Skip to main content

Advertisement

SpringerLink
Log in

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

  • Original Article
  • Published:
Purinergic Signalling Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

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

References

  1. Bonde C, Sarup A, Schousboe A, Gegelashvili G, Zimmer J, Noraberg J (2003) Neurotoxic and neuroprotective effects of the glutamate transporter inhibitor DL-threo-beta-benzyloxyaspartate (DL-TBOA) during physiological and ischemia-like conditions. Neurochem Int 43(4–5):371–380

    Article  CAS  PubMed  Google Scholar 

  2. Camacho A, Massieu L (2006) Role of glutamate transporters in the clearance and release of glutamate during ischemia and its relation to neuronal death. Arch Med Res 37(1):11–18

    Article  CAS  PubMed  Google Scholar 

  3. Trendelenburg G, Dirnagl U (2005) Neuroprotective role of astrocytes in cerebral ischemia: focus on ischemic preconditioning. Glia 50(4):307–320

    Article  PubMed  Google Scholar 

  4. Zhao Y, Rempe DA (2010) Targeting astrocytes for stroke therapy. Neurotherapeutics 7(4):439–451

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Danbolt NC (2001) Glutamate uptake. Prog Neurobiol 65(1):1–105

    Article  CAS  PubMed  Google Scholar 

  6. Schousboe A, Waagepetersen HS (2006) Glial modulation of GABAergic and glutamatergic neurotransmission. Curr Top Med Chem 6(10):929–934

    Article  CAS  PubMed  Google Scholar 

  7. Guillet BA et al (2005) Differential regulation by protein kinases of activity and cell surface expression of glutamate transporters in neuron-enriched cultures. Neurochem Int 46(4):337–346

    Article  CAS  PubMed  Google Scholar 

  8. Sheldon AL, Robinson MB (2007) The role of glutamate transporters in neurodegenerative diseases and potential opportunities for intervention. Neurochem Int 51(6–7):333–355

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Dienel GA (2013) Astrocytic energetics during excitatory neurotransmission: what are contributions of glutamate oxidation and glycolysis? Neurochem Int 63(4):244–258

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Trotti D, Danbolt NC, Volterra A (1998) Glutamate transporters are oxidant-vulnerable: a molecular link between oxidative and excitotoxic neurodegeneration? Trends Pharmacol Sci 19(8):328–334

    Article  CAS  PubMed  Google Scholar 

  11. Ciccarelli R, Ballerini P, Sabatino G, Rathbone MP, D’Onofrio M, Caciagli F, di Iorio P (2001) Involvement of astrocytes in purine-mediated reparative processes in the brain. Int J Dev Neurosci 19(4):395–414

    Article  CAS  PubMed  Google Scholar 

  12. Uemura Y, Miller JM, Matson WR, Beal MF (1991) Neurochemical analysis of focal ischemia in rats. Stroke 22(12):1548–1553

    Article  CAS  PubMed  Google Scholar 

  13. Ciccarelli R, di Iorio P, Giuliani P, D’Alimonte I, Ballerini P, Caciagli F, Rathbone MP (1999) Rat cultured astrocytes release guanine-based purines in basal conditions and after hypoxia/hypoglycemia. Glia 25(1):93–98

    Article  CAS  PubMed  Google Scholar 

  14. Tasca CI, Lanznaster D, Oliveira KA, Fernández-Dueñas V, Ciruela F (2018) Neuromodulatory effects of guanine-based purines in health and disease. Front Cell Neurosci 12:376

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Dal-Cim T, Martins WC, Santos ARS, Tasca CI (2011) Guanosine is neuroprotective against oxygen/glucose deprivation in hippocampal slices via large conductance Ca(2)+-activated K+ channels, phosphatidilinositol-3 kinase/protein kinase B pathway activation and glutamate uptake. Neuroscience 183:212–220

    Article  CAS  PubMed  Google Scholar 

  16. Dal-Cim T, Ludka FK, Martins WC, Reginato C, Parada E, Egea J, López MG, Tasca CI (2013) Guanosine controls inflammatory pathways to afford neuroprotection of hippocampal slices under oxygen and glucose deprivation conditions. J Neurochem 126(4):437–450

