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PubMed1984

Diurnal variations in amino acid incorporation into inner segment opsin.

Polyacrylamide gel and immunoelectrophoresis were used to measure the specific activity of nascent inner segment opsin following injections of labeled amino acids into frogs at different times during the diurnal cycle. Animals injected just after light onset showed the greatest incorporation of label into opsin, while animals injected during the night period had the lowest levels of isotope incorporation. As determined by rocket immunoelectrophoresis, there was a 13-fold decline in specific activity in animals injected during the late night hours in comparison with animals injected just after light onset. In contrast, rod outer segment rhodopsin-specific activity did not vary as greatly. Animals subjected to a 12-hour light-12-hour dark diurnal cycle incorporated only twice the radiolabeled amino acids into their rhodopsin compared with animals injected and maintained in constant dark for 24 hours. Taken together, these experiments suggest that adult Rana pipiens accumulate opsin within their inner segments during the dark phase of the diurnal cycle and that alterations of this pool effect changes in inner segment opsin specific activity. Differences in outer segment rhodopsin specific activity are probably due to averaged metabolic differences between constant dark and light-dark animals.

Absorption↗

PubMed1981

In vitro biosynthesis, core glycosylation, and membrane integration of opsin.

A membrane-integrated , core-glycosylated form of bovine opsin was synthesized in vitro when bovine retina mRNA was translated in a wheat germ cell-free system supplemented with dog pancreas microsomal vesicles; glycosylation and integration of opsin into membranes were coupled to translation. Proteolysis with themolysin was used to probe the orientation of opsin within the dog pancreas microsomal membrane, and to compare it with that of opsin in rod cell disk membranes isolated from bovine retina. Intact microsomal or disk vesicles were required for production of discrete, membrane-associated thermolysin fragments of opsin; no discrete opsin fragments were detected when membranes were incubated with thermolysin in the presence of the nonionic detergent, Triton X-100. The major opsin fragments produced by themosylin treatment of intact microsomal vesicles resembled those from disk vesicles in their size, oligosaccharide content, and order of appearance. In each case, the first cleavage of opsin took place at the COOH-terminus, generating a glycosylated fragment, O', which was only slightly smaller than intact opsin. Both the microsomal and disk membrane forms of O' were next cleaved internally; glycosylated fragments of similar sizes in both cases were detected which were derived from the NH(2)-terminal portion of O'. Several smaller NH(2)-terminal fragments of opsin were detected only in thermolysin-treated microsomal membranes, and not in disk membranes. The data suggest that the topology of opsin integrated into dog pancreas microsomal vesicles is similar to that in rod cell disk vesicles, although not identical. In each case, the glycosylated NH(2)-terminal region of opsin is located within the lumen of the vesicle, while discrete COOH-terminal and internal segments of opsin apparently emerge at the outer, cytoplasmic face of the membrane. Thus, opsin in the heterologous microsomal membrane, like its counterpart in the native disk membrane, may cross the bilayer at least three times. The internal domain of the polypeptide that emerges at the outer membrane surface is apparently more highly exposed in the case of opsin in microsomal membranes, evidenced by the additional internal thermolysin cleavage sites detected.

Animals↗

PubMed1993

Noncovalent occupancy of the retinal-binding pocket of opsin diminishes bleaching adaptation of retinal cones.

Bright light bleaches visual pigment and leads to a persistent desensitization of isolated rod and cone photoreceptors called bleaching adaptation. Bleaching adaptation results from the combined effects of pigment depletion and adaptational modulation of certain cellular reactions in the visual transduction cascade. Here, we present evidence that in solitary cone photoreceptors isolated from the salamander retina, the latter effect is due to the presence of free opsin in the outer segment. Also, we demonstrate that this "opsin adaptation" can be reversed by treating the cells with synthetic retinoids similar to 11-cis retinal but having polyene chains too short to form protonated Schiff base attachments to opsin.

Adaptation, Physiological↗

PubMed1995

Structure and function in rhodopsin: the fate of opsin formed upon the decay of light-activated metarhodopsin II in vitro.

We report that the light-activated bovine metarhodopsin II, upon decay, first forms opsin in the correctly folded form. The latter binds 11-cis-retinal and regenerates the native rhodopsin chromophore. However, when the opsin formed upon metarhodopsin II decay is kept in 0.1% dodecyl maltoside, it converts in a time-dependent manner to a form(s) that does not bind 11-cis-retinal. On subsequent addition of 11-cis-retinal, slow reversal of the non-retinal-binding forms to the correctly folded retinal-binding form has been demonstrated. We have studied the influence, on the above interconversions, of pH, phospholipids (rod outer segment and soybean), dithiothreitol, and a mixture of reduced and oxidized glutathione. Chromophore regeneration in the presence of 11-cis-retinal was highest at pH 6.0-6.3. The addition of dithiothreitol just before bleaching gave back only a small amount (7%) of rhodopsin on the subsequent addition of 11-cis-retinal, whereas the slow phase(s) of chromophore formation was completely abolished. The presence of a mixture of reduced and oxidized glutathione did not significantly affect the results. Addition of phospholipids, either from soybean or rod outer segment, prior to bleaching stabilized the initially formed opsin, resulting in much higher chromophore regeneration. However, addition of the phospholipids after conversion of the opsin to non-retinal-binding form(s) arrested the subsequent reversal of the opsin to the retinal-binding form.

