Intercellular Ca2+-transient propagation in normal and high glucose solutions in rat retinal epithelial (RPE-J) cells during mechanical stimulation

Abstract

We investigated the effect of high glucose and modulation of protein kinase C (PKC) on the intercellular propagation of Ca2+-waves in a rat retinal pigment epithelial cell line (RPE-J cells) in order to compare its properties with the properties previously investigated in primary LE-RPE cells. The intercellular propagation of the Ca2+-waves in RPE-J cells was analyzed by fluorescence imaging confocal microscopy and fluorescence recovery after photobleaching (FRAP). In control conditions the maximal normalized fluorescence in the mechanically stimulated (MS) cell and the propagation towards the neighboring RPE-J cells were similar to LE-RPE cells. As in LE-RPE cells, the propagation was reduced by the gap junction (GJ) blocker halothane, and FRAP experiments demonstrated the presence of functional GJ coupling. Similar to the effect in LE-RPE cells, the propagation of the Ca2+-transient was reduced by 25mM glucose. However, unlike LE-RPE cells, the neighboring RPE-J cells presented a Ca2+-rise of amplitude similar to that in normal glucose levels. PKC activation with 1μM PMA for 30min resulted in inhibition of the Ca2+-wave propagation, which could be overcome by PKC downregulation as in LE-RPE cells. Cells grown for 72h in a high glucose solution in which PKC activity was downregulated, did not develop the inhibitory effect on Ca2+-wave propagation that was induced by elevated glucose levels. However, the effects were not as pronounced as in LE-RPE cells. We concluded that despite marked similarities, the transduction and the modulation of intercellular propagation of the Ca2+-transients in RPE-J cells are not identical to the mechanisms in primary LE-RPE cells.