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PubMed1996

CD36 participates in the phagocytosis of rod outer segments by retinal pigment epithelium.

Mechanisms of phagocytosis are complex and incompletely understood. The retinal pigment epithelium provides an ideal system to study the specific aspects of phagocytosis since an important function of this cell is the ingestion of packets of membranous discs that are normally discarded at the apical ends of rod and cone cells during outer segment renewal. Here we provide evidence that rod outer segment phagocytosis by retinal pigment epithelium is mediated by CD36, a transmembrane glycoprotein which has been previously characterized on hematopoietic cells as a receptor for apoptotic neutrophils and oxidized low density lipoprotein. Immunocytochemical staining with monoclonal and polyclonal antibodies demonstrated CD36 expression by both human and rat retinal pigment epithelium in transverse cryostat sections of normal retina and in primary cultured cells. By western blot analysis of retinal pigment epithelial cell lysates, polyclonal and monoclonal antibodies to CD36 recognized an 88 kDa protein which comigrated with platelet CD36. Furthermore, the synthesis of CD36 mRNA by retinal pigment epithelium was confirmed by reverse transcriptase-PCR using specific CD36 oligonucleotides. The addition of CD36 antibodies to cultured retinal pigment epithelial cells reduced the binding and internalization of 125I-labeled rod outer segments by 60%. Immunofluorescence confocal microscopy confirmed that outer segment uptake was significantly diminished by an antibody to CD36. Moreover, we found that transfection of a human melanoma cell line with CD36 cDNA enabled these cells to bind and internalize isolated photoreceptor outer segments as seen by double immunofluorescent staining for surface bound and total cell-associated rod outer segments, and by measurement of cell-associated 125I-labeled rod outer segments. We conclude that the multifunctional scavenger receptor CD36 participates in the clearance of photoreceptor outer segments by retinal pigment epithelium and thus, participates in the visual process.

Aged↗
PubMed1989

Immunocytochemical localization of opsin in degenerating photoreceptors of RCS rats and rd and rds mice.

Opsin is normally sequestered predominantly in the outer segment disc and plasma membranes of adult photoreceptors. Absence of opsin from the inner segment plasma membrane in normal photoreceptors is probably not due to the inability of the inner segment plasma membrane to retain opsin. Rather, in the adult mammalian retina, if opsin is inserted at sites in the apical inner segment plasma membrane, in a fashion comparable to the pathway in amphibians, it is rapidly transported predominantly to the outer segment by unknown mechanisms. Dystrophic rds retinas, lacking an outer segment, display newly synthesized opsin throughout the plasma membrane. If opsin is transported to the inner segment plasma membrane as a specific insertional site, diffusion in the plane of the membrane may redistribute opsin throughout the plasma membrane which encloses the nucleus and the synaptic terminal. Alternatively, opsin may be inserted randomly throughout the entire cell's plasmalemma beneath the cilium. Selective transport to the outer segment may preferentially clear the inner segment of most of its opsin and nearly clear the perikaryal and synaptic terminal's plasmalemma in normal cells. In dystrophic retinas, however, as outer segments degenerate or fail to form, opsin is detected readily in the remaining plasma membrane sites. In the rd mouse, some of the opsin molecules in the inner segment plasma membrane might be newly synthesized while others may arise from molecules which reached the inner segment by back-diffusion from the outer segment at least at early stages in the degeneration while outer segments survive. The opsin in the plasma membrane which envelopes the residual rod nuclei and synaptic terminals in dystrophic retinas may account for the persisting light perception in retinas which have lost both the rod outer and inner segments. Dystrophic retinas, such as the rd mouse and RCS rats and possibly human RP retinas, in which cone nuclei survive long after rods disappear, might retain light perception because of cone photo-pigments in the outer nuclear and outer plexiform layers. To explore these questions further, the localization of other components of the transduction cascade and the determination of the efficiency of their coupling in dystrophic cells is necessary. We need to know where the cyclic GMP-sensitive sodium channels lie in these dystrophic cells and the cellular requirements for proximity of these components to generate a signal. Outer segment-free photoreceptors, bearing opsin in their plasma membranes, resemble other cells which have receptor-mediated alterations in membrane permeability to ions.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Characterization of photoreceptor cell differentiation in the rat retinal cell culture.

