Intercellular Ca(2+) wave propagation in human retinal pigment epithelium cells induced by mechanical stimulation.
Exp Eye Res
; 108: 129-39, 2013 Mar.
Article
em En
| MEDLINE
| ID: mdl-23352832
ABSTRACT
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Sinalização do Cálcio
/
Mecanotransdução Celular
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Células Epiteliais
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Epitélio Pigmentado da Retina
Limite:
Humans
Idioma:
En
Revista:
Exp Eye Res
Ano de publicação:
2013
Tipo de documento:
Article
País de afiliação:
Finlândia
País de publicação:
ENGLAND
/
ESCOCIA
/
GB
/
GREAT BRITAIN
/
INGLATERRA
/
REINO UNIDO
/
SCOTLAND
/
UK
/
UNITED KINGDOM