Influence of Hydroxyl Radicals on the Ca2+–Metabolism of the Retinal Pigment Epithelium

Abstract

 

Abstract:

 

An increased oxidative exposure of the retinal pigment epithelium (RPE) is hypothesized to play an important role in the genesis of age–related macular degeneration (AMD). Oxidative cell damage might be mediated by a disturbance of the Ca²⁺–homeostasis, subsequently leading to functional changes and apoptosis. In the present study, intracellular Ca²⁺–concentration is studied before and after exposing cultured RPE cells to oxidative stress.

 

All experiments were performed using the human RPE cell line ARPE–19. The intracellular Ca²⁺–content was determined by implementing the Fura–2 method. The ratio between the fluorescence intensities at 340 and 380nm is correlated to the Ca²⁺–concentration. The cells were incubated for 4 minutes with hydroxyl radicals that were created via the Fenton reaction using H₂O₂ and Fe³⁺ at different concentrations. Intracellular Ca²⁺–level was continuously tracked for up to 60 minutes after radical exposure.

 

Immediately after the onset of radical exposure the ARPE–19 cells showed a steep transient Ca²⁺–increase. The amplitude of this calcium–increase is dependent of the concentration of radicals used (table 1).

 

 

^{Afterwards the Ca2+–concentration decreased to a value below the original baseline but eventually started to rise again. In some cases subsequent cell death could be observed.}

 

Oxidative exposure of cultured RPE cells leads to an intracellular Ca²⁺ overload. Changes in intracellular Ca²⁺–levels could lead to various cellular dysfunctions. This includes altered phagocytosis activity as well as changes in gene expression pattern and apoptosis, which should be investigated in future studies.

 

 

Keywords: age-related macular degeneration • oxidation/oxidative or free radical damage • cell death/apoptosis