Nucleotides protect rat brain astrocytes against hydrogen peroxide toxicity and induce antioxidant defense via P2Y receptors

•2MeSADP, ATPγS, P2Y receptor agonists, protect astrocytes against oxidative stress.•P2Y receptors agonists maintain astrocytic survival and viability.•P2Y receptor agonists induce antioxidant enzymes, CAT, SOD2 and SOD3.•Total GSH content is increased by incubation with P2Y receptor agonists.•P2Y receptor agonists induce GCLM and 4F2hc, regulators of total GSH synthesis.

Consequences of neurodegenerative diseases or stroke also depend on astroglial survival during oxidative stress. P2Y receptors that are widely distributed in the central nervous system are suggested to be involved in cytoprotection. However, knowledge about the efficacy of protection by P2Y receptors and their involvement in antioxidant protective pathways is scarce. Here, we investigate the viability and reactive oxygen species (ROS) production after exposure of rat astrocytes to hydrogen peroxide. We determined the influence of treatment with the P2Y1 receptor-specific agonist 2-methyl-thio-ADP (2MeSADP) and the broad range P2Y receptor agonist adenosine 5’-(3-thiotriphosphate) (ATPγS). Preincubation (24-h before hydrogen peroxide application) and incubation with ATPγS and 2MeSADP protected astrocytes. The ROS production in hydrogen peroxide-treated astrocytes was reduced by pre- and co-incubation with ATPγS or 2MeSADP. Changes of levels of expression of antioxidant defense systems in astrocytes by treatment with P2Y receptor agonists were analyzed. Incubation with ATPγS and 2MeSADP increased mRNA levels of CAT encoding catalase and SOD2, encoding mitochondrial manganese dependent superoxide dismutase. ATPγS additionally increased mRNA levels of SOD3, encoding extracellular superoxide dismutase (ECSOD). Levels of total glutathione (GSH) increased in ATPγS/2MeSADP-treated astrocytes. mRNA levels of genes involved in GSH synthesis and in import of GSH precursors were analyzed after treatment with ATPγS and 2MeSADP. Both agonists significantly increased mRNA levels of a subunit of glutamate cysteine ligase, and a subunit of antiporter system xc−. Changes in mRNA levels of antioxidant enzymes and genes of GSH metabolism depend on rise of intracellular Ca2+ by P2Y receptor and basal activity of protein kinase A (PKA). SOD3 induction is suggested to depend on increased intracellular Ca2+, increased cyclic AMP levels and PKA activity. Thus, we confirm a role of purinergic signaling in astrocytic survival during oxidative stress by maintaining antioxidant defense, highlighting P2Y receptors as potential targets for cytoprotection.