Calcium overload is associated with lipofuscin formation in human retinal pigment epithelial cells fed with photoreceptor outer segments

Purpose To investigate the role of Ca2+ in lipofuscin formation in human retinal pigment epithelial (RPE) cells that phagocytize bovine photoreceptor outer segments (POSs). Methods Cultured human RPE cells fed with 2 x 10 7 per l bovine POS were treated with flunarizine, an antagonist of Ca2+ channel, or/and centrophenoxine, a lipofuscin scavenger. The Ca2+ changes and lipofuscin formation were measured with fluoresence dye Fluo-3/AM ester, laser scanning confocal microscopy (LSCM) and flow cytometry (FCM). The activity of RPE cells was measured by methyl thiazolyl tetrazolium (MTT) assay and argyrophilic nucleolar organizer regions (AgNORs) assay. Results The Ca2+ fluorescence intensity (CFI) of RPE cells fed with POS was significantly increased compared with the controls (165.36 +/- 29.92 U). It reached a peak with 777.33 +/- 63.86U (P < 0.01) at 12 h, and then decreased but still maintained a high level of 316.90 +/- 36.07U (P < 0.01) for 4 days. Flunarizine and centrophenoxine significantly decreased the Ca2+ overload to 227.18 +/- 14.00U at 12 h and 211.06 +/- 20.45U at 4 days. FCM confirmed these changes. The drugs also showed an inhibitory effect on the lipofuscin formation. The proliferation rate of the cells fed with POS increased significantly. Both drugs had inhibitory effects on the activity of the cultured cells. This tendency was confirmed by AgNORs assay. Conclusions The Ca2+ inflow initiated lipofuscin accumulation in RPE cells fed with POS. Flunarizine and centrophenoxine can decrease Ca2+ overload and lipofuscin formation in RPE cells, accompanied by maintaining cellular vitality. Eye (2011) 25, 519-527; doi:10.1038/eye.2011.7; published online 11 February 2011