The Effect Of Calcium Overload On The Lipofuscin Formation In Cultured Human Retinal Pigment Epithelial Cells

Background and purpose: The formation of lipofuscin (LPF) in human retinal pigment epithelial (RPE) cells results from RPE phagocytizing the membranous discs shed by photoreceptor outer segments (POS). The sediment of lipofuscin in RPE will change the structure and function of RPE cells and then lead to ocular region diseases, such as AMD and Stargard. Ca2+ is the second messenger of all vital movement in cells, but the inflow of too much Ca2+ may induce calcium overload which will cause the impairment and apoptosis of cells. Some researches indicated that calcium overload is the final pathway of cell death. Flunarizine (FNZ) is the fourth generation of antagonists of Ca2+ channel, which can stabilize the cellular membrane, reduce oxy-radical generation and protect the cells from too much Ca2+ influx. Centrophenoxine (CPH) is a stimulant of the central nervous system and has been used in clinic for many years. It can scavenge the lipofuscin (LPF) and oxy-radical in cells and then protect the cell membrane. The aim of this experiment was to investigate the role of calcium overload, the depressing effect and the impact of FNZ and CPH on cells'activity in the lipofuscin (LPF) formation in retinal pigment epithelial (RPE) cells co-cultured with bovine photoreceptor outer segments (POS).Methods: (1) The cultured human RPE cells were fed with 2×10¹⁰ /L bovine POS. During the formation of the lipofuscin, the change of Ca2+ at 12 h, 1, 2, 4 and 8 d. were analyzed with the fluoresence Ca2+ dye Fluo-3/AM, a laser scanning confocal microscope (LSCM) and flow cytometry(FCM). The activity of cells was measured with tetrazolium salt (MTT) assay and AgNORs for 12 h, 1, 2, 4 and 8 d.(2) The influence of varous concentrations of flunarizine (FNZ) on the change of Ca²⁺ was observed with fluoresence Ca2+ dye Fluo-3/AM, LSCM and FCM and the cells'activity was tested with MTT and AgNORs at 12 h, 1, 2, 4 and 8 d.(3) The influence of flunarizine (FNZ), centrophenoxine (CPH) and combination of them on the change of Ca2+ were tested with fluoresence Ca2+ dye Fluo-3/AM, LSCM and FCM and the cells'activity was measured with MTT and AgNORs at 12 h, 1, 2, 4 and 8 d, respectively.Results: (1) LSCM showed that calcium fluorescence intensity of RPE cells co-cultured with POS reached the peak with 777.33±63.86 U (vs, in control, 165.36±29.92 U; P<0.01) at 12 h and then decreased but maintained at a high level of 334.12±30.55 U for 8 d. The mean autofluoresence intensity of the lipofuscin in RPE cells was 350.24±26.49 U (P<0.01) for 8 d compared to 105.51±15.39 U in controls. FCM revealed that calcium fluorescence intensity of controlled RPE cells was 11.1±0.36 and arrived its maximum value of 69.3±0.75 (P<0.01) for 12 h after co-cultured with POS. Then it declined and maintained at the level of 42.3±0.44 (P<0.01). The autofluoresence intensity of the lipofuscin in RPE cells increased with the time and reached 23.5±0.36 on day 8, which was significantly different from the level of the control group of 3.6±0.24 (P<0.01). MTT assay showed that the proliferation rate of the cells fed with POS reached the peak with 84.4% for 1 d and then decreased. AgNORs test demonstrated that the number of AgNORs in RPE cells fed with POS was evidently larger than that of controls, which was maximized on day 2 with a value of 4.2 (P<0.01) and decreased to 3.6 on day 8.(2) LSCM and FCM showed that FNZ group had inhibition effect on the Ca²⁺ inflow of RPE cells co-cultured with POS at various time points (P<0.05). The autofluoresence intensities of the lipofuscin in RPE cells on the 4th and 8th day were significantly different from the controll group, but it was not follow the dose-dependent manner. The 25μmol/L of FNZ had the greatest effect. MTT assay and AgNORs test revealed that the varous concentrations of FNZ had inhibition effect on the activity of the co-cultured cells on day 1 and the larger the dosage, the greater the effect.(3) LSCM and FCM showed that the FNZ and the combination of FNZ and CPH could prevent the calcium overload to some extent (P<0.01) and the effect of group with FNZ and CPH was more evident. In addition, they also had some inhibition effect on the formation of lipofuscin. The autofluoresence intensity of group with FNZ and CPH was 125.01±18.79 U on day 8 (vs, 350.24±26.49 U of the controls; P<0.01). MTT assay and AgNORs test demonstrated that the varous concentration of FNZ had inhibition effect to some degree on the activity of the co-cultured cells during 1 d to 2 d. The combination of FNZ and CPH had the greatest effect and cells'vitality remained growth in the period of 4 d to 8 d. Conclusions: In the process of lipofuscin formation in RPE cells fed with POS, inflow of Ca²⁺ may play an important role in the generation and accumulation of LPF. FNZ and CPH could depress the calcium overload and the lipofuscin formation in RPE cells and then maintain a stable vitality of cell growth.The innovation of our study is that this experiment investigated the role of calcium overload, the depressing effect and the impact of FNZ and CPH on cells'activity in the LPF formation in RPE cells co-cultured with bovine POS. It has not been found in the literature up to our knowledge.