Photoreceptor cell differentiation in the rat retina was studied in vivo and in vitro, using an immunohistochemical method to demonstrate opsin-like immunoreactivity. Cells in a dissociated monolayer culture expressed some properties characteristic of rat rod cells developing in vivo, including a ciliary structure and opsin-like immunoreactivity. Immunoblot analysis revealed that cultured retinal cells synthesize a polypeptide with the same molecular weight as that synthesized by the intact retina. Although the outer segment (OS) was not present in the culture, immunoreactive cells possessed a ciliary structure. Opsin-like immunoreactivity was found on the plasma membrane, including the cilia. The neuritic extensions were also intensely stained. In mature rod cells of the intact rat retina, opsin was detected only on the OS but, during development, it was found both in the somatic region of the rod cells and on the differentiating OS. During maturation of rod cells opsin immunoreactivity seemed to shift to the OS from other locations. However, some "displaced" photoreceptor cells, found in the inner nuclear layer and extending fibers bipolarly, retained immunoreactivity throughout their structure. The absence of polarized distribution of opsin in these cells is considered to be due to an abnormal environment, which may also be the case with cultured retinal cells. The present culture conditions will offer a useful model system to understand the cellular mechanism of the hereditary retinal dystrophy of rodent animals in which photoreceptor cells selectively degenerate.

Animals↗
PubMed1999

Influence of UVA light stress on photoreceptor cell metabolism: decreased rates of rhodopsin regeneration and opsin synthesis.

There is considerable evidence indicating that rhodopsin is the chromophore mediating light damage to the rat retina caused by exposure to mid-visible wavelengths. Retinal damage is, however, more effectively produced by short-wavelength light, and little is known about the initiating events for this damage class. The present study sought to determine the involvement of rhodopsin bleaching in short-wavelength damage by examining rhodopsin levels and opsin synthesis at early time points following acute ultraviolet-A (UVA) exposures of the pigmented rat eye. A gradual decline in rhodopsin to 8% of the level in non-exposed control eyes occurred over a 1 hr exposure to 1500 microW cm-2of UVA light. When animals were placed in darkness following this exposure, rhodopsin had recovered to only 27% of control levels by 2 hr post-exposure indicating a very slow rate of regeneration. For later time points, animals were returned to dim cyclic light and by 2 days following exposure, rhodopsin levels had risen to 57% of control. In contrast, opsin levels at this same time point were unaffected by UVA exposure. Other observations indicating the UVA exposure affected photoreceptor cell metabolism included a 27% decrease in the rate of opsin synthesis between 1 and 2 days following exposure, and a 69% reduction in the rate of rod outer segment disk renewal during the initial 3 days following exposure. These data show that UVA light stress in the retina causes a gradual bleaching of rhodopsin followed by a slow rate of recovery and altered photoreceptor cell metabolism. These results are consistent with the concept that rhodopsin mediates UVA-induced retinal damage and the possible mechanisms by which this might occur are discussed in relation to alternative hypotheses currently in the literature.

Animals↗
PubMed1983

Defective ingestion of rod outer segments by cultured dystrophic rat pigment epithelial cells.

A new procedure for assaying the phagocytosis of rod outer segments (ROS) by cultured rat pigment epithelial (PE) cells has been developed. Using an ROS antiserum and a double immunofluorescent labeling procedure, ROS attached to the external surfaces of these cells can be distinguished from those that have already been ingested. We have used this procedure to study the phagocytosis of ROS by PE cells isolated from normal rats and rats with inherited retinal dystrophy (RCS rats). With this approach we have been able to show that the attachment of ROS to the external surfaces of dystrophic PE cells does take place to a normal extent. However, only a small number of these bound ROS are subsequently ingested, demonstrating that the ingestion phase of phagocytosis is defective. After a 4-hr incubation during which ROS are continuously present, normal rat PE cells ingest about 80% of the ROS that have bound to the cell surfaces. In contrast, after this time period, less than 20% of the ROS bound to the dystrophic PE cells have been ingested. These results, as well as the results of pulse-chase experiments in which ROS are rinsed away after two hours and the incubation continued without further addition of ROS, have demonstrated that normal PE cells rapidly ingest most of the bound ROS, whereas the dystrophic PE cells show no such rapid ingestion. Both cell types, however, are able to slowly ingest additional bound ROS with time.

Animals↗
PubMed1992

Lipids of frog retinal pigment epithelium: comparison with rod outer segments, retina, plasma and red blood cells.

