Long-term activation of adenosine receptors regulates the survival and blocks glutamate-mediated neurotoxicity in cultures of retinal neurons

LONG-TERM ACTIVATION OF ADENOSINE RECEPTORS REGULATES THE SURVIVAL AND BLOCKS GLUTAMATE-MEDIATED NEUROTOXICITY IN CULTURES OF RETINAL NEURONS^*

ROBERTO PAES-DE-CARVALHO AND JAINNE MARTINS FERREIRA

Department of Neurobiology and Program of Neuroimmunology, Institute of Biology, Federal Fluminense University, Niterói, Brazil

Presented by FERNANDO G. DE MELLO

Adenosine (ado), a neuromodulator in the CNS, is taken up and released from developing chick retinal neurons in culture, suggesting a role for this molecule during retinal development. In the present work we show that preincubation of retinal neurons in culture with ado prevents cell death induced when refeeding the cultures with fresh medium or by exposure of cultures to glutamate. Cells dissociated by trypsinization from 8-day-old chick embryo retinas were seeded ( 8.3 ´ 10² cells /mm² ) on poly-L-ornithine coated dishes and incubated for 3 days at C in Medium 199 or BME. Under these conditions neurons and photoreceptors differentiate in the absence of glial cells and extensive cell contacts. The number of neurons decreases 50-70% after refeeding the cultures with fresh medium, an effect blocked when cultures are previously treated with ado + EHNA (ado deaminase inhibitor) or with Nitrobenzylthioinosine (NBI, ado uptake blocker) for hours. Ado deaminase also promotes cell death even without changing the medium, indicating that endogenous ado is necessary for cell survival. CGS21680, an A2a receptor agonist, but not CHA, an A1 agonist, is able to block cell death, an effect mimicked by 8-bromo cyclic AMP. Refeeding the cultures with a conditioned medium collected during the first 3 days of culture does not promote cell death. The addition of glutamate to cultures incubated in BME, but not in Medium 199, induces the death of about 80% of neurons that was blocked by preincubation with MK-801, an NMDA antagonist, and also with ado EHNA, NBI, the A2a adenosine receptor agonists CGS21680 and DPMA, or the cyclic AMP analogs 8-Bromo cAMP and Sp-cAMP. Maximal death is reached after 8 hours and in concentrations of glutamate as low as 50 mM as determined measuring the remaining LDH intracellular activity. The protective effect by ado is small when added 1 hour before glutamate and is maximal when added 24 hours or longer periods before the amino acid, indicating that the protection is mediated by the synthesis and/or release of trophic factors. The results show that ado promotes the survival and blocks glutamate toxicity in retinal neurons through the long-term activation of A2a receptors and elevation of intracellular cyclic AMP levels. — ( June 27, 2000 )

Publication Dates

• Publication in this collection
  05 Oct 2000
• Date of issue
  Sept 2000