Animals↗

PubMed1988

A diurnal rhythm in opsin content of Rana pipiens rod inner segments.

Quantitative electron microscope immunocytochemistry, employing an antibody specific to opsin, was used to evaluate the amount and location of opsin in Rana pipiens rod photoreceptors throughout a 24 hr light/dark cycle. We found a distinct diurnal rhythm in the density of anti-opsin labeling of the rough endoplasmic reticulum (RER) and Golgi apparatus in the myoid region of the rod inner segment. Opsin labeling of these organelles was lowest at light onset, increasing thereafter by three- to four-fold, and remained high until 2 hr into the dark phase. A fall in labeling density occurred within the following 4 hr, and remained low for the remainder of the dark phase. Our finding of a diurnal rhythm regulating inner segment opsin transport in Rana pipiens contrasts with published observations on outer segment membrane turnover, since it has been shown that the rates of disc formation and disc shedding are governed by environmental lighting alone in this species. These results imply that there is opsin pooling in the inner segment during the first 14 hr of a 24 hr light/dark cycle; thereafter the loss of inner segment opsin due to mobilization of this protein from the Golgi exceeds the rate of formation of new opsin. There was no evidence of accumulation of opsin-containing vesicles near the cilium or in the ellipsoid just prior to light onset. At light onset, prominent opsin labeling was identified at the proximal portion of the outer segment in regions separate from the disc stack. In two separate experiments, additional groups of frogs were killed around the time of light onset and were examined by conventional transmission electron microscopy. Disordered disc membranes were seen at the base of the outer segment which were not in register with the disc stack. These disordered membranes were observed as early as 2 hr before light onset, and were no longer observed by 1 hr after light onset. We suggest that these disordered membranes reflect a step in the biogenesis of new discs, serving as a pool of membrane that forms during the later part of the dark cycle. It appears that light onset triggers the ordering of neatly registered discs from this new membrane, rather than assembly of new membrane from pooled transport vesicles in the inner segment.

Animals↗

PubMed1993

Photoreceptor recovery in retinoid-deprived rats after vitamin A replenishment.

Dietary deficiency in the retinoid precursors of the visual pigment chromophore 11-cis retinal results in the synthesis of photoreceptor outer segments containing opsin in excess of the vitamin A available for rhodopsin regeneration. This suggests that vitamin A-free opsin may be incorporated into newly synthesized outer segment disc membranes. If this opsin is functionally intact, it should be possible convert it to rhodopsin in vivo by providing the appropriate retinoids, and the resulting rhodopsin should should be able to mediate visual transduction. Experiments were conducted to evaluate this possibility and to identify the rate-limiting steps in photoreceptor recovery from retinoid depletion. Rates were maintained on diets either containing or lacking retinoid precursors of 11-cis retinal for 23 weeks, at which time outer segment opsin content greatly exceeded the availability of visual cycle retinoids in the retina. The retinoid-deprived animals were then each given a single intramuscular injection of all-trans retinol. At various time intervals after retinol administration, electroretinograms (ERGs) were recorded on some rats, and retinal rhodopsin contents were determined in others. At similar time intervals, blood and retinal pigment epithelial (RPE) retinoid levels and photoreceptor outer segment size were also determined. No significant increase in retinal rhodopsin content was observed up to 8 hr after injection, despite the fact that by 3 hr, blood retinol levels had recovered to more than 30% of normal. By 1 day after injection, however, rhodopsin levels had recovered to 30% of normal and ERG responses showed increases in visual sensitivity commensurate with the recovery of rhodopsin. The lag in rhodopsin recovery was apparently due to delayed uptake of retinol from the blood by the RPE. Photoreceptor outer segment size was reduced by over 50% in the retinoid- deprived rats and did not begin to recover by 1 day. By 1 week, however, outer segment size had returned to an average of 65% of normal. Commensurate with this regrowth of the outer segments, both rhodopsin levels and visual sensitivity increased between 1 and 7 days after vitamin A administration. Because the rates of recovery in rhodopsin levels and visual sensitivity greatly exceeded the normal rate of new opsin synthesis at short time intervals after vitamin A repletion, it appears that the opsin incorporated into the disc membranes of retinoid-deprived rats is able to form functional rhodopsin in vivo when the chromophore is supplied. Regrowth of the outer segments back to their normal size is required for full recovery of visual sensitivity.

Animals↗

PubMed1986

Rod outer segment lipids in vitamin A-adequate and -deficient rats.

