Intercellular Ca(2+) wave propagation in human retinal pigment epithelium cells induced by mechanical stimulation.
Ca(2+) signaling is vitally important in cellular physiological processes and various drugs also affect Ca(2+) signaling. Thus, knowledge of Ca(2+) dynamics is important toward understanding cell biology, as well as the development of drug-testing assays. ARPE-19 cells are widely used for modeling human retinal pigment epithelium functions and drug-testing, but intercellular communication has not been assessed in these cells. In this study, we investigated intercellular Ca(2+) communication induced by mechanical stimulation in ARPE-19 cells. An intercellular Ca(2+) wave was induced in ARPE-19 monolayer by point mechanical stimulation of a single cell. Dynamic changes of intracellular Ca(2+) concentration ([Ca(2+)](i)) in the monolayer were tracked with fluorescence microscopy imaging using Ca(2+)-sensitive fluorescent dye fura-2 in presence and absence of extracellular Ca(2+), after depletion of intracellular Ca(2+) stores with thapsigargin, and after application of gap junction blocker α-glycyrrhetinic acid and P2-receptor blocker suramin. Normalized fluorescence values, reflecting amplitude of [Ca(2+)](i) increase, and percentage of responsive cells were calculated to quantitatively characterize Ca(2+) wave propagation. Mechanical stimulation of a single cell within a confluent monolayer of ARPE-19 cells initiated an increase in [Ca(2+)](i), which propagated to neighboring cells in a wave-like manner. Ca(2+) wave propagated to up to 14 cell tiers in control conditions. The absence of extracellular Ca(2+) reduced [Ca(2+)](i) increase in the cells close to the site of mechanical stimulation, whereas the depletion of intracellular Ca(2+) stores with thapsigargin blocked the wave spreading to distant cells. The gap junction blocker α-glycyrrhetinic acid reduced [Ca(2+)](i) increase in the cell tiers close to the site of mechanical stimulation, indicating involvement of gap junctions in Ca(2+) wave propagation. The P2-receptor blocker suramin reduced the percentage of responsive cells participating in Ca(2+) wave spreading beyond the fourth cell tier, showing the necessity of P2-receptors for Ca(2+) wave propagation. In disconnected, i.e., subconfluent, ARPE-19 cell clusters Ca(2+) wave spreading was considerably less efficient compared to that in confluent ARPE-19 monolayer at the same distances. ARPE-19 cells showed repeatable and robust Ca(2+) dynamics after mechanical stimulus. The ARPE-19 cells exhibited two different mechanisms of Ca(2+) wave propagation dependent on the cell location: in the cells close to the site of mechanical stimulation the Ca(2+) wave propagated mainly through gap junctions and required Ca(2+) from both intracellular Ca(2+) stores and extracellular media, while farther away the propagation was more dependent on the purinergic receptors and did not require extracellular Ca(2+). The proposed method could provide a tool to assess the drug-induced changes in intercellular communication in in vitro assays in human retinal pigment epithelial cells.
Department of Biomedical Engineering, Tampere University of Technology, PL 692, 33101 Tampere, Finland. amna.abukhamidakh@tut.fi
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Abu Khamidakh
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Juuti-Uusitalo
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Larsson
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H
Skottman
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J
Hyttinen
D020013:Calcium Signaling / Q000187:drug effects; D002460:Cell Line; D048430:Cell Shape; D002470:Cell Survival; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D005456:Fluorescent Dyes; D016257:Fura-2; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism; D006034:Glycyrrhetinic Acid / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D040542:Mechanotransduction, Cellular / Q000187:drug effects; D008856:Microscopy, Fluorescence; D010812:Physical Stimulation; D058919:Purinergic P2 Receptor Antagonists / Q000494:pharmacology; D018048:Receptors, Purinergic P2 / Q000187:drug effects / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000187:drug effects / Q000378:metabolism*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D013498:Suramin / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D013498:Suramin; D019284:Thapsigargin; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D006034:Glycyrrhetinic Acid; D016257:Fura-2