The glycerolipid and fatty acid compositions of frog retinal pigment epithelium (RPE) were determined and compared with rod outer segments (ROS), retina, plasma, and red blood cells (RBC). The glycerolipid class composition of RPE was similar to RBC and ROS or retina, with phosphatidylcholine and phosphatidylethanolamine being the major components. The fatty acid composition of RPE differed substantially from that of plasma or RBC; the former contained much higher levels of C-20 and C-22 polyunsaturated fatty acids (PUFAs), such as 20:4n-6 and 22:6n-3, but less C-18 mono-, dienoic, and trienoic acids. The difference between RPE and ROS or retina with respect to fatty acid profile was also dramatic; RPE had relatively less 22:6n-3, but more 20:4n-6 and 18:2n-6, than ROS or retina. These results suggest that frog RPE cells may selectively take up C-20 and C-22 PUFAs from the circulation, but preferentially deliver 22:6n-3 to the ROS and retina. Fatty acid analyses show that 20:4n-6 and 22:6n-3 were unevenly distributed among RPE glycerolipids; phosphatidic acid, diglyceride, triglyceride, and phosphatidylserine are relatively more enriched in 22:6n-3 compared with 20:4n-6. This information might imply that these two PUFAs are metabolized differently inside the frog RPE cells.

Animals↗
PubMed1987

Kinetic studies of rod outer segment binding and ingestion by cultured rat RPE cells.

Retinal pigment epithelial (RPE) cells selectively phagocytize rod outer segments (ROS) by a process which may be mediated by specific cell surface receptors. We have studied the kinetics of this process using rat RPE cells grown in tissue culture. By cooling RPE cells to 17 degrees C, the binding and ingestion phases of phagocytosis can be separated. Maximum ROS binding with minimum ingestion occurs at 17 degrees C; above 17 degrees C the rate of ingestion increases markedly. Thus it is possible to measure the kinetics of ROS binding to RPE cells at 17 degrees C and of ROS ingestion at 37 degrees C. At 17 degrees C, ROS binding is saturable, both with respect to time and to ROS concentration. ROS ingestion saturates after 4 hr of incubation at 37 degrees C, after which the cells are refractory to further ROS ingestion for 1-2 hr. During this recovery period, rapid digestion of the internalized ROS takes place. Cycloheximide, when present at a concentration (2 x 10(-5) M) which inhibits protein synthesis by 92%, has no effect on ROS phagocytosis or on the recovery of ROS ingestion at 37 degrees C. This suggests that if receptors mediate the ingestion of ROS by RPE cells, they are not degraded after the ROS are internalized. Dystrophic rat (RCS-p+) RPE cells exhibit normal binding, but very limited ingestion of ROS at 37 degrees C. The rate and amount of ROS binding to these cells at 37 degrees C is comparable with that occurring to normal cells at 17 degrees C. These observations support the hypothesis that there are a limited number of receptors which are specific for ROS binding on the surface of normal and dystrophic rat RPE cells.

Animals↗
PubMed1997

Vitronectin is responsible for serum-stimulated uptake of rod outer segments by cultured retinal pigment epithelial cells.

PURPOSE: To examine whether the vitronectin (VN) in serum is responsible for the serum stimulation of phagocytosis in the rod outer segment (ROS) by cultured retinal pigment epithelial (RPE) cells. METHODS: Vitronectin was removed from fetal bovine serum by heparin-agarose affinity chromatography. Concentrations in normal and depleted serum were determined by enzyme-linked immunosorbent assay, using a polyclonal antibody against bovine VN and commercially prepared human VN as a standard. A monoclonal antibody against human alpha v beta 5 was used in localization and in blocking experiments. Rod outer segment phagocytosis was measured using a flow cytometric assay. RESULTS: Affinity chromatography removed 95% of the VN from serum as determined by enzyme-linked immunosorbent assay. Vitronectin-depleted serum did not stimulate ROS phagocytosis by RPE cells. Commercially prepared VN added to serum-free medium stimulated ROS phagocytosis in a dose-dependent manner. Pretreatment of RPE cells with an antibody against alpha v beta 5, an integrin receptor for VN, had no effect on phagocytosis in the absence of serum but completely blocked the serum stimulation of ROS phagocytosis. Antibody against alpha v beta 5 demonstrated a variable labeling pattern on the cultured RPE cell surface with morphologically distinct cell clusters exhibiting less labeling. Those cell clusters exhibiting less receptor labeling also showed less uptake of fluorescent-labeled ROS. CONCLUSIONS: Vitronectin is the component responsible for serum stimulation of ROS uptake, and this uptake appears to be mediated by an alpha v beta 5 integrin. Although clearly important in vitro, a role for VN in ROS uptake by RPE cells in situ remains to be determined.

