Guanosine prevents oxidative damage and glutamate uptake impairment induced by oxygen/glucose deprivation in cortical astrocyte cultures: involvement of A1 and A2A adenosine receptors and PI3K, MEK, and PKC pathways
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Glial cells are involved in multiple cerebral functions that profoundly influence brain tissue viability during ischemia, and astrocytes are the main source of extracellular purines as adenosine and guanosine. The endogenous guanine-based nucleoside guanosine is a neuromodulator implicated in important processes in the brain, such as modulation of glutamatergic transmission and protection against oxidative and inflammatory damage. We evaluated if the neuroprotective effect of guanosine is also observed in cultured cortical astrocytes subjected to oxygen/glucose deprivation (OGD) and reoxygenation. We also assessed the involvement of A1 and A2A adenosine receptors and phosphatidylinositol-3 kinase (PI3K), MAPK, and protein kinase C (PKC) signaling pathways on the guanosine effects. OGD/reoxygenation decreased cell viability and glutamate uptake and increased reactive oxygen species (ROS) production in cultured astrocytes. Guanosine treatment prevented these OGD-induced damaging effects. Dipropyl-cyclopentyl-xanthine (an adenosine A1 receptor antagonist) and 4-[2-[[6-amino-9-(N-ethyl-β-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl] benzenepropanoic acid hydrochloride (an adenosine A2A receptor agonist) abolished guanosine-induced protective effects on ROS production, glutamate uptake, and cell viability. The PI3K pathway inhibitor 2-morpholin-4-yl-8-phenylchromen-4-one, the extracellular-signal regulated kinase kinase (MEK) inhibitor 2′-amino-3′-methoxyflavone, or the PKC inhibitor chelerythrine abolished the guanosine effect of preventing OGD-induced cells viability reduction. PI3K inhibition partially prevented the guanosine effect of reducing ROS production, whereas MEK and PKC inhibitions prevented the guanosine effect of restoring glutamate uptake. The total immunocontent of the main astrocytic glutamate transporter glutamate transporter-1 (GLT-1) was not altered by OGD and guanosine. However, MEK and PKC inhibitions also abolished the guanosine effect of increasing cell-surface expression of GLT-1 in astrocytes subjected to OGD. Then, guanosine prevents oxidative damage and stimulates astrocytic glutamate uptake during ischemic events via adenosine A1 and A2A receptors and modulation of survival signaling pathways, contributing to microenvironment homeostasis that culminates in neuroprotection.
KeywordsGuanosine Astrocytes Oxygen/glucose deprivation Glutamate uptake GLT-1
Adenosine A1 receptor
Adenosine A2A receptor
4-[2-[[6-Amino-9-(N-ethyl-β-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl] benzenepropanoic acid hydrochloride
Extracellular signal-regulated kinase
Hank’s balanced salt solution
Krebs-Ringer bicarbonate buffer
Mitogen-activated protein kinase
Extracellular-signal regulated kinase kinase
Protein kinase C
Reactive oxygen species
All authors have materially participated in the research and/or article preparation.
Research was supported by grants from the Brazilian funding agencies: CAPES (Coordenação do Pessoal de Ensino Superior)—Project CAPES-PVE 052/2012; CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico)—Project INCT for Excitotoxicity and Neuroprotection; and FAPESC (Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina)—Project NENASC. C.I.T. is recipient of CNPq productivity fellowship.
Compliance with ethical standards
The procedures used in the present study complied with the guidelines on animal care of the UFSC Ethics Committee on the Use of Animals (CEUA), which follows the Principles of laboratory animal care from NIH (2011).
Conflict of interest
The authors declare that they have no conflict of interest.
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