Apoptotic Death of Retinal Ganglion Cells Following P2X7 Receptor Stimulation Involves Activation of L–type Ca2+ Channels and Not Pore Formation

Abstract

Abstract: : Purpose:Previous work from our laboratory has indicated that the non–specific P2 receptor agonist BzATP can elevate the intracellular Ca²⁺ of retinal ganglion cells and kill them. This study determines the specific receptor responsible for these responses and examines the mechanisms leading to cell death. Methods: Ca²⁺ levels inside dissociated neonatal ganglion cells were monitored with fura–2. Cell viability was determined by injecting aminostilbamidine into the superior colliculus of neonatal rats, culturing dissociated retinal cells for 24 hrs in the presence and absence of drugs and counting the number of fluorescently labeled ganglion cells remaining. Yo–Pro–1 uptake was assessed in labeled ganglion cells using fluorescent microscopy. Apoptosis in labeled ganglion cells purified with the panning method was detected with the CaspACE FITC–VAD–FMK in situ marker after 24 hrs in BzATP. Results: Brief applications of 50 µM BzATP to retinal ganglion cells led to reproducible elevations in Ca²⁺. Removal of extracellular Ca²⁺ prevented this response. Brilliant Blue G (BBG) inhibited the Ca²⁺ response to BzATP at 0.1, 1 and 10 µM, consistent only with activity of P2X₇ receptors. BzATP reduced the number of viable ganglion cells, and led to activation of caspase, suggesting cell loss involved apoptosis. Cell death was attenuated by 0.1, 1 and 10 µM BBG. However, 30 µM suramin did not block the effects of BzATP, providing further confirmation that only P2X₇ receptors were responsible for cell death. Exposure to BzATP for up to 30 min did not enhance permeability of ganglion cells to Yo–Pro–1, suggesting the formation of large pores detected by others in retinal microglial cells was not occurring. However, cell survival was considerably enhanced by co–incubation with the L–type Ca²⁺ channel blocker nifedipine. Conclusions: Pharmacologic and physiologic analysis confirms that BzATP acts at P2X₇ receptors to raise Ca²⁺ and kill retinal ganglion cells. Cell death following receptor stimulation does not involve pore formation, but may instead involve a downstream activation of L–type Ca²⁺ channels.

Keywords: cell death/apoptosis • excitatory neurotransmitters • ganglion cells