title: Calcium overload is associated with lipofuscin formation in human retinal pigment epithelial cells fed with photoreceptor outer segments.
abstract: OBJECTIVE
  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 × 10⁷per 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 ± 63.86 U (P<0.01) at 12 h, and then decreased but still maintained a high level of 316.90 ± 36.07 U (P<0.01) for 4 days. Flunarizine and centrophenoxine significantly decreased the Ca²(+) overload to 227.18 ± 14.00 U at 12 h and 211.06 ± 20.45 U 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.
authors.affiliation: Department of Ophthalmology, Eye Institute of Chinese PLA, Xijing Hospital, The Fourth Military University, Xian, Shaanxi, China.
authors.forename: L
authors.initials: L
authors.lastname: Zhang
authors.forename: Y-N
authors.initials: YN
authors.lastname: Hui
authors.forename: Y-S
authors.initials: YS
authors.lastname: Wang
authors.forename: J-X
authors.initials: JX
authors.lastname: Ma
authors.forename: J-B
authors.initials: JB
authors.lastname: Wang
authors.forename: L-N
authors.initials: LN
authors.lastname: Ma
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002478:Cells, Cultured; D005021:Ethylamines / Q000494:pharmacology; D005434:Flow Cytometry; D005444:Flunarizine / Q000494:pharmacology; D005453:Fluorescence; D006801:Humans; D008062:Lipofuscin / Q000096:biosynthesis*; D010642:Phenoxyacetates / Q000494:pharmacology; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000502:physiology*
chemical_list: D002121:Calcium Channel Blockers; D005021:Ethylamines; D008062:Lipofuscin; D010642:Phenoxyacetates; C541393:meclophenoxate; D005444:Flunarizine; D002118:Calcium