Stimulation of P2X7 Receptors in a Pulsatile Manner Causes Lethal Ca2+ Overload in Human Retinal Neurons

Abstract

Purpose: : Stimulation of the ionotropic purinoceptor, P2X₇, has been implicated in apoptotic cell death and neurotoxicity. In this study, Ca²⁺ imaging was used to investigate the effect of the P2X₇ agonist, BzATP, on cultured human retinal neurons.

Methods: : Human cell cultures were obtained from donor retinal tissue. Ca²⁺ imaging on neurons was carried out using ratiometric fluorescent dye techniques. The cells were perfused in an examination bath and changes in cytosolic Ca²⁺ concentrations were monitored using real time epifluorescent techniques.

Results: : A 30 second application of BzATP (50µM) led to a transient increase in cytosolic Ca²⁺. When BzATP (50µM) was applied in a pulsatile manner (5 x repeat cycles of: 30 seconds on / 30 seconds off), the cytosolic Ca²⁺ increased and remained elevated throughout the exposure period with spikes occurring during the pulses. Ca²⁺ then returned to baseline levels. Subsequently, 66.7% of the neurons had a sustained increase in baseline Ca²⁺ (n = 5 experiments from 3 different donors). This increase was associated with morphological changes consistent with cell death. From a total of 60 cells monitored, 40 cells showed Ca²⁺ overload. When pulsed BzATP (50µM) was applied in the presence of the P2X₇ antagonist, PPADS (100µM), 92.3% of the neurons survived with no appreciable increase in the baseline Ca²⁺ concentration after 90 minutes (n = 5 experiments from 2 different donors). From a total of 39 cells monitored, 36 cells survived.

Conclusions: : Pulsed BzATP can lead to a lethal Ca²⁺ overload in cultured human retinal neurons. This neurotoxic effect is decreased by a P2X₇ antagonist. Neuroprotection strategies in human retinal cultures may have direct implications in glaucoma.

Keywords: neuroprotection • apoptosis/cell death • neurotransmitters/neurotransmitter systems