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C.A. Burnett, J. Sanderson, V. Tovell, D.C. Broadway; Stimulation of P2X7 Receptors in a Pulsatile Manner Causes Lethal Ca2+ Overload in Human Retinal Neurons . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4809.
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© ARVO (1962-2015); The Authors (2016-present)
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.
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.
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.
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.
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