Animals↗
PubMed1992

The cGMP-gated channel of rod outer segments is not localized in bipolar cells of the mammalian retina.

It has recently been suggested (Nature, 346 (1990) 269-271) that ON-bipolar cells express the same biochemical cascade and guanosine 3',5'-cyclic monophosphate (cGMP)-gated cation channel as rod outer segments. An antibody directed against the cGMP-gated channel of bovine rod outer segments was applied to cryostat sections of rat and cat retinae. No immunocytochemical labelling was found in bipolar cells. Therefore, if those cells express a cGMP-gated channel, it must be immunologically different to the 63 kDa protein constituting the cGMP-gated channel of the outer segment.

Animals↗
PubMed1984

An ultrastructural study of retinal photoreceptor degeneration associated with bronchial carcinoma.

We studied both eyes of a 66-year-old man with retinal degeneration and oat cell carcinoma of the bronchus. Retinal degeneration was most marked peripheral to the parafovea where photoreceptor cells and their outer segments were absent. Within the parafovea, photoreceptor cells remained but rod outer segments were absent and cone outer segments were fragmented and disorganized. The retinal pigment epithelium contained many immature melanin granules within melanolysosomes, suggesting abnormal melanin synthesis and resorption. We suggest that a pharmacologically active substance resembling a hormone produced by the tumor increased melanin synthesis in the pigment epithelium and that the increased melanin content in these cells compromised their ability to phagocytose and maintain normal turnover of photoreceptor outer segments. We believe these changes led to photoreceptor outer segment loss and subsequent degeneration of the photoreceptor cells.

Aged↗
PubMed1998

Phagocytosis of rod outer segments by human iris pigment epithelial cells in vitro.

BACKGROUND: We set out to evaluate the growth potential of human iris pigment epithelial (hIPE) cells in vitro, to establish whether these cells acquire the ability to phagocytose rod outer segments (ROS) and to compare the phagocytic activity of hIPE to that of human retinal pigment epithelial (hRPE) cells. METHODS: hIPE and hRPE cells were isolated and cultured from human donor eyes and surgical specimens and growth characteristics were analyzed. HIPE and hRPE of an eye of a 46-year-old donor were used for the phagocytosis assay. Phagocytosis was evaluated by adding ROS isolated from porcine retina to cultures of hIPE and hRPE, which had been labeled with the pH-sensitive fluorescent dye, carboxy-SNAFL. After 4 h the number of ingested ROS was counted with a light microscope. For each cell type phagosomes in 500 cells were counted. The epithelial characteristics of the cells used in this study were evidenced by their morphology. RESULTS: Morphologically cultured hIPE are indistinguishable from the hRPE cultured from the same donor eye and show a similar pattern of cytokeratin distribution. Cultured hIPE acquire the ability to phagocytose ROS at a level slightly lower than hRPE; hIPE contained 0.76 phagosomes per cell, hRPE 0.99 phagosomes per cell. CONCLUSION: The morphology of hIPE in culture and the acquisition of the phagocytic phenotype indicate that these cells have the ability to differentiate into cells that have characteristics in common with hRPE. The acquisition of phagocytic activity suggests that it is feasible to culture hIPE from surgical iridectomies and that these cultured cells can be transplanted into the subretinal space in individuals with retinal degenerations.

Cells, Cultured↗
PubMed1996

Kinetics of rod outer segment phagocytosis by cultured retinal pigment epithelial cells. Relationship to cell morphology.