    Article  CAS  PubMed  Google Scholar 

  17. Thomaz DT, Dal-Cim TA, Martins WC, Cunha MP, Lanznaster D, de Bem AF, Tasca CI (2016) Guanosine prevents nitroxidative stress and recovers mitochondrial membrane potential disruption in hippocampal slices subjected to oxygen/glucose deprivation. Purinergic Signal 12(4):707–718

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Dal-Cim T, Martins WC, Thomaz DT, Coelho V, Poluceno GG, Lanznaster D, Vandresen-Filho S, Tasca CI (2016) Neuroprotection promoted by guanosine depends on glutamine synthetase and glutamate transporters activity in hippocampal slices subjected to oxygen/glucose deprivation. Neurotox Res 29(4):460–468

    Article  CAS  PubMed  Google Scholar 

  19. Lanznaster D, Dal-Cim T, Piermartiri TCB, Tasca CI (2016) Guanosine: a neuromodulator with therapeutic potential in brain disorders. Aging Dis 7(5):657–679

    Article  PubMed  PubMed Central  Google Scholar 

  20. Mendes-de-Aguiar CB et al (2008) Thyroid hormone increases astrocytic glutamate uptake and protects astrocytes and neurons against glutamate toxicity. J Neurosci Res 86(14):3117–3125

    Article  CAS  PubMed  Google Scholar 

  21. Pocock JM, Nicholls DG (1998) Exocytotic and nonexocytotic modes of glutamate release from cultured cerebellar granule cells during chemical ischaemia. J Neurochem 70(2):806–813

    Article  CAS  PubMed  Google Scholar 

  22. Hurtado O, Lizasoain I, Fernández-Tomé P, Álvarez-Barrientos A, Leza JC, Lorenzo P, Moro MA (2002) TACE/ADAM17-TNF-alpha pathway in rat cortical cultures after exposure to oxygen-glucose deprivation or glutamate. J Cereb Blood Flow Metab 22(5):576–585

    Article  CAS  PubMed  Google Scholar 

  23. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65(1–2):55–63

    Article  CAS  PubMed  Google Scholar 

  24. Molz S, Dal-Cim T, Tasca CI (2009) Guanosine-5′-monophosphate induces cell death in rat hippocampal slices via ionotropic glutamate receptors activation and glutamate uptake inhibition. Neurochem Int 55(7):703–709

    Article  CAS  PubMed  Google Scholar 

  25. Dal-Cim T, Molz S, Egea J, Parada E, Romero A, Budni J, Martín de Saavedra MD, Barrio L, Tasca CI, López MG (2012) Guanosine protects human neuroblastoma SH-SY5Y cells against mitochondrial oxidative stress by inducing heme oxigenase-1 via PI3K/Akt/GSK-3beta pathway. Neurochem Int 61(3):397–404

    Article  CAS  PubMed  Google Scholar 

  26. Constantino LC, Binder LB, Vandresen-Filho S, Viola GG, Ludka FK, Lopes MW, Leal RB, Tasca CI (2018) Role of phosphatidylinositol-3 kinase pathway in NMDA preconditioning: different mechanisms for seizures and hippocampal neuronal degeneration induced by quinolinic acid. Neurotox Res 34(3):452–462

    Article  CAS  PubMed  Google Scholar 

  27. Lowry OH et al (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193(1):265–275

    Article  CAS  PubMed  Google Scholar 

  28. Peterson GL (1977) A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem 83(2):346–356

    Article  CAS  PubMed  Google Scholar 

  29. Gegelashvili G et al (2000) The high-affinity glutamate transporters GLT1, GLAST, and EAAT4 are regulated via different signalling mechanisms. Neurochem Int 37(2–3):163–170

    Article  CAS  PubMed  Google Scholar 

  30. Decker H, Francisco SS, Mendes-de-Aguiar CBN, Romão LF, Boeck CR, Trentin AG, Moura-Neto V, Tasca CI (2007) Guanine derivatives modulate extracellular matrix proteins organization and improve neuron-astrocyte co-culture. J Neurosci Res 85(9):1943–1951

