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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

  • Tharine Dal-Cim
  • Gabriela G. Poluceno
  • Débora Lanznaster
  • Karen A. de Oliveira
  • Claudia B. Nedel
  • Carla I. TascaEmail author
Original Article
  • 32 Downloads

Abstract

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.

Keywords

Guanosine Astrocytes Oxygen/glucose deprivation Glutamate uptake GLT-1 

Abbreviations

A1R

Adenosine A1 receptor

A2AR

Adenosine A2A receptor

CGS21680

4-[2-[[6-Amino-9-(N-ethyl-β-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl] benzenepropanoic acid hydrochloride

DPCPX

Dipropyl-cyclopentyl-xanthine

ERK

Extracellular signal-regulated kinase

GLT-1

Glutamate transporter-1

GUO

Guanosine

HBSS

Hank’s balanced salt solution

KRB

Krebs-Ringer bicarbonate buffer

LY294002

2-Morpholin-4-yl-8-phenylchromen-4-one

MAPK

Mitogen-activated protein kinase

MEK

Extracellular-signal regulated kinase kinase

MTT

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

OGD

Oxygen/glucose deprivation

PD98059

2′-Amino-3′-methoxyflavone

PI3K

Phosphatidylinositol-3 kinase

PKC

Protein kinase C

ROS

Reactive oxygen species

Notes

Author contributions

All authors have materially participated in the research and/or article preparation.

Funding information

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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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Departamento de Bioquímica, Centro de Ciências BiológicasUniversidade Federal de Santa CatarinaFlorianopolisBrazil
  2. 2.Programa de Pós-graduação em Neurociências, Centro de Ciências BiológicasUniversidade Federal de Santa CatarinaFlorianopolisBrazil
  3. 3.Programa de Pós-graduação em Bioquímica, Centro de Ciências BiológicasUniversidade Federal de Santa CatarinaFlorianopolisBrazil
  4. 4.Departamento de Biologia Celular, Embriologia e Genética, Centro de Ciências BiológicasUniversidade Federal de Santa CatarinaFlorianopolisBrazil

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