PURPOSE: To study phenotypic variation in primary cultures of rat retinal pigment epithelium (RPE) and to correlate cell morphology with rates of binding and ingestion of rod outer segments (ROS). METHOD: Replicate cultures were prepared using RPE cell sheets isolated with Dispase from Royal College of Surgeons normal (RCS rdy+ p+) and dystrophic (RCS p+) rats. Retinal pigment epithelial morphology was analyzed, and phagocytosis was assessed by fluorescence microscopy in cultures fixed at 2-hour intervals from 3 to 19 hours after continuous incubations with fluorescein isothiocyanate (FITC)-stained ROS. RESULTS: A wide range of RPE cell size, shape, and pigmentation was present at confluence; however, distinct morphologic subtypes were recognized, defined as types 1 to 3, and studied separately. In both normal and dystrophic cultures, the extent and rate of ROS binding varied with RPE phenotype. In normal cultures, highly spread pigmented binucleate cells (type 3) bound and rapidly ingested multiple ROS per cell starting at 3 hours and reached a peak at 9 hours. Lightly pigmented daughter cells (type 2) bound and ingested far fewer ROS per cell than did type 3 RPE, which had not divided. Patches of hexagonally packed cells with in vivo morphology (type 1) bound large numbers of ROS per cell only after prolonged (9- to 11-hour) incubations and ingested them synchronously. Comparison of normal versus dystrophic RPE subtypes 1 to 3 revealed the known ingestion defect in all three mutant phenotypes but indicated delayed ROS binding in type 2 and type 3 cells as well. CONCLUSIONS: Kinetics of ROS binding and ingestion differ markedly among phenotypic variants of RPE cells typically found in primary cultures at confluence. Thus, accurate quantitation requires comparison of equivalent microscopic fields or like RPE subtypes, and the heterogeneous responses of various RPE subtypes should be considered when interpreting phagocytic data obtained from entire cultures at a particular time.

Animals↗
PubMed1992

Changes in retinal pigment epithelial cell autofluorescence and protein expression associated with phagocytosis of rod outer segments in vitro.

The accumulation of autofluorescent lipofuscin was quantified in cultured human retinal pigment epithelial (RPE) cells phagocytosing bovine rod outer segments (BROS) and the expression of proteins in these cells was investigated. Results showed a steady increase in autofluorescence of RPE cells over a 4-week period as measured by fluorophotometric flow cytometry. A significantly greater increase in autofluorescence was found in the cultured RPE cells from a 7-year-old donor compared with those from a 47-year-old donor. Within both groups the BROS-challenged cells had significantly higher fluorescence readings than the control cells which were not challenged. Autoradiography of 35S-labelled proteins separated by polyacrylamide gel electrophoresis (PAGE) revealed a small distinct band at 102 kDa in BROS-challenged RPE cells of both bovine and human origin that did not appear in control or microsphere-phagocytosing RPE cells. The intensity of the signal was unrelated to the duration of the challenge period.

Aging↗
PubMed1997

Porcine iris pigment epithelial cells can take up retinal outer segments.

This study investigates the ability of iris epithelial cells (IPE) to ingest rod outer segments (ROS) and compares the amount of phagocytosis of porcine RPE and IPE cells by the use of a pH sensitive fluorescent dye (carboxy SNAFL) at the light microscopic level. The dye allowed investigation of ingestion separately from binding of rod outer segments. In a second set of experiments, after exposing ferritin-labeled ROS to the cultured cells, phagosomes were also counted in electron microscopic sections. Additionally immunocytochemical staining was performed with IPE and RPE cells. Both cell types stained positive with polyclonal NaK-ATPase antibodies against the alpha 1 subunit from rat brain and kidney. The epithelial nature of the cultured cells was determined by monoclonal anti-human-cytokeratin antibodies. Moreover, the ultrastructure of the cells revealed high amounts of phagosomes smaller than 1 micron in diameter present in both RPE and IPE cells. The iron label of the phagosomes was determined by EELS spectra taken from individual phagosomes. Electron and light microscopic quantification shows that cultured IPE cells have 64% of the phagocytic capacity of the RPE with respect to phagosomes larger than 1 micron in diameter.

Animals↗
PubMed1994

RCS rat retinal rod outer segment membranes exhibit different cholesterol distributions than those of normal rats.

Royal College of Surgeons (RCS) rats exhibit an hereditary defect in phagocytosis of the tips of the photoreceptor cell rod outer segments (ROS) which leads to degeneration of the retinal visual cells. The lipid composition of outer segment membranes of these rats was analysed and compared to those of normal rats to determine whether there are differences between the normal and mutant rat ROS. The cholesterol distribution in ROS disk membranes from normal and RCS rats was investigated using a digitonin induced change in membrane density. Normal rat ROS disks varied in cholesterol to phospholipid mole ratio from 0.36 to 0.03. The disk membranes from RCS rats, however, do not exhibit the same marked cholesterol heterogeneity. The mean molar ratio of cholesterol to phospholipid in the disk membranes of normal rats is 0.11 while that found in the RCS rats is 0.14. The ROS plasma membrane of dystrophic rats also has a lower cholesterol to phospholipid ratio (0.20) than is found in the normal rat (0.40). The phospholipid headgroup composition of RCS disks and plasma membrane were determined. RCS disks were shown to differ from those of normal animals. The cholesterol content of ROS disks may be governed by the phospholipid composition.

