P2Y 2 Receptor Up-Regulation Induced by Guanosine or UTP in Rat Brain Cultured Astrocytes

Abstract

Among P2 metabotropic ATP receptors, P2Y₂ subtype seems to be peculiar as its upregulation triggers important biological events in different cells types. In non-stimulated cells including astrocytes, P2Y₂ receptors are usually expressed at levels lower than P2Y₁ sites, however the promoter region of the P2Y₂ receptors has not yet been studied and little is known about the mechanisms underlying the regulation of the expression of this ATP receptor. We showed that not only UTP and ATP are the most potent and naturally occurring agonist for P2Y₂ sites, but also guanosine induced an up-regulation of astrocyte P2Y₂ receptor mRNA evaluated by Northern blot analysis. We also focused our attention on this nucleoside since in our previous studies it was reported to be released by cultured astrocytes and to exert different neuroprotective effects. UTP and guanosine-evoked P2Y₂ receptor up-regulation in rat brain cultured astrocytes was linked to an increased P2Y₂-mediated intracellular calcium response, thus suggesting an increased P2Y₂ activity. Actinomycin D, a RNA polymerase inhibitor, abrogated both UTP and guanosine-mediated P2Y₂ up-regulation, thus indicating that de novo transcription was required. The effect of UTP and guanosine was also evaluated in astrocytes pretreated with different inhibitors of signal transduction pathways including ERK, PKC and PKA reported to be involved in the regulation of other cell surface receptor mRNAs. The results show that ERK1-2/MAPK pathway play a key role in the P2Y₂ receptor up-regulation mediated by either UTP or guanosine. Moreover, our data suggest that PKA is also involved in guanosine-induced transcriptional activation of P2Y₂ mRNA and that increased intracellular calcium levels and PKC activation may also mediate P2Y₂ receptor up-regulation triggered by UTP. The extracellular release of ATP under physiological and pathological conditions has been widely studied. On the contrary, little is known about the release of pyrimidines and in particular of UTP. Here we show that astrocytes are able to release UTP, either at rest or during and following hypoxia/hypoglicemia obtained by submitting the cells to glucose-oxygen deprivation (OGD). Interestingly, also P2Y₂ receptor mRNA increased by about two-fold the control values when the cultures were submitted to OGD. It has been recently reported that P2Y receptors can play a protective role in astrocytes, thus either guanosine administration or increased extracellular concentrations of guanosine and UTP reached locally following CNS injury may increase P2Y₂-mediated biological events aimed at promoting a protective astrocyte response.