The role of ATP and adenosine in the brain under normoxic and ischemic conditions

By taking advantage of some recently synthesized compounds that are able to block ecto-ATPase activity, we demonstrated that adenosine triphosphate (ATP) in the hippocampus exerts an inhibitory action independent of its degradation to adenosine. In addition, tonic activation of P2 receptors contributes to the normally recorded excitatory neurotransmission. The role of P2 receptors becomes critical during ischemia when extracellular ATP concentrations increase. Under such conditions, P2 antagonism is protective. Although ATP exerts a detrimental role under ischemia, it also exerts a trophic role in terms of cell division and differentiation. We recently reported that ATP is spontaneously released from human mesenchymal stem cells (hMSCs) in culture. Moreover, it decreases hMSC proliferation rate at early stages of culture. Increased hMSC differentiation could account for an ATP-induced decrease in cell proliferation. ATP as a homeostatic regulator might exert a different effect on cell trophism according to the rate of its efflux and receptor expression during the cell life cycle. During ischemia, adenosine formed by intracellular ATP escapes from cells through the equilibrative transporter. The protective role of adenosine A1 receptors during ischemia is well accepted. However, the use of selective A1 agonists is hampered by unwanted peripheral effects, thus attention has been focused on A2A and A3 receptors. The protective effects of A2A antagonists in brain ischemia may be largely due to reduced glutamate outflow from neurones and glial cells. Reduced activation of p38 mitogen-activated protein kinases that are involved in neuronal death through transcriptional mechanisms may also contribute to protection by A2A antagonism. Evidence that A3 receptor antagonism may be protective after ischemia is also reported

Role of the metabotropic P2Y(4) receptor during hypoglycemia: cross talk with the ionotropic NMDAR1 receptor

It is well established that both extracellular ATP and glutamate exert a critical role during metabolic impairment, that several P2 receptor subunits are directly involved in this action and that a strong relationship exists between glutamatergic and purinergic signals. Therefore, here we studied the molecular behavior of the purinergic metabotropic P2Y4 and the glutamatergic ionotropic NMDAR1 receptors during hypoglycemic cell death. We find that these proteins are oppositely modulated during glucose starvation (P2Y4 is induced, whereas NMDAR1 is inhibited) and that both P2 and NMDA antagonists can restore basal protein expression levels. Moreover, double immunofluorescence experiments with confocal laser microscopy reveal co-localization at the membrane level between the P2Y4 and NMDAR1 receptors, in both homologous (cerebellar granule neurons) and heterologous (Hek-293) cellular systems. This is furthermore confirmed by co-immunoprecipitation experiments. Finally, when we express the P2Y4 receptor in the heterologous SH-SY5Y neuronal cell line, hypoglycemia then causes severe cell death and simultaneous downregulation of the NMDAR1 protein. In summary, our work establishes a potential molecular interplay between P2Y4 and NMDAR1 receptors during glucose deprivation and the causative role of the P2Y 4 during cell death. © 2004 Elsevier Inc. All rights reserved

The metabotropic P2Y4 receptor participates in the commitment to differentiation and cell death of human neuroblastoma SH-SY5Y cells

Extracellular nucleotides exert a variety of biological actions through different subtypes of P2 receptors. Here we characterized in the human neuroblastoma SH-SY5Y cells the simultaneous presence of various P2 receptors, belonging to the P2X ionotropic and P2Y metabotropic (2,4,6,7) and P2Y(1,2,4,6) families. Western blot analysis detected the P2X(1,) but not the P2X(3) and P2Y(12) receptors. We then investigated which biological effects were mediated by the P2Y(4) subtype and its physiological pyrimidine agonist UTP. We found that neuronal differentiation of the SH-SY5Y cells with dibutiryl-cAMP increased the expression of the P2Y(4) protein and that UTP itself was able to positively interfere with neuritogenesis. Moreover, transient transfection and activation of P2Y(4) also facilitated neuritogenesis in SH-SY5Y cells, as detected by morphological phase contrast analysis and confocal examination of neurofilament proteins NFL. This was concurrent with increased transcription of immediate-early genes linked to differentiation such as cdk-5 and NeuroD6, and activity of AP-1 transcription family members such as c-fos, fos-B, and jun-D. Nevertheless, a prolonged activation of the P2Y(4) receptor by UTP also induced cell death, both in naive, differentiated, and P2Y(4)-transfected SH-SY5Y cells, as measured by direct count of intact nuclei and cytofluorimetric analysis of damaged DNA. Taken together, our data indicate that the high expression and activation of the P2Y(4) receptor participates in the neuronal differentiation and commitment to death of SH-SY5Y cells. (C) 2004 Elsevier Inc. All rights reserved

