Expression of Orai genes and ICRAC activation in the human retinal pigment epithelium

Background The retinal pigment epithelium (RPE) fulfills a large variety of tasks that are important for visual function. Many of these tasks, such as phagocytosis, growth factor secretion, or transepithelial ion transport, are regulated by increases in intracellular Ca2+ as second-messenger. Despite the multitude of Ca2+-dependently regulated functions, only few Ca2+ channels have been described so far in the RPE to couple Ca2+ conductance and Ca2+ signaling. Methods RT-PCR experiments with mRNA of freshly isolated RPE cells as well as from the RPE cell line ARPE-19 and measurements of the intracellular free Ca2+ concentration were performed. Results The RT-PCR experiments revealed the expression of the ICRAC channel proteins Orai 1, 2, and 3 and their stimulators Stim-1 and Stim-2. The classic maneuver to stimulate capacitive Ca2+ entry (depletion of Ca2+ stores by 1 ìM thapsigargin under extracellular Ca2+-free conditions and then re-adding extracellular Ca2+) led to an increase in intracellular free Ca2+, which could be blocked by application of a high concentration of 2-APB (75 ìM) either before or during induction of capacitive Ca2+ entry. On the other hand, application of a low concentration of 2-APB (2 ìM) led to enhancement of the Ca2+ increase induced by capacitive Ca2+ entry. Depletion of cytosolic Ca2+ stores by administration of an extracellular divalent cation-free solution led to an increase in the whole-cell conductance. Conclusions With these data we show a new Ca2+ entry pathway linked to the Ca2+/inositolphosphate secondmessenger system in RPE cells which help to further understand regulatory pathways of agonists. The expression of Orai channels enables the RPE cells to generate sustained or repetitive Ca2+ signals as they are known to be induced by different stimuli like ATP, bFGF, and the stimulation with photoreceptor outer segments.