Activation of P2X 7 Receptors Stimulates the Expression of P2Y 2 Receptor mRNA in Astrocytes Cultured from Rat Brain

Abstract

Under pathological conditions brain cells release ATP at concentrations reported to activate P2X₇ ionotropic receptor subtypes expressed in both neuronal and glial cells. In the present study we report that the most potent P2X₇ receptor agonist BzATP stimulates the expression of the metabotropic ATP receptor P2Y₂ in cultured rat brain astrocytes. In other cell types several kinds of stimulation, including stress or injury, induce P2Y₂ expression that, in turn, is involved in different cell reactions. Similarly, it has recently been found that in astrocytes and astrocytoma cells P2Y₂ sites can trigger neuroprotective pathways through the activation of several mechanisms, including the induction of genes for antiapoptotic factors, neurotrophins, growth factors and neuropeptides. Here we present evidence that P2Y₂ mRNA expression in cultured astrocytes peaks 6 h after BzATP exposure and returns to basal levels after 24 h. This effect was mimicked by high ATP concentrations (1 mM) and was abolished by P2X₇-antagonists oATP and BBG. The BzATP-evoked P2Y₂ receptor up-regulation in cultured astrocytes was coupled to an increased UTP-mediated intracellular calcium response. This effect was inhibited by oATP and BBG and by P2Y₂siRNA, thus supporting evidence of increased P2Y₂ activity. To further investigate the mechanisms by which P2X₇ receptors mediated the P2Y₂ mRNA up-regulation, the cells were pre-treated with the chelating agent EGTA, or with inhibitors of mitogen-activated kinase (MAPK) (PD98059) or protein kinase C, (GF109203X). Each inhibitor significantly reduced the extent to which BzATP induced P2Y₂ mRNA. Both BzATP and ATP (1 mM) increased ERK1/2 activation. P2X₇-induced ERK1/2 phosphorylation was unaffected by pre-treatment of astrocytes with EGTA whereas it was inhibited by GF109203X. Phorbol-12-myristate-13-acetate (PMA), an activator of PKCs, rapidly increased ERK1/2 activation. We conclude that activation of P2X₇ receptors in astrocytes enhances P2Y₂ mRNA expression by a mechanism involving both calcium influx and PKC/MAPK signalling pathways.