    Article  CAS  PubMed  Google Scholar 

  31. Lukaszevicz AC, Sampaïo N, Guégan C, Benchoua A, Couriaud C, Chevalier E, Sola B, Lacombe P, Onténiente B (2002) High sensitivity of protoplasmic cortical astroglia to focal ischemia. J Cereb Blood Flow Metab 22(3):289–298

    Article  CAS  PubMed  Google Scholar 

  32. Giffard RG, Swanson RA (2005) Ischemia-induced programmed cell death in astrocytes. Glia 50(4):299–306

    Article  PubMed  Google Scholar 

  33. Quincozes-Santos A, Bobermin LD, Souza DG, Bellaver B, Gonçalves CA, Souza DO (2014) Guanosine protects C6 astroglial cells against azide-induced oxidative damage: a putative role of heme oxygenase 1. J Neurochem 130(1):61–74

    Article  CAS  PubMed  Google Scholar 

  34. Giuliani P et al (2015) Guanosine protects glial cells against 6-hydroxydopamine toxicity. Adv Exp Med Biol 837:23–33

    Article  PubMed  Google Scholar 

  35. Bellaver B, Souza DG, Bobermin LD, Gonçalves CA, Souza DO, Quincozes-Santos A (2015) Guanosine inhibits LPS-induced pro-inflammatory response and oxidative stress in hippocampal astrocytes through the heme oxygenase-1 pathway. Purinergic Signal 11(4):571–580

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Souza DG, Bellaver B, Bobermin LD, Souza DO, Quincozes-Santos A (2016) Anti-aging effects of guanosine in glial cells. Purinergic Signal 12(4):697–706

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Nanetti L, Raffaelli F, Vignini A, Perozzi C, Silvestrini M, Bartolini M, Provinciali L, Mazzanti L (2011) Oxidative stress in ischaemic stroke. Eur J Clin Investig 41(12):1318–1322

    Article  CAS  Google Scholar 

  38. Hansel G, Ramos DB, Delgado CA, Souza DG, Almeida RF, Portela LV, Quincozes-Santos A, Souza DO (2014) The potential therapeutic effect of guanosine after cortical focal ischemia in rats. PLoS One 9(2):e90693

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Hansel G, Tonon AC, Guella FL, Pettenuzzo LF, Duarte T, Duarte MMMF, Oses JP, Achaval M, Souza DO (2015) Guanosine protects against cortical focal ischemia. Involvement of inflammatory response. Mol Neurobiol 52(3):1791–1803

    Article  CAS  PubMed  Google Scholar 

  40. de Oliveira DL et al (2004) Quinolinic acid promotes seizures and decreases glutamate uptake in young rats: reversal by orally administered guanosine. Brain Res 1018(1):48–54

    Article  PubMed  CAS  Google Scholar 

  41. Moretto MB, Arteni NS, Lavinsky D, Netto CA, Rocha JBT, Souza DO, Wofchuk S (2005) Hypoxic-ischemic insult decreases glutamate uptake by hippocampal slices from neonatal rats: prevention by guanosine. Exp Neurol 195(2):400–406

    Article  CAS  PubMed  Google Scholar 

  42. Molz S, Dal-Cim T, Budni J, Martín-de-Saavedra MD, Egea J, Romero A, del Barrio L, Rodrigues ALS, López MG, Tasca CI (2011) Neuroprotective effect of guanosine against glutamate-induced cell death in rat hippocampal slices is mediated by the phosphatidylinositol-3 kinase/Akt/glycogen synthase kinase 3beta pathway activation and inducible nitric oxide synthase inhibition. J Neurosci Res 89(9):1400–1408

    Article  CAS  PubMed  Google Scholar 

  43. Di Liberto V et al (2016) The guanine-based purinergic system: the tale of an orphan neuromodulation. Front Pharmacol 7:158

