Expression of Orai Genes and I(CRAC) 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 I(CRAC) 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 μM 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 μM) either before or during induction of capacitive Ca²(+) entry. On the other hand, application of a low concentration of 2-APB (2 μM) 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 second-messenger 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.