Release of soluble and vesicular purine nucleoside phosphorylase from rat astrocytes and microglia induced by pro-inflammatory stimulation with extracellular ATP via P2X7 receptors. (May 2018)

• Record Type: Journal Article
• Authors: Peña-Altamira, Luis Emiliano; Polazzi, Elisabetta; Giuliani, Patricia; Beraudi, Alina; Massenzio, Francesca; Mengoni, Ilaria; Poli, Alessandro; Zuccarini, Mariachiara; Ciccarelli, Renata; Di Iorio, Patrizia; Virgili, Marco; Monti, Barbara; Caciagli, Francesco
• Is Part Of: Neurochemistry international. Volume 115(2017)
• Page Start: 37
• Page End: 49
• Publication Date: 2018-05
• Subjects: Brain cell cultures -- Extracellular purine nucleoside phosphorylase (PNP) -- P2X7 receptor -- Mechanism of release -- Lysosomal vesicles; A-CM astrocyte derived-conditioned medium -- BME Basal Medium Eagle -- Bz-ATP 3′-O-benzoyl-benzoyl-ATP -- CGNs cerebellar granule neurons -- CNS central nervous system -- CSF cerebrospinal fluid -- JNK c-Jun N-terminal kinase -- DTT dithiothreitol -- ELISA enzyme linked immunosorbent assay -- EVs extracellular vesicles -- FBS fetal bovine serum -- GFAP glial fibrillary acidic protein -- GUA guanine -- GUO guanosine -- IL-1β interleukin-1β -- LAMP-1 Lysosome-Associated Membrane Protein-1 -- LPS lipopolysaccharide -- LDH lactate dehydrogenase -- MAPK mitogen-activated protein kinase -- M-CM microglia derived-conditioned medium -- N-CM neuron derived-conditioned medium -- PBS phosphate buffered saline -- PF pellet fraction -- PNP purine nucleoside phosphorylase -- P2X7R P2X7 receptor -- RT room temperature -- SDS sodium dodecyl sulphate -- SF soluble fraction -- VAMP7 Vesicle-associated membrane protein 7 -- XOR xanthine oxidoreductase; ATP: CID 5957 -- LPS: CID 11970143 -- Bz-ATP: CID 115205 -- SB202190: CID 5353940 -- SP600125: CID 8515 -- A438079: CID 11673921; Neurochemistry -- Periodicals; Neurochemistry -- Periodicals; Neurochimie -- Périodiques; Neurochemistry; Periodicals; 612.804205
• DOI: 10.1016/j.neuint.2017.10.010
• Languages: English
• ISSNs: 0197-0186

Abstract

Abstract: Purine nucleoside phosphorylase (PNP), a crucial enzyme in purine metabolism which converts ribonucleosides into purine bases, has mainly been found inside glial cells. Since we recently demonstrated that PNP is released from rat C6 glioma cells, we then wondered whether this occurs in normal brain cells. Using rat primary cultures of microglia, astrocytes and cerebellar granule neurons, we found that in basal condition all these cells constitutively released a metabolically active PNP with Km values very similar to those measured in C6 glioma cells. However, the enzyme expression/release was greater in microglia or astrocytes that in neurons. Moreover, we exposed primary brain cell cultures to pro-inflammatory agents such as lipopolysaccharide (LPS) or ATP alone or in combination. LPS alone caused an increased interleukin-1β (IL-1β) secretion mainly from microglia and no modification in the PNP release, even from neurons in which it enhanced cell death. In contrast, ATP administered alone to glial cells at high micromolar concentrations significantly stimulated the release of PNP within 1 h, an effect not modified by LPS presence, whereas IL-1β secretion was stimulated by ATP only in cells primed for 2 h with LPS. In both cases ATP effect was mediated by P2X7 receptor (P2X7 R), since it was mimicked by cell exposure to Bz-ATP, an agonist of P2X7 R, and blocked by cell pre-treatment with the P2X7 R antagonist A438079. Interestingly, ATP-induced PNP release from glial cells partly occurred through the secretion of lysosomal vesicles in the extracellular medium. Thus, during inflammatory cerebral events PNP secretion promoted by extracellular ATP accumulation might concur to control extracellular purine signals. Further studies could elucidate whether, in these conditions, a consensual activity of enzymes downstream of PNP in the purine metabolic cascade avoids accumulation of extracellular purine bases that might concur to brain injury by unusual formation of reactive oxygen species. Graphical abstract: Highlights: Purine Nucleoside Phosphorylase (PNP) is usually regarded as a cytosolic enzyme. PNP is also constitutively released from cultured neurons and glial cells. PNP release from glial cells is stimulated by ATP but not by lipopolysaccharide. ATP-enhanced PNP release is mediated by P2X7 receptors. ATP-induced PNP release partly occurs through secretion of lysosomal vesicles.