کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
4011324 1602614 2013 11 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Intercellular Ca2+ wave propagation in human retinal pigment epithelium cells induced by mechanical stimulation
موضوعات مرتبط
علوم زیستی و بیوفناوری ایمنی شناسی و میکروب شناسی ایمونولوژی و میکروب شناسی (عمومی)
پیش نمایش صفحه اول مقاله
Intercellular Ca2+ wave propagation in human retinal pigment epithelium cells induced by mechanical stimulation
چکیده انگلیسی

Ca2+ signaling is vitally important in cellular physiological processes and various drugs also affect Ca2+ signaling. Thus, knowledge of Ca2+ 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 Ca2+ communication induced by mechanical stimulation in ARPE-19 cells. An intercellular Ca2+ wave was induced in ARPE-19 monolayer by point mechanical stimulation of a single cell. Dynamic changes of intracellular Ca2+ concentration ([Ca2+]i) in the monolayer were tracked with fluorescence microscopy imaging using Ca2+-sensitive fluorescent dye fura-2 in presence and absence of extracellular Ca2+, after depletion of intracellular Ca2+ stores with thapsigargin, and after application of gap junction blocker α-glycyrrhetinic acid and P2-receptor blocker suramin. Normalized fluorescence values, reflecting amplitude of [Ca2+]i increase, and percentage of responsive cells were calculated to quantitatively characterize Ca2+ wave propagation. Mechanical stimulation of a single cell within a confluent monolayer of ARPE-19 cells initiated an increase in [Ca2+]i, which propagated to neighboring cells in a wave-like manner. Ca2+ wave propagated to up to 14 cell tiers in control conditions. The absence of extracellular Ca2+ reduced [Ca2+]i increase in the cells close to the site of mechanical stimulation, whereas the depletion of intracellular Ca2+ stores with thapsigargin blocked the wave spreading to distant cells. The gap junction blocker α-glycyrrhetinic acid reduced [Ca2+]i increase in the cell tiers close to the site of mechanical stimulation, indicating involvement of gap junctions in Ca2+ wave propagation. The P2-receptor blocker suramin reduced the percentage of responsive cells participating in Ca2+ wave spreading beyond the fourth cell tier, showing the necessity of P2-receptors for Ca2+ wave propagation. In disconnected, i.e., subconfluent, ARPE-19 cell clusters Ca2+ 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 Ca2+ dynamics after mechanical stimulus. The ARPE-19 cells exhibited two different mechanisms of Ca2+ wave propagation dependent on the cell location: in the cells close to the site of mechanical stimulation the Ca2+ wave propagated mainly through gap junctions and required Ca2+ from both intracellular Ca2+ stores and extracellular media, while farther away the propagation was more dependent on the purinergic receptors and did not require extracellular Ca2+. 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.


► Single cell mechanical stimulation induced a repeatable Ca2+ wave in ARPE-19 cells.
► Two different propagation pathways of Ca2+ waves were identified.
► The Ca2+ wave spread mainly via gap junctions in cells close to the stimulation site.
► In distant cells the Ca2+ wave propagation was dependent on purinergic receptors.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Experimental Eye Research - Volume 108, March 2013, Pages 129–139
نویسندگان
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