The inhibition of CA2+ influx induced by hypericin in cultured human retinal pigment epithelial cells analyzed by confocal imaging.
OBJECTIVE
Hypericin, a specific inhibitor of protein kinase C, has been reported to have potential as a therapeutic drug for proliferative vitreoretinopathy (PVR) in vitro and in vivo. In the present studies, we analyzed the dynamic changes in Ca2+ influx and free intracellular Ca2+ concentration ([Ca2+]i) of cultured human retinal pigment epithelial (RPE) cells after stimulation with hypericin in an attempt to elucidate its mechanism as a therapeutic drug for PVR.


METHODS
RPE cells were plated in a special plastic dish and then stimulated with 100 nM phorbol-12-myristate-13-acetate (PMA) and/or 6 hypericin concentrations (0.5, 1, 2, 3, 4 and 5 microM), after which Ca2+ influx and [Ca2+]i were determined using the fluorescence Ca2+ dye fluo-3 AM and laser scanning confocal microscopy.


RESULTS
The fluorescence in resting RPE cells was strong and distributed throughout the cells. The nucleus appeared more fluorescent than the cytoplasm. After stimulation with 0.5 microM hypericin, no obvious change of Ca2+ influx and [Ca2+]i was observed. In contrast, stimulation with higher concentrations of hypericin (1-5 microM) led to a rapid decrease in Ca2+ influx and [Ca2+]i, which was significantly different from those detected without hypericin (control experiments). In addition, no significant differences in [Ca2+]i were found between 1 and 5 microM hypericin used. Stimulation with hypericin, which was applied immediately after preincubation with PMA for 24 h did not further change Ca2+ influx and [Ca2+]i.


CONCLUSIONS
In RPE cells, high concentrations of hypericin (1-5 microM) significantly inhibit Ca2+ influx and induce a decrease in [Ca2+]i. Therefore, hypericin has potential as a therapeutic drug for PVR maybe through its inhibition of the Ca2+ influx pathway.
Gao
QianYing
Q
Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
Ge
Jian
J
D000814:Aniline Compounds / Q000378:metabolism; D000873:Anthracenes; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D018613:Microscopy, Confocal; D010569:Perylene / Q000031:analogs & derivatives* / Q000494:pharmacology; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014966:Xanthenes / Q000378:metabolism
D000814:Aniline Compounds; D000873:Anthracenes; D004791:Enzyme Inhibitors; D014966:Xanthenes; C059715:Fluo-3; D010569:Perylene; C004965:hypericin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium