Purpose:
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 (ARMD). Oxidative cell damage might be mediated by a disturbance of the Ca2+-homeostasis, which leads to functional changes and apoptosis. In the present study, the reverse mode of the NCX1 is studies as a potential Ca2+ entry route after hydroxyl radical (OH- ) stimulation.
Methods:
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 cells were exposed for 5 minutes with OH- twice with an interval of 1h between. OH- radicals were created via the Fenton reaction using H2O2 and Fe3+. One of both OH- exposures was done in presence and one in absence of KB-R7943, which blocks the reverse mode of the NCX1, or nifedipine, a L-type Ca2+ channel blocker. For control purposes the cells were exposed twice to OH- without adding any pharmacological compound (sham control).
Results:
: Immediately after the onset of radical exposure the ARPE-19 cells showed a steep transient Ca2+-peak. The amplitude of this Ca2+-increase is significantly reduced in the presence of KB-R7943, while in the nifedipine and the sham control group the second Ca2+-peak amplitude is not reduced but even slightly increased (for quantitative results please refer to the table).
Conclusions:
Oxidative exposure of cultured RPE cells leads to a transient increased intracellular Ca2+ load. This increase is mediated by the reverse mode of the NCX1. This might be a potential target for future therapy since oxidative stress is thought to be one of the major risk factors for ARMD and disturbed Ca2+ handling might lead to various cellular dysfunctions. The latter may include altered phagocytosis activity as well as changes in gene expression pattern and apoptosis.
Keywords: calcium • retinal pigment epithelium • oxidation/oxidative or free radical damage