Identification of intracellular phospholipases A2 in the human eye: involvement in phagocytosis of photoreceptor outer segments.
OBJECTIVE
To identify intracellular phospholipases A(2) (PLA(2)) in the human retina and to explore the role of these enzymes in human retinal pigment epithelium (RPE) phagocytosis of photoreceptor outer segments (POS).


METHODS
PCR amplification and Western blot analysis were used to identify mRNA and protein expression of intracellular PLA(2) subtypes in the retinal pigment epithelial cell line ARPE-19. Immunohistochemical staining of normal human eye sections was performed to reveal the cellular location of the enzymes. A model of RPE phagocytosis of POS was used to explore the role of intracellular PLA(2) in phagocytosis. An activity assay was used to evaluate PLA(2) activity, and inhibitors of specific PLA(2) were applied to evaluate the role of PLA(2) in RPE phagocytosis.


RESULTS
Genes encoding calcium-independent (i)PLA(2), group VIA; calcium-dependent cytosolic (c)PLA(2), groups IVA, IVB, and IVC; and iPLA(2), group VIB, were identified in the human RPE cell line ARPE-19. Furthermore, protein of iPLA(2)-VIA, cPLA(2)-IVA, and iPLA(2)-VIB were identified in ARPE-19 cells and in various parts of the normal human eye. iPLA(2)-VIA protein levels were upregulated during phagocytosis, and iPLA(2)-VIA activity was found to be specifically increased 12 hours after ARPE-19 cells were fed with POS. Finally, RPE phagocytosis was inhibited by the iPLA(2)-VIA inhibitor bromoenol lactone.


CONCLUSIONS
Various intracellular PLA(2) subtypes are present in the human retina. iPLA(2)-VIA may play an important role in the regulation of RPE phagocytosis of POS and may also be involved in the regulation of photoreceptor cell renewal.
Kolko
Miriam
M
Eye Pathology Institute, University of Copenhagen, Copenhagen, Denmark. mkolko@dadlnet.dk
Wang
Jinmei
J
Zhan
Chen
C
Poulsen
Kristian A
KA
Prause
Jan Ulrik
JU
Nissen
Mogens Holst
MH
Heegaard
Steffen
S
Bazan
Nicolas G
NG
D015153:Blotting, Western; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005123:Eye / Q000378:metabolism*; D006801:Humans; D007124:Immunoenzyme Techniques; D018613:Microscopy, Confocal; D010587:Phagocytosis / Q000502:physiology*; D010741:Phospholipases A / Q000235:genetics / Q000378:metabolism*; D010857:Pigment Epithelium of Eye / Q000502:physiology*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000502:physiology*; D014018:Tissue Distribution
D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D010741:Phospholipases A