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O. Strauss, S. Wimmers; Expression of Orai Genes and Icrac Activation in the Human RPE. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1833.
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© ARVO (1962-2015); The Authors (2016-present)
The retinal pigment epithelium (RPE) fulfills a large variety of tasks which 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.
RT-PCR with mRNA from freshly isolated human RPE cells and from the human RPE cell line ARPE-19 was used to identify expression of Ca2+-conducting ion channels. Membrane currents of APRE-19 cells were measured in the whole-cell configuration under K+-free conditions. Fura-2 Ca2+-imaging in ARPE-19 cells was used to record changes in intracellular free Ca2+.
RT-PCR from mRNA of freshly isolated RPE cells as well from the RPE cell line ARPE-19 revealed the expression of the Icrac channel proteins Orai 1, 2 and 3 as well as their stimulators stim-1 and stim-2. Using the classic maneuver to stimulate capacitative 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 capacitative Ca2+ entry. On the other hand application of a low concentration of 2-APB (2 µM) led to an enhancement of the Ca2+ increase induced by capacitative Ca2+ entry. Depletion of cytosolic Ca2+ stores by administering an extracellular divalent cation-free solution led to an increase in the whole-cell conductance monitored by voltage-ramps between -130 mV and +50 mV.
Human RPE cells express all known three members of the Orai gene family and the two members of the stim family. Depletion of cytosolic Ca2+ stores increased the membrane conductance in RPE cells leading to increases in intracellular free Ca2+ with characteristics of Icrac channels. With these data we show a new Ca2+ entry mechanism linked to the Ca2+/inositolphosphate second-messenger system in RPE cells which help to further understand regulatory pathways of agonists such acetylcholine, ATP or IGF-1.
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