Animals↗
PubMed1987

The influence of carbohydrates on the binding of rod outer-segment (ROS) disc membranes and intact ROS by the cells of the retinal pigment epithelium of the embryonic chick.

The role of carbohydrates in mediating the interaction of rhodopsin-containing membranes with retinal pigment epithelium (RPE) cells was investigated by studying the influence of various monosaccharides on their binding by RPE cells of the embryonic chick maintained in cell culture. Rod outer-segment (ROS) disc membranes were selected as a model rhodopsin-containing membrane system for these studies in view of their high concentration of rhodopsin and the relative purity with which they can be isolated. Disc membranes, frozen and thawed in order to expose the carbohydrate groups of rhodopsin which are oriented intraluminally in situ, were incubated with monolayers of RPE cells under various conditions, and the binding of the membranes by the cells was quantitated by radioimmunoassay for rhodopsin. Cell-membrane association was also verified by indirect immunofluorescence microscopy. The surface accessibility of the sugars in frozen-thawed discs was verified by succinyl concanavalin A-binding studies. From 15- to 20-fold increase in carbohydrate-reactive sites was obtained after freezing and thawing the discs. The RPE cell-membrane binding process was saturable, and time- and temperature-dependent. By means of competition studies carried out in the presence of high concentrations of various monosaccharides, and also by comparing the binding of disc membranes whose carbohydrate groups were either exposed (frozen-thawed) on the surface or inaccessible (native), it was concluded that the carbohydrates of rhodopsin, mannose and N-acetylglucosamine, were not involved in the interaction with the RPE. The possibility was also examined that enzymatically galactosylated rhodopsin might serve as a site for recognition by the RPE cell. The binding of ROS disc membranes modified in this manner was not enhanced, indicating that the presence of galactose groups on rhodopsin did not serve as a site for recognition by the RPE. The influence of monosaccharides on the binding of intact ROS by the RPE cells was also investigated. Similar to the results with the disc membranes, the process was not blocked by the presence in the incubation medium of high concentrations (up to 30,000-fold higher than that of rhodopsin) of mannose or GlcNAc, as with the disc membranes, or by glucose or galactose. Thus, from these studies it is concluded that a lectin-like carbohydrate-recognition process may not be involved in the interaction between rhodopsin-containing membranes and the RPE cells.

Animals↗
PubMed2006

Phospholipase D from photoreceptor rod outer segments is a downstream effector of RhoA: evidence of a light-dependent mechanism.

Photoreceptor cells contain rod outer segments (ROS) which are specialized light-sensitive organelles. The biological function of ROS is to generate a photoresponse, which occurs via the classic transducin-mediated pathway. Moreover, ROS undergo light-regulated membrane turnover and protein translocation whose mechanisms have not been fully elucidated to date. Phospholipase D (PLD) is a key enzyme involved in lipid signal transduction and membrane trafficking. We have previously reported that PLD activity is present in purified ROS (Salvador, G.A., Giusto, N.M., 1998. Characterization of phospholipase D activity in bovine photoreceptor membranes. Lipids 33, 853-860). We now demonstrate that ROS PLD activity is enhanced by phosphatidylinositol bisphosphate (PIP2) and cytosolic factors in a GTP dependent-manner. Western blot analysis demonstrates the presence of PLD1 isoform in purified ROS. In ROS obtained from dark-adapted retinas (DROS), PIP2-dependent PLD activity was higher than that observed in ROS obtained from light-adapted retinas (LROS). In addition, experiments carried out in the presence of C3 toxin inhibited PLD activity from DROS whereas pertussis toxin did not affect the enzyme activity. Western blot analysis demonstrates the presence of RhoA, a PLD upstream-regulator. Moreover, RhoA levels were higher in DROS with respect to those in LROS. The present study reports evidence of the involvement of the small G-protein, RhoA, in ROS PLD regulation. Our data strongly suggest that RhoA regulates ROS PLD activity under a light-dependent mechanism.

Adaptation, Ocular↗