Activation of the P2X7 Receptor on Retinal Ganglion Cells Delivers Glutamate and Relieves the Mg2+ Block to Activate the NMDA Receptor

Abstract

Purpose:: Stimulation of the P2X₇ receptor for ATP can kill retinal ganglion cells. Cell death is prevented by the NMDA antagonists NMDA, APV and memantine, implying involvement of the NMDA receptor. Stimulation of the P2X₇ receptor causes a release of glutamate from isolated ganglion cells, consistent with the downstream involvement of NMDA receptors. We hypothesize that activation of the P2X₇ receptor also depolarizes the ganglion cell, relieving the voltage-dependent block of the NMDA channel by Mg²⁺. This study investigated the relationship between the NMDA receptor, the P2X₇ receptor and Mg²⁺.

Methods:: Intracellular calcium measurements were made from unlabeled isolated immunopanned rat pup retinal ganglion cells cultured for 24 h and loaded with fura-2. Cell viability was assessed by determining the percentage of labeled ganglion cells present after 24 h culture with drugs. NMDA was always given with co-factor glycine.

Results:: In the absence of Mg²⁺, NMDA stimulated a Ca²⁺ elevation in 92% of retinal ganglion cells. Addition of physiologic levels of Mg²⁺ (0.5 mM) eliminated the Ca²⁺ elevation in response to NMDA. However, MK-801 substantially inhibited the Ca²⁺ transient evoked after stimulation of the P2X₇ receptor in the presence of Mg²⁺, suggesting the channel could open if the P2X₇ receptor was activated first. This was supported by the inability of NMDA alone to kill cells, while MK-801 stopped the cell death triggered by P2X₇ receptor agonist BzATP. The independence of the two receptors was determined pharmacologically, with Brilliant Blue G inhibiting the response to BzATP, but having no effect on the response to NMDA. The response to P2X₇ receptor activation was reduced by bafilomycin-A, consistent with a vesicular release of glutamate.

Conclusions:: This study suggests that stimulation of the P2X₇ receptor releases glutamate and depolarizes the membrane to relieve the Mg²⁺ block of the NMDA receptor. This one-two combination would make the P2X₇ receptor well positioned to activate the NMDA receptor under conditions of either potentiation or excitotoxicity.

Keywords: ganglion cells • excitatory neurotransmitters • neurotransmitters/neurotransmitter systems