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Intra- and intercellular Ca²⁺ signaling in retinal pigment epithelial cells during mechanical stimulation

Laboratory of Physiology, KULeuven, B-3000 Leuven, Belgium

¹Correspondence: Bernard Himpens, Laboratory of Physiology, Herestraat 49, KULeuven, B-3000 Leuven, Belgium. E-mail: Bernard.Himpens{at}med.kuleuven.ac.be

The intercellular communication (IC) was investigated between cultured rat retinal pigment epithelial (RPE) cells isolated from Long-Evans (LE) or dystrophic Royal College of Surgeons (RCS) rats and grown in solutions containing normal and high glucose concentrations, or after modulation of protein kinase C (PKC). This was performed by studying the conduction of the free Ca²⁺-concentration ([Ca2+]_i) wave elicited by mechanical stimulation and by analyzing the fluorescence recovery after photobleaching (FRAP). Mechanical stimulation of LE-RPE cells triggers Ca²⁺ influx, mediated by stretch-sensitive cation channels followed by intracellular Ca²⁺ release. A regenerative [Ca²⁺]_i wave was found with a lower propagation rate in RCS-RPE cells. This rate could be increased by PKC down-regulation. Mechanical stimulation caused a [Ca²⁺]_i increase in the mechanically stimulated (MS) cell followed after a delay by a [Ca²⁺]_i rise in the adjacent cell layers. The intercellular [Ca²⁺]_i wave propagation could be blocked by gap junction blockers such as halothane or PKC activation. An inhibition of the [Ca²⁺]_i-wave propagation similar to that induced by halothane could be observed in cells grown in solutions containing 14 mM or higher concentrations of glucose. PKC down-regulated cells grown in glucose-rich medium did not develop this inhibitory effect on gap junction communication (GJC). FRAP experiments confirmed that the observed changes were consistent with a PKC-mediated inhibitory effect of high glucose concentrations on GJC.

Key Words: RCS rats • gap junctions • intercellular communication • protein phosphorylation • calcium wave • confocal imaging • nucleus • cytoplasm • nucleo-cytoplasmic gradient • cytoskeleton

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