portugal para prosseguir formação e ou investigação. Procura-se encontrar na sua experiência elos de ligação explicativos sobre as motivações e os processos que os trazem para Portugal, assim como as expetativas e os projetos que comportam para os seus futuros e que incluem, ou não, este país. Temos em conta, especialmente, a forma como essa narrativa transporta sentidos identitários decorrentes das formas de relacionamento intercultural e político entre Portugal e Brasil e formas de cooperação implícitas, assim como mapas representacionais acerca dos lugares de eleição para desenvolvimento de carreiras científicas e académicas. A nossa pesquisa incide sobre as informações recolhidas através de um inquérito por questionário e entrevistas realizadas junto de estudantes e bolseiros brasileiros em Portugal.We present an exploratory study that investigated biographical stories of Brazilians who choose to continue their education or develop research in Portugal. We sought to find in their experiences explanatory links connecting the motivations and processes that bring them to Portugal, as well as the expectations and projects that they hold for the future, which may include, or not, this country. We take into account, particularly, the way this narrative carries senses of identity arising from the forms of intercultural and political relationship between Portugal and Brazil, as well as implicit forms of cooperation and representations about the places chosen for the development of scientific and academic careers. Our research draws on information collected through a survey based on questionnaires and interviews with Brazilian students and scholarship holders in Portugal.(undefined

Papel fisiológico de los nucleótidos extracelulares en el sistema nervioso central: señalización vía receptores P2X y P2Y

In the last few years nucleotide receptors, the ionotropic P2X1-7 subunits and the metabotropic P2Y1, 2, 4, 6, 11, 12, 13, 14, have acquired an excepcional importance due to their strategic location in organs and tissues, their great variety along with the complexity of the associated signalling pathways and the first evidence of the serious alterations entailed in their dysfunctions. Our group has been pioneer in the characterization of these receptors in the nervous system, where we defined their location and functionality. The abundant presence, at a presynaptic level, of P2X3 and P2X7 should be emphasized, where by means of calcium intake they induce neurotransmitter exocytosis, such as glutamate, GABA, catecholamines and acetylcholine among others, as described in previous works by our group. In addition, they induce an extensive remodeling of the terminal’s cytoskeleton and exocytotic mechanisms through CaMKII and they can interact widely with other ionotropic and metabotropic receptors co-existing in nearby areas. Neural cells also exhibit the presence of most P2Y receptors signalling through a large variety of intracellular cascades. Recently we have demostrated that P2Y metabotropic receptors of the sub-family activated by ADP, especially P2Y13, are connected with the signalling towards GSK3 and â-catenin, opening new ways of understading the nucleotide function in survival and maintenance of neural cells. In addition both P2X and P2Y receptors play a role in early developmental stages and neural maturation where their function has to be fully understanded. Nucleotide receptors are also very abundant in glial cells, and our group has shown that most P2Y receptors are present and fully functional in cultured astrocytes, where, depending on the subtype receptor they activate a large variety of signalling cascades.En los ultimos anos los receptores de nucleotidos, receptores ionotropicos P2X1-7 y metabotropicos P2Y1, 2, 4, 6, 11, 12, 13, 14, han adquirido una importancia excepcional debido a su localizacion estrategica en organos y tejidos, a su gran variedad junto con la complejidad de vias de senalizacion a las que estan asociados y a las primeras evidencias de importantes alteraciones debidas a su mal funcionamiento. Nuestro grupo ha sido pionero en la caracterizacion estos receptores en el sistema nervioso, donde definimos su localizacion y su funcionalidad. La abundante presencia, a nivel presinaptico, de las subunidades P2X3 y P2X7 debe ser resaltada, donde gracias a la entrada de calcio inducen la exocitosis de varios neurotransmisores, como glutamato, GABA, catecolaminas y acetilcolina entre otros, como ha sido descrito por nuestro grupo en trabajos previos. Ademas, estos receptores inducen una profunda remodelacion del citoesqueleto de las terminales nerviosas y de los mecanismos exocitoticos a traves de la CaMKII y pueden interactuar con otros receptores ionotropicos y metabotropicos co-existentes en sus cercanias. La mayoria de los receptores P2Y tambien estan presentes en las celulas nerviosas, activando vias de senalizacion a traves de una gran variedad de cascadas intracelulares. Recientemente hemos demostrado que los receptores metabotropicos P2Y pertenecientes a la sub-familia de receptores activados por ADP, especialmente el P2Y13, estan conectados con la senalizacion hacia GSK3 y ƒÀ-catenina, lo que abre nuevas vias para la comprension de la funcion de los nucleotidos en la supervivencia y el mantenimiento de las celulas nerviosas. Ademas, tanto los receptores P2X como los P2Y juegan un papel en los estadios iniciales del desarrollo y en la maduracion neuronal donde su funcion aun ha de ser plenamente comprendida. Los receptores de nucleotidos son tambien muy abundantes en las celulas gliales, y nuestro grupo ha demostrado que la mayoría de los receptores P2Y están presentes y son plenamente funcionales en astrocitos en cultivo, donde, dependiendo del subtipo de receptor, activan una gran variedad de cascadas de señalización