Weanling albino rats were fed a vitamin-A-adequate diet or vitamin-A-deficient diet and maintained in a cyclic light or dark environment for up to 14 weeks. One half of the rats were supplemented with additional dietary linolenic acid in the form of linseed oil. The lipid composition and rhodopsin-opsin contents of isolated rod outer segments were determined after 6-7 weeks or 12-14 weeks on diet. This study shows that feeding rats a standard vitamin A-adequate or -deficient diet results in an age-dependent loss of omega three docosahexaenoic acid and a concomitant increase in omega six docosapentanoic acid in the rod outer segments. The loss of docosahexaenoate appears to be caused by insufficient dietary omega three fatty acids. The increase in omega six docosapentanoic acid appears to arise from the high concentration of linoleic acid in standard diets containing either cottonseed, or peanut oil or supplemental corn oil. Feeding rats diets supplemented with linseed oil, however, results in a rod outer-segment lipid profile which is the same as for chow-fed animals. The same effects were seen in the fatty-acid profile of lipids from liver, although the content of polyunsaturates was much lower than in rod outer segments. Vitamin A deficiency, itself, does not lead to changes in the fatty-acid composition of either the rod outer segments or liver. After 6-7 weeks on A+ or A- diet, rhodopsin levels were, as expected, higher in dark-reared rats than in cyclic-light animals. Although the rhodopsin levels in dark-reared vitamin A-adequate rats were significantly higher than in vitamin A-deficient animals, measurements of the lipid to opsin ratio of rod outer segments indicate that the rods of vitamin A-deficient rats are not markedly different than those of vitamin A-adequate rats. It is concluded that these diets may be useful in providing a means for evaluating the role of docosahexaenoic acid in visual cell death from damaging light.

Age Factors↗

PubMed1992

Topography of opsin within disk and plasma membranes revealed by a rapid-freeze deep-etch technique.

Rod outer segments in fresh rat retinas were examined by a rapid-freeze, deep-etch technique to explore how membrane proteins are organized at the macromolecular level. Cross-fractures revealed that intradiscal membranes are adherent to each other except at the rim. When an isolated fresh retina was incubated in a hypotonic solution for a few minutes, the interdiscal space was expanded and the cytoplasmic surface of the disk membrane was found to be covered with protrusions except at the rim. A few particles were scattered among the protrusions and were attached to the cytoplasmic surface. Since the distribution density of the cytoplasmic surface protrusions was similar to that of the P-face particles, which are known to reflect opsins, the protrusions were considered to be portions of opsins extending into the cytoplasm. The intradiscal surfaces in chemically-fixed retinas were rather smooth and were labelled with anti-opsin antibodies and wheat germ agglutinin. The true surfaces of the plasma membrane were found to be similar in fine structure to those of the disk. A model of the macromolecular organization of rod outer segments is proposed on the basis of these observations. The model shows apposed opsins within a disk membrane adhering to one another except at the rim. These opsins, as well as those in the plasma membrane, are minimally exposed to the extracellular surface, but protrude deeply into the cytoplasm.

Animals↗

PubMed1991

Opsin distribution and protein incorporation in photoreceptors after experimental retinal detachment.

The distribution of opsin was examined immunocytochemically after experimental retinal detachment in adult cats. Retinal detachments were produced by injecting fluid between the retinal pigment epithelium and neural retina. One to 60 days later the animals were killed. Tissue areas from detached and attached retinal regions from the eye with the detached retina, as well as normal (control) retinas, were processed for post-embedding light and electron microscopic immunocytochemistry. In normal and attached retinal regions, anti-opsin labeled the outer segments and Golgi apparatus most heavily, although the entire photoreceptor plasma membrane was labeled at a low level. Beginning at 2 days after retinal detachment, immunolabeling increased in the photoreceptor inner segment, cell body and synaptic terminal plasma membranes. This pattern of anti-opsin labeling continued at all intervals up through the 60-day detachment time-point. Injection of radiolabeled amino acid in detachments from 1 to 30 days show that radiolabeled protein is still transported to the truncated outer segments of the photoreceptor cells. In addition, these outer segment disks label with anti-opsin. These data imply that opsin continues to be transported and incorporated into the outer segments of photoreceptors showing severe degeneration as a result of long-term detachment from the RPE.

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↗

PubMed1976

Distribution of enzyme activities in subcellular fractions of bovine retina.

Centrifugation of homogenates of bovine retinas to isopycnic equilibrium in sucrose density gradients yielded three partially overlapping bands of particles which were, in the order of increasing density: (a) photoreceptor cell (rod) outer segments; (b) plasma membranes, lysosomes, and large fragments of endoplasmic reticulum; and (c) mitochondria. The only enzyme activity investigated which had a peak coinciding only with outer segment fractions was guanylate cyclase. Enzyme activities with peaks in both the outer segment and denser fractions included 5'-nucleotidase and cyclic GMP phosphodiesterase. Enzyme activities with peaks only in the denser fractions included sodium and potassium ion-activated ATPase ((Na+ + K+)-ATPase), adenylate cyclase, cyclic AMP phosphodiesterase, beta-glucosidase, beta-galactosidase, and succinate-dependent cytochrome c reductase. These results suggest that some of the activities once thought to be present in rod outer segments are actually present in particles from elsewhere in the retina which contaminate rod outer segment preparations.

3',5'-Cyclic-AMP Phosphodiesterases↗

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↗