Adenosine Protects Retinal Ganglion Cells from Cytotoxic Death following P2X7 Receptor Stimulation

Abstract

Abstract: : Purpose: To study the effect of ATP P2X₇ receptor agonist BzATP and adenosine on retinal ganglion cells (RGCs). Methods: 1) Cell viability studies: RGCs were backlabled by injecting Fluoro-Gold dye into the superior colliculus of Long Evan rat pups on PD2-6. Retinas were dissociated from PD6-15 and plated onto poly-L-lysine coated coverslips. After culturing for 24 hours with the drugs added to the medium, fluorescent cells present in 40 fields on each coverslip were counted in a masked fashion. 2) Intracellular Ca²⁺ measurements: Unlabled RGCs grown on coverslips for 24 hours were loaded with 10µM fura-2 and Ca²⁺ levels in RGCs were measured ratiometrically using a fluorescent imaging system. Results: BzATP killed up to 30% of RGCs at a concentration of 50µM when incubated in mixed retinal culture for 24 hours. Cell loss was concentration-dependent and increased with 100 µM and 500 µM BzATP. Cell loss was reduced by a 30 min preincubation with adenosine (300µM-1mM) and co-incubation of adenosine with BzATP. BzATP produced large elevations in cell calcium which frequently remained elevated after BzATP removal. Adenosine itself did not affect Ca²⁺ levels but pre- and co-treatment did attenuate the BzATP-triggered elevations and reduced the sustained Ca²⁺ elevation. Conclusions: Stimulation of P2X₇ receptors can elevate Ca²⁺ and kill RGCs. Adenosine can stop the P2X₇-triggered Ca²⁺ overload and prevent cell death.

Keywords: adenosine • ganglion cells • neuroprotection