Microglia P2Y6 receptors mediate nitric oxide release and astrocyte apoptosis

Abstract

BackgroundDuring 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.MethodsCo-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 P2Y6 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.ResultsUTP, UDP (0.001 to 1 mM) or PSB 0474 (0.01 to 10 μM) inhibited cell proliferation up to 43 ± 2% (n = 10, P <0.05), an effect prevented by the selective P2Y6 receptor antagonist MRS 2578 (1 μM). 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 μM) increased NO release up to 199 ± 20% (n = 4, P <0.05), an effect dependent on P2Y6 receptors-PLC-PKC pathway activation, indicating that this pathway mediates NO release. Western blot and immunocytochemistry analysis indicated that P2Y6 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 μM). UDP-mediated NO release induced apoptosis in astrocytes, but not in microglia.ConclusionsIn 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.

Microglia modulate respiratory rhythm generation and autoresuscitation

“…Thus, the inhibition or depletion of microglia would reduce this tonic excitatory modulation, which would result in the reduction in respiratory rhythm generation (Doi and Ramirez, ; Peña‐Ortega, ; Toporikova and Butera, ). There are several mediators released by microglia that might be playing this role, including BDNF (Gomes et al, ), glutamate (Wu et al, ), D‐serine (Wu et al, ), ATP (Imura et al, ), hydrogen peroxide (Zhang et al, ), nitric oxide (Quintas et al, ), histamine (Katoh et al, ), or even prostaglandin E2 (Zhang et al, ; Wang et al, ). We suggest that the reduction in one or several of these microglia‐released modulators, upon microglia inhibition or depletion, might be affecting respiratory rhythm generation (Hutchinson et al, ; Peña‐Ortega, ).…”

Heterogeneous astrocytes: Active players in CNS

“…In addition, the microglia-derived cytokines also influence the activities of astrocytes, such as proliferation and reactivity (Bezzi et al 2001;Pascual et al 2012). Activated microglia is shown to control the rate of astrocyte proliferation and prevent astrogliosis by releasing NO (Quintas et al 2014). In rat retinitis pigmentosa model, microglia-Mϋller glia crosstalk exerts the neuroprotective effects in CNS diseases (Arroba et al 2014).…”

Lipopolysaccharide-Induced Apoptosis of Astrocytes: Therapeutic Intervention by Minocycline

“…that contribute to the pathogenesis of various neurodegenerative diseases (Sofroniew and Vinters 2010;Claycomb et al 2013). Although there are reports indicating NO-mediated apoptosis of astrocytes in rat primary astrocyte culture exposed to a combination of LPS and inflammatory cytokines but not by either alone (Suk et al 2001;Caruso et al 2007;Quintas et al 2014). In vivo studies stating intracerebroventricular (into lateral ventricle) LPS-induced astrocytic response in the hippocampus are scanty.…”