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  44. Volpini R, Marucci G, Buccioni M, Dal Ben D, Lambertucci C, Lammi C, Mishra RC, Thomas A, Cristalli G (2011) Evidence for the existence of a specific g protein-coupled receptor activated by guanosine. ChemMedChem 6(6):1074–1080

    Article  CAS  PubMed  Google Scholar 

  45. Zelenaia O, Schlag BD, Gochenauer GE, Ganel R, Song W, Beesley JS, Grinspan JB, Rothstein JD, Robinson MB (2000) Epidermal growth factor receptor agonists increase expression of glutamate transporter GLT-1 in astrocytes through pathways dependent on phosphatidylinositol 3-kinase and transcription factor NF-kappaB. Mol Pharmacol 57(4):667–678

    Article  CAS  PubMed  Google Scholar 

  46. Crossthwaite AJ, Hasan S, Williams RJ (2002) Hydrogen peroxide-mediated phosphorylation of ERK1/2, Akt/PKB and JNK in cortical neurones: dependence on Ca(2+) and PI3-kinase. J Neurochem 80(1):24–35

    Article  CAS  PubMed  Google Scholar 

  47. Brazil DP, Yang ZZ, Hemmings BA (2004) Advances in protein kinase B signalling: AKTion on multiple fronts. Trends Biochem Sci 29(5):233–242

    Article  CAS  PubMed  Google Scholar 

  48. Niizuma K, Yoshioka H, Chen H, Kim GS, Jung JE, Katsu M, Okami N, Chan PH (2010) Mitochondrial and apoptotic neuronal death signaling pathways in cerebral ischemia. Biochim Biophys Acta 1802(1):92–99

    Article  CAS  PubMed  Google Scholar 

  49. Krizman-Genda E, González MI, Zelenaia O, Robinson MB (2005) Evidence that Akt mediates platelet-derived growth factor-dependent increases in activity and surface expression of the neuronal glutamate transporter, EAAC1. Neuropharmacology 49(6):872–882

    Article  CAS  PubMed  Google Scholar 

  50. Sims KD, Straff DJ, Robinson MB (2000) Platelet-derived growth factor rapidly increases activity and cell surface expression of the EAAC1 subtype of glutamate transporter through activation of phosphatidylinositol 3-kinase. J Biol Chem 275(7):5228–5237

    Article  CAS  PubMed  Google Scholar 

  51. Frizzo ME, Frizzo JK, Amadio S, Rodrigues JM, Perry ML, Bernardi G, Volonté C (2007) Extracellular adenosine triphosphate induces glutamate transporter-1 expression in hippocampus. Hippocampus 17(4):305–315

    Article  CAS  PubMed  Google Scholar 

Download references

Funding

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.

Author information

Author notes

  1. Tharine Dal-Cim and Gabriela Poluceno contributed equally to this work.

Authors and Affiliations

  1. Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Trindade, Florianopolis, SC, 88040-900, Brazil

    Tharine Dal-Cim, Gabriela G. Poluceno, Débora Lanznaster, Karen A. de Oliveira & Carla I. Tasca

  2. Programa de Pós-graduação em Neurociências, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianopolis, SC, Brazil

    Tharine Dal-Cim, Gabriela G. Poluceno, Débora Lanznaster & Carla I. Tasca

  3. Programa de Pós-graduação em Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianopolis, SC, Brazil

    Karen A. de Oliveira & Carla I. Tasca

  4. Departamento de Biologia Celular, Embriologia e Genética, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Florianopolis, SC, Brazil

    Claudia B. Nedel

Authors
  1. Tharine Dal-Cim
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gabriela G. Poluceno
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Débora Lanznaster
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Karen A. de Oliveira
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Claudia B. Nedel
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Carla I. Tasca
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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

Corresponding author

Correspondence to Carla I. Tasca.

Ethics declarations

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.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Dal-Cim, T., Poluceno, G.G., Lanznaster, D. et al. 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. Purinergic Signalling 15, 465–476 (2019). https://doi.org/10.1007/s11302-019-09679-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11302-019-09679-w

Keywords

Search

Navigation