Intercellular Ca2+ Wave Propagation In Human Retinal Pigment Epithelium Cells Induced By Mechanical Stimulation

Abstract

Purpose: : Ca²⁺ signaling plays an important role in cellular physiological processes, and various drugs have an influence on Ca²⁺ signaling, thus the information on Ca²⁺ dynamics is important in development of drug-testing assays. ARPE-19 cells are widely used for modeling human retinal pigment epithelium (RPE) functions and drug-testing, however, the Ca²⁺ wave propagation has not been assessed from these cells. In this study we investigated intercellular Ca²⁺ communications induced by mechanical stimulation.

Methods: : 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 using Ca²⁺-sensitive fluorescent dye fura-2 in presence and absence of extracellular Ca²⁺,after depletion of intracellular Ca²⁺ stores, and after application of gap junction (GJ) blockers quinidine and α-glycyrrhetinic acid. Intracellular Ca²⁺ stores were depleted with thapsigargin. Fluorescence images were then used to analyze the dynamics of Ca²⁺ wave propagation. Immunofluorescence staining was used to show the presence of GJ.

Results: : Mechanical stimulation 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 amplitude of [Ca²⁺]_i rise was attenuated with the increase of the distance from mechanically stimulated cell. The absence of extracellular Ca²⁺ had no influence on Ca²⁺ wave propagation, but the depletion of intracellular Ca²⁺ stores with thapsigargin blocked Ca²⁺ wave propagation. GJ blockers, quinidine and α-glycyrrhetinic acid, inhibited Ca²⁺ wave propagation, former being more efficient than latter. The propagation of Ca²⁺ wave after mechanical stimulation in disconnected, i.e. unconfluent, ARPE-19 cell clusters was significantly less efficient comparing to the wave propagation in confluent ARPE-19 monolayer at the same distances.

Conclusions: : Mechanical stimulation induces Ca²⁺ wave in human ARPE-19 cells, which occurs due to the release of Ca²⁺ from intracellular Ca²⁺ stores. Extracellular Ca²⁺ is not required for Ca²⁺ wave propagation. Signaling molecules diffuse both through GJ and extracellular medium with GJ being more prominent pathway.

Keywords: calcium • retinal pigment epithelium