Background: During cerebral inflammation uracil nucleotides leak to the extracellular medium and activate glial pyrimidine receptors contributing to the development of a reactive phenotype. Chronically activated microglia acquire an anti-inflammatory phenotype that favors neuronal differentiation, but the impact of these microglia on astrogliosis is unknown. We investigated the contribution of pyrimidine receptors to microglia-astrocyte signaling in a chronic model of inflammation and its impact on astrogliosis. Methods: Co-cultures of astrocytes and microglia were chronically treated with lipopolysaccharide (LPS) and incubated with uracil nucleotides for 48 h. The effect of nucleotides was evaluated in methyl-[3H]-thymidine incorporation. Western blot and immunofluorescence was performed to detect the expression of P2Y(6) receptors and the inducible form of nitric oxide synthase (iNOS). Nitric oxide (NO) release was quantified through Griess reaction. Cell death was also investigated by the LDH assay and by the TUNEL assay or Hoechst 33258 staining. Results: UTP, UDP (0.001 to 1 mM) or PSB 0474 (0.01 to 10 mu M) inhibited cell proliferation up to 43 +/- 2% (n = 10, P lt; 0.05), an effect prevented by the selective P2Y(6) receptor antagonist MRS 2578 (1 mu M). UTP was rapidly metabolized into UDP, which had a longer half-life. The inhibitory effect of UDP (1 mM) was abolished by phospholipase C (PLC), protein kinase C (PKC) and nitric oxide synthase (NOS) inhibitors. Both UDP (1 mM) and PSB 0474 (10 mu M) increased NO release up to 199 +/- 20% (n = 4, P lt; 0.05), an effect dependent on P2Y(6) receptors-PLC-PKC pathway activation, indicating that this pathway mediates NO release. Western blot and immunocytochemistry analysis indicated that P2Y(6) receptors were expressed in the cultures being mainly localized in microglia. Moreover, the expression of iNOS was mainly observed in microglia and was upregulated by UDP (1 mM) or PSB 0474 (10 mu M). UDP-mediated NO release induced apoptosis in astrocytes, but not in microglia. Conclusions: In LPS treated co-cultures of astrocytes and microglia, UTP is rapidly converted into UDP, which activates P2Y6 receptors inducing the release of NO by microglia that causes astrocyte apoptosis, thus controlling their rate of proliferation and preventing an excessive astrogliosis.