gms | German Medical Science

An increased oxidative exposure of the retinal pigment epithelium (RPE) plays an important role in the genesis of age-related macular degeneration (AMD). A major damaging factor could be the disturbance of the Ca²⁺-homeostasis, subsequently leading to functional changes and apoptosis. This work uses cultured RPE-cells to investigate the effect of Hydroxyl radicals on the Ca²⁺-homeostasis.

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 of 340 and 380nm is correlated to the Ca²⁺-concentration. The cells were incubated for 2 minutes with Hydroxyl radicals that were created via the Fenton reaction using H₂O₂ and Fe³⁺ and continuously observed for 60 minutes. The survival rate of the RPE cells is determined by using the Life-Dead-Assay from MoBiTec (Göttingen, Germany).

Immediately after radical exposure the ARPE-19 cells showed a steep transient Ca²⁺-increase. Afterwards the Ca²⁺-concentration decreased but stabilized at a permanently elevated value well above the baseline. A progressive death rate of RPE cells could be detected with the Life-Dead-Assay between 1 to 6 hours after radical exposure. Addition of 10mM Ascorbic acid inhibited the increase of the radical induced intracellular Ca²⁺-concentration.

Oxidative exposure of cultured RPE cells leads to an intracellular Ca²⁺ overload. The delayed cell death after radical exposure is rather caused by a selective damage to the Ca²⁺-homeostasis then by some unspecified destruction of the cell membrane. Changes in intracellular Ca²⁺-levels could lead to cellular dysfunctions because Ca²⁺ is a strong apoptosis factor as well as an important second messenger.