Neuronal Death Evoked by the P2X7 Receptor Mediated by the NMDA Receptor

Abstract

Purpose: : Stimulation of the P2X₇ receptor for ATP kills retinal ganglion cells. The mechanism linking the P2X₇ receptor to neuronal death is not known, although this death is not due the pore formation found in peripheral cells. Here we ask whether activation of the P2X₇ receptor leads to secondary activation of the NMDA receptor that culminates in excitotoxic death of rat retinal ganglion cells.

Methods: : Cell viability was assessed by determining the percentage of labeled rat pup RGCs cells present after 24 hrs culture with drugs. Intracellular calcium measurements were made from unlabeled rat neonatal RGCs cultured for 24 hrs and loaded with fura–2. Glutamate measurements were derrived from fluorescence of NADH after conversion with glutamate dehydrogenase.

Results: : The large elevations in ganglion cell Ca²⁺ levels triggered by the P2X₇ agonist BzATP were blocked by NMDA–receptor antagonists MK–801, AP5 and memantine. These antagonists reduced the response in both isolated retinal ganglion cells and those present in mixed retinal cell populations, indicating the response did not require astrocytes or additional neurons. The cell death caused by BzATP was prevented by all three NMDA antagonists, confirming a necessary role for the NMDA receptor in this death. BzATP led to large, reversible and repeatable releases of glutamate from isolated ganglion cells, implying NMDA receptors were activated by glutamate released by P2X₇ receptor activation. The co–localization of both P2X₇ and NMDA receptors on >65% of ganglion cells supports the presence of a lethal autocrine loop in a subpopulation of cells.

Conclusions: : Stimulation of the P2X₇ receptor on retinal ganglion cells triggers a release of glutamate that acts on NMDA receptors and leads to death. ATP is thus a potential upstream trigger for glutamate release in the retina.

Keywords: retina: neurochemistry • ganglion cells • excitatory neurotransmitters