NUDT2 Disruption Elevates Diadenosine Tetraphosphate (Ap4A) and Down-Regulates Immune Response and Cancer Promotion Genes.

Regulation of gene expression is one of several roles proposed for the stress-induced nucleotide diadenosine tetraphosphate (Ap4A). We have examined this directly by a comparative RNA-Seq analysis of KBM-7 chronic myelogenous leukemia cells and KBM-7 cells in which the NUDT2 Ap4A hydrolase gene had been disrupted (NuKO cells), causing a 175-fold increase in intracellular Ap4A. 6,288 differentially expressed genes were identified with P < 0.05. Of these, 980 were up-regulated and 705 down-regulated in NuKO cells with a fold-change ≥ 2. Ingenuity® Pathway Analysis (IPA®) was used to assign these genes to known canonical pathways and functional networks. Pathways associated with interferon responses, pattern recognition receptors and inflammation scored highly in the down-regulated set of genes while functions associated with MHC class II antigens were prominent among the up-regulated genes, which otherwise showed little organization into major functional gene sets. Tryptophan catabolism was also strongly down-regulated as were numerous genes known to be involved in tumor promotion in other systems, with roles in the epithelial-mesenchymal transition, proliferation, invasion and metastasis. Conversely, some pro-apoptotic genes were up-regulated. Major upstream factors predicted by IPA® for gene down-regulation included NFκB, STAT1/2, IRF3/4 and SP1 but no major factors controlling gene up-regulation were identified. Potential mechanisms for gene regulation mediated by Ap4A and/or NUDT2 disruption include binding of Ap4A to the HINT1 co-repressor, autocrine activation of purinoceptors by Ap4A, chromatin remodeling, effects of NUDT2 loss on transcript stability, and inhibition of ATP-dependent regulatory factors such as protein kinases by Ap4A. Existing evidence favors the last of these as the most probable mechanism. Regardless, our results suggest that the NUDT2 protein could be a novel cancer chemotherapeutic target, with its inhibition potentially exerting strong anti-tumor effects via multiple pathways involving metastasis, invasion, immunosuppression and apoptosis

A deletion and a duplication in distal 22q11.2 deletion syndrome region. Clinical implications and review

<p>Abstract</p> <p>Background</p> <p>Individuals affected with DiGeorge and Velocardiofacial syndromes present with both phenotypic diversity and variable expressivity. The most frequent clinical features include conotruncal congenital heart defects, velopharyngeal insufficiency, hypocalcemia and a characteristic craniofacial dysmorphism. The etiology in most patients is a 3 Mb recurrent deletion in region 22q11.2. However, cases of infrequent deletions and duplications with different sizes and locations have also been reported, generally with a milder, slightly different phenotype for duplications but with no clear genotype-phenotype correlation to date.</p> <p>Methods</p> <p>We present a 7 month-old male patient with surgically corrected ASD and multiple VSDs, and dysmorphic facial features not clearly suggestive of 22q11.2 deletion syndrome, and a newborn male infant with cleft lip and palate and upslanting palpebral fissures. Karyotype, FISH, MLPA, microsatellite markers segregation studies and SNP genotyping by array-CGH were performed in both patients and parents.</p> <p>Results</p> <p>Karyotype and FISH with probe N25 were normal for both patients. MLPA analysis detected a partial <it>de novo </it>1.1 Mb deletion in one patient and a novel partial familial 0.4 Mb duplication in the other. Both of these alterations were located at a distal position within the commonly deleted region in 22q11.2. These rearrangements were confirmed and accurately characterized by microsatellite marker segregation studies and SNP array genotyping.</p> <p>Conclusion</p> <p>The phenotypic diversity found for deletions and duplications supports a lack of genotype-phenotype correlation in the vicinity of the LCRC-LCRD interval of the 22q11.2 chromosomal region, whereas the high presence of duplications in normal individuals supports their role as polymorphisms. We suggest that any hypothetical correlation between the clinical phenotype and the size and location of these alterations may be masked by other genetic and/or epigenetic modifying factors.</p