Presence of a Light-independent Phospholipase A2 in Bovine Retina but Not in Rod Outer Segments

Abstract

262, 163-168). This led that author to conclude that ROS PLA 2 could be involved in the phototransduction process, and raised the possibility of receptor-mediated activation of PLA 2 via G-proteins in cell types other than rods. However, the biochemical characteristics and the role of this PLA 2 have not been fully elucidated. We have tried to reproduce some of the results previously reported in order to further characterize this enzyme. We have found that, in our hands, there is neither light-dependent nor GTP-dependent PLA 2 activity in intact purified ROS. We also failed to detect PLA 1 activity in those ROS preparations. Nevertheless, we detected significant amounts of PLA 2 activity in two subretinal fractions adjacent to ROS: RPE (enriched with retinal pigment epithelial cells) and P200 (presumably containing neuronal cells, Mü ller cells, and rod inner segments). The enzyme present both in RPE and P200 is light-and GTP-independent, Ca 2؉ -and Mg 2؉ -independent, and seems to be optimally active in the alkaline pH range. Our results suggest that there is, if any, vanishingly little PLA 2 or PLA 1 activity in intact purified ROS and that the activity levels previously reported in the literature could have been due to a contamination by either RPE or P200. This is supported by our observation that some contaminated ROS preparations were "PLA 2 active."In the mammalian eye, rod outer segments (ROS) 1 consist of a stack of 1000 -2000 disks which contain the visual pigment, rhodopsin. ROS are thus responsible for the phototransduction process. It has been clearly shown that following absorption of light, photoexcited rhodopsin binds to and activates transducin (T␣␤␥), which is a member of the heterotrimeric GTP-binding protein family. During the activation of transducin, the GDP molecule (normally associated with the inactive state of the protein) is exchanged for GTP. As a consequence, transducin dissociates into T␣ and T␤␥ subunits. T␣ is well known to activate a cGMP-phosphodiesterase whose activity eventually leads to hyperpolarization of the rod through closure of Na ϩ / Ca 2ϩ cGMP-dependent channels (see Refs. 1-4 for reviews). Jelsema reported in 1987 (5) that phospholipase A 2 (PLA 2 ) activity was present in "crude ROS" and "partially purified ROS," and that this activity was stimulated by light and a non-hydrolyzable analog of GTP, GTP␥S. Moreover, Jelsema and Axelrod (6) demonstrated that T␤␥ was responsible for the activation of this ROS PLA 2 . These results led them to conclude that ROS PLA 2 could be involved in the phototransduction process. However, conflicting results have been reported since that time. In fact, although Zimmerman and Keys (8) detected phospholipase A activity in their ROS preparations, the activity that they measured, either PLA 2 or PLA 1 , was neither light-dependent nor GTP-dependent. It was rather stimulated by ATP and coenzyme A (CoA). Moreover, the maximum activity that they observed was approximately 1 order of magnitude lower than that reported by Jelsema (5). In addi...

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

“…The present finding of genes encoding various sPLA 2 s in the retina strongly supports the previous hypotheses of PLA 2 as a significant player in retinal maintenance and during pathologic conditions (Bazan, 1976;Aveldano de Caldironi et al, 1981;Bazan et al, 1986;Birkle and Bazan, 1989;Jacob et al, 1996Jacob et al, , 1998Rego et al, 1996;Bonne et al, 1998;Giusto et al, 2000). In addition to the detection of genes encoding sPLA 2 s, we demonstrated the location of sPLA 2 -IB.…”

Identification of a Lipase-linked Cell Membrane Receptor for Pigment Epithelium-derived Factor

“…The distribution in inner segments of photoreceptors and ganglion cells of both PEDF-R-and Fl-PEDF-binding sites is in agreement with the PEDF action and ligand/receptor interactions in the retina (41). Our observations are also in agreement with those of Jacob et al (56). They detected significant amounts of PLA 2 activity against 2-arachidonyl phospholipids in two subretinal fractions adjacent to the rod outer segments, the RPE, and another one presumably containing neuronal cells, Muller cells, and rod inner segments but not in rod outer segments.…”

Bovine retinal pigment epithelium contains novel types of phospholipase A2

“…RPE was prepared from fresh bovine eyes as described previously [17] and was either used immediately or divided into aliquots and stored at k80 mC.…”

“…In the future, this classification will certainly be refined, since recent results from Thomson and Clark [14], Buhl et al [15] and Soubeyrand et al [16] suggest that other groups or subgroups could be needed to describe newly discovered and cloned PLA # . We have recently demonstrated [17] that PLA # activity is present in bovine retinal pigment epithelium (RPE), a subretinal fraction that is enriched with RPE cells. We have shown that PLA # activity present in RPE homogenate was active at alkaline (4-guanidino)butane ; GTP[S], guanosine 5h-[γthio]triphosphate ; lysoPC, lysophosphatidylcholine ; OG, n-octyl glucoside ; PAPE, 1-palmitoyl-2-arachidonoylphosphatidylethanolamine ; PE, phosphatidylethanolamine ; PLA 2 , phospholipase A 2 ; cPLA 2 , cytosolic PLA 2 ; iPLA 2 , Ca 2 + -independent PLA 2 ; sPLA 2 , secretory PLA 2 ; PLC, phospholipase C ; 10PyPM, 1-O-hexadecyl-2-[pyrene 10 ]decanoyl-sn-3-phosphomethanol ; 10Py-decanoic acid, [Pyrene 10 ]decanoic acid ; RPE, retinal pigment epithelium ; bf-TLC, ' back-and-forth ' TLC.…”

“…pH [17]. In order to characterize, for the first time, the minimum number of PLA # enzymes present in RPE, as well as their properties, we tested the effects of H # SO % , guanosine 5h-[γthio]triphosphate (GTP[S]) and ATP on RPE homogenate.…”