P2 receptor modulation and cytotoxic function in cultured CNS neurons

In this study we investigate the presence, modulation and biological function of P2 receptors and extracellular ATP in cultured cerebellar granule neurons. As we demonstrate by RT-PCR and western blotting, both P2X and P2Y receptor subtypes are expressed and furthermore regulated as a function of neuronal maturation. In early primary Cultures, mRNA for most of the P2 receptor subtypes, except P2X(6), are found, while in older cultures only P2X(3), P2Y(1) and P2Y(6) mRNA persist. In contrast, P2 receptor proteins are more prominent in mature neurons, with the exception of P2Y(1). We also report that extracellular ATP acts as a cell death mediator for fully differentiated and mature granule neurons, for dissociated striatal primary cells and hippocampal organotypic cultures, inducing both apoptotic and necrotic features of degeneration. ATP causes cell death with EC50 in the 20-50 muM range within few minutes of exposure and with a time lapse of at most two hours. Additional agonists for P2 receptors induce toxic effects, whereas selected antagonists are protective. Cellular swelling, lactic dehydrogenase release and nuclei fragmentation are among the features of ATP-evoked cell death, which also include direct P2 receptor modulation. Comparably to P2 receptor antagonists previously shown preventing glutamate-toxicity, here we report that competitive and non-competitive NMDA receptor antagonists inhibit the detrimental consequences of extracellular ATP. Due to the massive extracellular release of purine nucleotides and nucleosides often Occurring during a toxic insult, our data indicate that extracellular ATP can now be included among the potential causes of CNS neurodegenerative events. (C) 2002 Elsevier Science Ltd. All rights reserved

Up-regulation of P2X(2), P2X(4) receptor and ischemic cell death: Prevention by P2 antagonists

In the present work we examined the involvement of selected P2X receptors for extracellular ATP in the onset of neuronal cell death caused by glucose/oxygen deprivation. The in vitro studies of organotypic cultures from hippocampus evidenced that P2X(2) and P2X(4) were up-regulated by glucose/oxygen deprivation. Moreover, we showed that ischemic conditions induced specific neuronal loss not only in hippocampal, but also in cortical and striatal organotypic cultures and the P2 receptor antagonists basilen blue and suramin prevented these detrimental effects. In the in vivo experiments we confirmed the induction of P2X receptors in the hippocampus of gerbils subjected to bilateral common carotid occlusion. In particular, P2X(2) and P2X(4) proteins became significantly up-regulated, although to different extent and in different cellular phenotypes. The induction was confined to the pyramidal cell layer of the CA1 subfield and to the transition zone of the CA2 subfield and it was coincident with the area of neuronal damage. P2X(2) was expressed in neuronal cell bodies and fibers in the CA1 pyramidal cell layer and in the strata oriens and radiatum. Intense P2X(4) immunofluorescence was localized to microglia cells. Our results indicate a direct involvement of P2X receptors in the mechanisms sustaining cell death evoked by metabolism impairment and suggest the use of selected P2 antagonists as effective neuroprotecting agents. (C) 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved

Regional expression of P2Y4 receptors in the rat central nervous system

P2Y receptors are G protein-coupled receptors composed of eight known subunits (P2Y1, 2, 4, 6, 11, 12, 13, 14), which are involved in different functions in neural tissue. The present study investigates the expression pattern of P2Y4 receptors in the rat central nervous system (CNS) using immunohistochemistry and in situ hybridization. The specificity of the immunostaining has been verified by preabsorption, Western blot, and combined use of immunohistochemistry and in situ hybridization. Neurons expressing P2Y4 receptors were distributed widely in the rat CNS. Heavy P2Y4 receptor immunostaining was observed in the magnocellular neuroendocrine neurons of the hypothalamus, red nucleus, pontine nuclei, mesencephalic trigeminal nucleus, motor trigeminal nucleus, ambiguous nucleus, inferior olive, hypoglossal nucleus, and dorsal motor vagus nucleus. Both neurons and astrocytes express P2Y4 receptors. P2Y4 receptor immunostaining signals were mainly confined to cell bodies and dendrites of neurons, suggesting that P2Y4 receptors are mainly involved in regulating postsynaptic events. In the hypothalamus, all the vasopressin (VP) and oxytocin (OT) neurons and all the orexin A neurons were immunoreactive for P2Y4 receptors. All the neurons expressing P2Y4 receptors were found to express N-methyl-d-aspartate receptor 1 (NR1). These data suggest that purines and pyrimidines might be involved in regulation of the release of the neuropeptides VP, OT, and orexin in the rat hypothalamus via P2Y4 receptors. Further, the physiological and pathophysiological functions of the neurons may operate through coupling between P2Y4 receptors and NR1