ObjectiveComputational models of calcium (Ca2+) signaling have been constructed for several cell types. There are, however, no such models for retinal pigment epithelium (RPE). Our aim was to construct a Ca2+ signaling model for RPE based on our experimental data of mechanically induced Ca2+ wave in the in vitro model of RPE, the ARPE-19 monolayer.MethodsWe combined six essential Ca2+ signaling components into a model: stretch-sensitive Ca2+ channels (SSCCs), P2Y2 receptors, IP3 receptors, ryanodine receptors, Ca2+ pumps, and gap junctions. The cells in our epithelial model are connected to each other to enable transport of signaling molecules. Parameterization was done by tuning the above model components so that the simulated Ca2+ waves reproduced our control experimental data and data where gap junctions were blocked.ResultsOur model was able to explain Ca2+ signaling in ARPE-19 cells, and the basic mechanism was found to be as follows: 1) Cells near the stimulus site are likely to conduct Ca2+ through plasma membrane SSCCs and gap junctions conduct the Ca2+ and IP3 between cells further away. 2) Most likely the stimulated cell secretes ligand to the extracellular space where the ligand diffusion mediates the Ca2+ signal so that the ligand concentration decreases with distance. 3) The phosphorylation of the IP3 receptor defines the cell’s sensitivity to the extracellular ligand attenuating the Ca2+ signal in the distance.ConclusionsThe developed model was able to simulate an array of experimental data including drug effects. Furthermore, our simulations predict that suramin may interfere ligand binding on P2Y2 receptors or accelerate P2Y2 receptor phosphorylation, which may partially be the reason for Ca2+ wave attenuation by suramin. Being the first RPE Ca2+ signaling model created based on experimental data on ARPE-19 cell line, the model offers a platform for further modeling of native RPE functions.

PurposeThe aim of this study was to investigate changes in spontaneous Ca2+ activity and mechanically induced intercellular Ca2+ communication in human embryonic stem cells‐derived retinal pigment epithelium (hESC‐RPE) during maturation.MethodsIn this study, we assessed Ca2+ activity in hESC‐RPE cells cultured for 9 or 28 days. Ca2+ imaging was done using a Ca2+‐sensitive fluorescence dye fluo‐4‐AM. Spontaneous Ca2+ activity and mechanically induced intercellular Ca2+ communication were recorded in control conditions, in the absence of extracellular Ca2+, after depletion of intracellular Ca2+ stores with thapsigargin, in presence of a gap junction blocker α‐glycyrrhetinic acid and a P2‐receptor blocker suramin.Results9 days cells exhibited twice lower spontaneous Ca2+ activity and 4‐fold higher mechanically induced intercellular Ca2+ communication compared to 28 days cells. Absence of extracellular Ca2+ reduced spontaneous Ca2+ activity in 9 days cells and almost completely inhibited it in 28 days cells, while having no effect on mechanically induced intercellular Ca2+ communication. Depletion of intracellular Ca2+ stores abolished spontaneous Ca2+ activity in 9 days and 28 days cells, as well as mechanically induced intercellular Ca2+ communication in 28 days cells, while not affecting the latter in 9 days cells. Blockade of gap junctions and P2‐receptors had no effect on spontaneous Ca2+ activity or mechanically induced intercellular Ca2+ communication in cells from both time points.ConclusionsOur results show that hESC‐RPE cells undergo significant Ca2+ signaling re‐arrangements during maturation: the cells increase spontaneous Ca2+ activity, while decreasing their intercellular Ca2+ communication.

Purpose Ca2+ signaling is relevant to most biological functions. In retinal pigment epithelium (RPE) a significant Ca2+ wave is produced by mechanical stimulation. To understand this process in detail, we modeled Ca2+ wave propagation in ARPE‐19 cells. Methods Mechanically induced Ca2+ wave was recorded from ARPE‐19 cells by Ca2+ imaging. Based on the measurements, a mathematical model was constructed. The model assessed Ca2+ wave propagation by assuming that cells were experiencing different conditions depending on their location with respect to the stimulation site. Results The model describes Ca2+ metabolism after stimulation as follows: 1) Cells near the stimulus site are likely to conduct Ca2+ through plasma membrane stretch‐sensitive Ca2+ channels and gap junctions. 2) The extracellular ligand and inositol 1,4,5‐trisphosphate (IP3) diffusion through gap junctions mediate the signal in all locations of the monolayer, ligand concentration decreasing with distance. 3) The kinase activity targeted to IP3 receptor defines the sensitivity of the cell to the ligand. The model predicts suramin drug effects on P2Y2 receptors suggesting that suramin accelerates the phosphorylation rate of the receptors by enhancing their desensitization. Conclusion Our model is the first mathematical model of Ca2+ signaling in ARPE‐19 cells. The model enables the analysis of the Ca2+ signal propagation mechanisms, and predicts new pathways of suramin drug effects.

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