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

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

Dissecting the Purinergic Signaling Puzzle

Purinergic signaling regulates a plethora of physiological processes and is an expanding research field [...

INFLUENCIA DE LA FLUIDEZ DE LA MEMBRANA SOBRE LA ATPasa DEPENDIENTE DE Ca2+ DEL RETÍCULO SARCOPLÁSMICO DE MÚSCULO ESQUELÉTICO

The ATPase from sarcoplasmic reticulum was reconstituted using either DMPC or DPPC in order to obtain a lipid-protein system with a single lipidic component and only one type of protein. The effects of the lipidic microenvironment on the protein have been studied through changes in enzyme activity at different temperatures and with different phospholipid composition of the membrane. The results obtained after the dependence of ATPase activity on temperature reveal an increase of activation energy at the transition temperature of the phospholipid (Te) and also at a temperature lower than Te corresponding to the fusion of lipid-protein patches. ATPase reconstituted with DPPC did not follow a Michaelis-Menten type of kinetic model in which activation by excess of substrate (ATP) is proposed. A study of the proposed kinetic parameters showed that all of them notably increased at approximately 30°C. being this temperature below Te for DPPC (41ºC). This is thetemperature at which the protein-rich lipid-protein patches melted in the DPPC-ATPase samples.Se ha utilizado DMPC o DPPC para reconstituir Ca2+ -ATPasa de retículo sarcoplásmico y así disponer de un sistema lípido-proteico que contenga un único tipo de fosfolípido y un único tipo de proteína. Para estudiar el efecto del entorno lipídico sobre la proteína se han medido los cambios que tienen lugar en la actividad enzimática al variar la temperatura y la composición lipídica de la membrana. Los datos obtenidos mediante la variación de la actividad ATPasa con la temperatura revelan un aumento de la energía de activación a la temperatura de transición del fosfolípido y también a una temperatura inferior, a la cual tiene lugar la fusión de agregados lípido-proteicos. La dependencia de la actividad de la Ca2+ -ATPasa reconstituida con DPPC con la concentración de sustrato (ATP) no sigue el modelo de Michaelis-Menten. Se propone un modelo cinético en el cual tiene lugar una activación por exceso de sustrato. Al estudiar la variación de los parámetros cinéticos asociados al modelo propuesto encontramos que todos ellos aumentan considerablemente a aproximadamente 30ºC. temperatura por debajo de la temperatura de transición del DPPC (41ºC ). A esta temperatura tiene lugar la fusión de los agregados lipoproteicos ricos en proteína cuando el lípido utilizado es el DPPC

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

Mechanisms of protein kinase D activation in response to P2Y2 and P2X7 receptors in primary astrocytes

Protein kinase D (PKD) is a family of serine/threonine kinases that can be activated by many stimuli via protein kinase C in a variety of cells. This is the first report where PKD activation and localization is studied in glial cells. Herein, we demonstrate that P2Y2 and P2X7 receptor stimulation of primary rat cerebellar astrocytes rapidly increases PKD1/2 phosphorylation and activity. P2Y2 receptor response evokes a PKD1/2 activation that is dependent on a pertussis toxin-insensitive G protein, phospholipase C (PLC)-mediated generation of diacylglycerol, and protein kinase C. This mechanism is similar to the one described for other G-protein coupled receptors. In contrast, the way the ionotropic P2X7 receptor activates PKD1/2 is significantly different. Importantly, this response is not dependent on calcium entry, but depends on the activity of several phospholipases, including phosphoinositide-phospholipase C (PI-PLC), phosphatidylcholine-phospholipase C (PC-PLC) and also phospholipase D (PLD). Immunoblot and confocal microscopy analysis show that PKD1/2 activation by nucleotides is transient. The active kinase first moves to and concentrates in certain plasma membrane domains. Then, phosphorylated-PKD1/2 translocates to intracellular vesicles, where it remains active. All together, our results open the perspective of PKD1/2 being involved in many physiological functions where nucleotides play important roles not only in astrocytes but in other cell types bearing these receptors. © 2010 Wiley-Liss, Inc.Grant sponsor: Ministerio de Educaci on y Ciencia; Grant number: BFU2008-02699; Grant sponsor: Consolider-Ingenio-SD2008-00005 (‘‘The Spanish Ion Channel Initiative’’); Grant sponsor: Comunidad de Madrid; Grant number: S-SAL-0253-2006; Grant sponsor: Fundacion Marcelino Botin to M.T. Miras-Portugal; Grant sponsor: Ministerio de Ciencia e Innovaci on; Grant number: SAF2008-01951; Grant sponsor: Comunidad de Madrid; Grant number: S-SAL-0202-2006-01; Grant sponsors: Instituto de Salud Carlos III (CIBERNED, Spain) to T. Iglesias.Peer Reviewe