Bovine retinal pigment epithelium contains novel types of phospholipase A2

Abstract

We have recently demonstrated the presence of phospholipase A2 (PLA2) activity in cells from bovine retinal pigment epithelium (RPE) [Jacob et al. (1996) J. Biol. Chem. 271, 19209-19218]. We report here our results on the characterization of this RPE-PLA2 activity. We show that RPE probably contains two types of PLA2 enzyme, as indicated by the results obtained with different PLA2-active fractions eluted from cation-exchange columns and treated with Ca2+/EGTA, dithiothreitol, p-bromophenacyl bromide or heat. These results, in addition to those from PLA2 assays using different substrates, also suggest that RPE-PLA2 enzymes are different from the well-known secretory, cytoplasmic and Ca2+-independent forms. Sequential extraction of RPE with (1) isotonic, (2) hypertonic and (3) detergent-containing PBS argues for the presence of weakly membrane-associated enzymes. Control experiments using 'back and forth' TLC allowed us to discriminate between PLA2 and phospholipase C/diacylglycerol lipase activity and confirmed that, in our assay conditions, the release of fatty acids was indeed due to PLA2 enzymes. These results, together with those obtained by treating RPE homogenates with H2SO4, guanosine 5'-[gamma-thio]triphosphate, ATP and different protease inhibitors, permitted us to make the first characterization of these RPE-PLA2 enzymes. We conclude that RPE contains novel types of PLA2 that are different from the secretory, cytoplasmic and Ca2+-independent forms.

Identification of Intracellular Phospholipases A2in the Human Eye: Involvement in Phagocytosis of Photoreceptor Outer Segments

“…However, no specific PLA 2 has been identified in these tissues, and the current knowledge is based on indirect evidence of substrate-specific PLA 2 activity. 16,17,27 Based on the previous findings, the purpose of this study was to identify intracellular PLA 2 in the human eye, with emphasis on the outer retinal layers, and to evaluate the role of intracellular PLA 2 in RPE phagocytosis of POS. Intracellular PLA 2 expression was found in various parts of the eye, as outlined in Table 2.…”

“…9 There is only limited evidence of the involvement of PLA 2 in RPE phagocytosis. However, RPE has been shown to elicit PLA 2 activity, 16,17 and RPE phagocytosis of POS has been shown to induce prostaglandins by COX activation. 18 Preliminary findings have revealed the highest abundance of the high-molecular-weight PLA 2 in the human RPE cell line ARPE-19 compared with the low-molecular-weight sPLA 2 .…”

Human retinal pigment epithelium secretes a phospholipase A2 and contains two novel intracellular phospholipases A2

“…Mutations in genes like RPE65 that are specific to human RPE (hRPE) cells were shown to lead to hereditary retinal degeneration (Marlhens et al 1997(Marlhens et al , 1998. Two novel intracellular phospholipase A2 (PLA2) enzymes were recently characterized in bovine RPE and shown to be different from known PLA2s (Jacob et al 1997). These enzymes could thus be specific to RPE and, consequently, the characterization of PLA2 enzymes from hRPE cells is of particular importance, as they could be involved in different types of retinal degeneration.…”

“…The iPLA2 class is very heterogeneous and has been further divided into three groups, based on their biochemical characteristics (Ackermann and Dennis 1995). Interestingly, novel types of mammalian PLA2s have also been recently characterized, suggesting that the PLA2 classification will have to be refined and that new classes or groups of PLA2s might be needed (Buhl et al 1995;Jacob et al 1997;Soubeyrand et al 1997;Thomson and Clark 1995).…”

Expression and location of mRNAs encoding multiple forms of secretory phospholipase A2 in the rat retina

“…sPLA 2 activity has been described for the retina (Castagnet and Giusto, 1993;Hanasaki and Arita, 1999), and there seems to be enhanced sPLA 2 activity in retinal ischemia and light damage (Jung and Reme, 1994;Rego et al, 1996). Furthermore, different PLA 2 s are present in the retinal pigment epithelium (Jacob et al, 1997;Van Themsche et al, 2001). To date, no PLA 2 -specific receptors have been cloned or characterized in the retina.…”