[16] Methods for isolating and fractionating pigment epithelial cells

Feeney-Burns, L; Berman, E.R.

doi:10.1016/s0076-6879(82)81018-5

Cited by 19 publications

(7 citation statements)

References 21 publications

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“…Culturing choroid and sclera together with tritiated retinol for 2 hr and then isolating a nucleus-enriched fraction (Feeney-Burns and Berman, 1982) of the sclera, we found that this fraction was highly enriched in all-trans-retinoic acid. Although the scleral punch represents less than 1 % of the total volume in culture (scleral volume was 3n8 µl of a total volume of 500 µl), it accumulated more than 50 % of the all-trans-retinoic acid released by the choroidal tissue into the medium (290p50 femtomoles ; Table I and Fig.…”

Section: Retinoic Acid Alters Scleral Proteoglycan Formationmentioning

confidence: 89%

Choroidal Retinoic Acid Synthesis: A Possible Mediator between Refractive Error and Compensatory Eye Growth

Mertz

Wallman

2000

Experimental Eye Research

135

122

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Section: Retinoic Acid Alters Scleral Proteoglycan Formationmentioning

confidence: 89%

Choroidal Retinoic Acid Synthesis: A Possible Mediator between Refractive Error and Compensatory Eye Growth

Mertz

Wallman

2000

Experimental Eye Research

135

122

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“…Only undamaged eyes were used. Retinas and RPE were prepared as described [Feeney-Burns and Berman, 1982;Lolley, 1982]. Briefly, eyes were opened with a pair of scissors, the retina was removed with a flat spatula and the eyecup was rinsed with 3 ml of ice-cold RPE buffer (20 mM Tris/HCl, pH 7.5, 400 ml saccharose, 2 mM EDTA, 1 mM DTT, 1 mM benzidine, and 0.1 mM phenylmethylsulfonylfluoride).…”

Section: Tissue Preparationmentioning

confidence: 99%

Adenylyl Cyclase Type 7 Is the Predominant Isoform in the Bovine Retinal Pigment Epithelium

Beitz

Völkel²,

Guo³

et al. 1998

Cells Tissues Organs

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The retinal pigment epithelium (RPE) fulfills important supporting tasks to maintain the visual functions of the sensorineural retina. One major signalling mechanism by which adjacent tissues impinge on the RPE is the adenylyl cyclase (AC)/cAMP pathway. In the RPE, cAMP seems to modulate unique functions such as the phagocytosis of discs shed from the rod outer segments, transport of vitamin A or the ion and fluid control in the subretinal space. We analyzed the AC expression pattern in the retina and the RPE and found AC type 7 to be almost the only isoform expressed in the RPE. We cloned AC type 7 from a cDNA library established with fresh bovine RPE, expressed this isoform in eukaryotic cells and characterized some of its properties.

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“…Dissection was made under dim red light according to a modification of the procedure described by Feeney-Burns and Berman (27). Briefly, 64 eyes were cut along the ora serrata.…”

Section: Isolation Of Ros By Homogenizing (H-ros)-darkmentioning

confidence: 99%

“…Preparation of RPE-RPE cells were purified according to a modification of the method described by Feeney-Burns and Berman (27). After the retinas were cut from the optic disc, eyecups were gently filled with buffer A containing 2 mM EGTA and incubated at room temperature for 15 min.…”

Section: Isolation Of Ros By Hand Shaking (Hs-ros)-dark-adaptedmentioning

confidence: 99%

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

Jacob

Weech

Salesse

1996

Journal of Biological Chemistry

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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...

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[16] Methods for isolating and fractionating pigment epithelial cells

Cited by 19 publications

References 21 publications

Choroidal Retinoic Acid Synthesis: A Possible Mediator between Refractive Error and Compensatory Eye Growth

Choroidal Retinoic Acid Synthesis: A Possible Mediator between Refractive Error and Compensatory Eye Growth

Adenylyl Cyclase Type 7 Is the Predominant Isoform in the Bovine Retinal Pigment Epithelium

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

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