title: Photosensitized oxidative stress to ARPE-19 cells decreases protein receptors that mediate photoreceptor outer segment phagocytosis.
abstract: OBJECTIVE
  To determine whether previously shown photodynamic (PD)-induced inhibition of specific photoreceptor outer segment (POS) phagocytosis by ARPE-19 cells is associated with reductions in receptor proteins mediating POS phagocytosis, and if PD treatment with merocyanine-540 (MC-540) produces additional effects leading to its inhibition of nonspecific phagocytosis.
  
  
  METHODS
  ARPE-19 cells preloaded with MC-540 or rose bengal (RB) were sublethally irradiated with green light. Phagocytosis of POS was measured by flow cytometry and POS receptor proteins (Mer tyrosine kinase receptor [MerTK] and integrin subunits αv and β5) and β-actin were quantified by Western blotting at 0.5 and 24 hours after irradiation, with comparison to samples from nonsensitized control cultures. The intact integrin heterodimer αvβ5 was quantified by immunoprecipitation followed by blotting. The distribution of N-cadherin, ZO-1, and F-actin was visualized by fluorescence microscopy.
  
  
  RESULTS
  Mild PD stress mediated by both photosensitizers that elicits no significant morphologic changes produces transient and recoverable reductions in MerTK. The individual αv and β5 integrin subunits are also reduced but only partially recover. However, there is sufficient recovery to support full recovery of the functional heterodimer. Light stress mediated by MC-540 also reduced levels of actin, which is known to participate in the internalization of particles regardless of type.
  
  
  CONCLUSIONS
  After PD treatment POS receptor protein abundance and phagocytosis show a coincident in time reduction then recovery suggesting that diminution in receptor proteins contributes to the phagocytic defect. The additional inhibition of nonspecific phagocytosis by MC-540-mediated stress may result from more widespread effects on cytosolic proteins. The data imply that phagocytosis receptors in RPE cells are sensitive to oxidative modification, raising the possibility that chronic oxidative stress in situ may reduce the efficiency of the RPE's role in photoreceptor turnover, thereby contributing to retinal degenerations.
authors: Department of Ophthalmology, Eye Institute, Medical College of Wisconsin, Milwaukee, Wisconsin 53226-4812, USA.
authors: Magdalena M
authors: MM
authors: Olchawa
authors: Anja M
authors: AM
authors: Herrnreiter
authors: Christine M B
authors: CM
authors: Skumatz
authors: Mariusz
authors: M
authors: Zareba
authors: Tadeusz J
authors: TJ
authors: Sarna
authors: Janice M
authors: JM
authors: Burke
mesh_terms: D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D005434:Flow Cytometry; D006801:Humans; D039564:Integrin alphaV / Q000378:metabolism; D039641:Integrin beta Chains / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects*; D010587:Phagocytosis / Q000187:drug effects*; D010778:Photochemotherapy / Q000009:adverse effects*; D017319:Photosensitizing Agents / Q000494:pharmacology*; D011744:Pyrimidinones / Q000494:pharmacology*; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D055214:Retinal Photoreceptor Cell Outer Segment / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism
chemical_list: D039564:Integrin alphaV; D039641:Integrin beta Chains; D017319:Photosensitizing Agents; D011744:Pyrimidinones; C084344:integrin beta5; C003954:merocyanine dye; D020794:Receptor Protein-Tyrosine Kinases