Uptake of Retinol and Retinoic Acid from Serum Retinol-binding Protein by Retinal Pigment Epithelial Cells *

Isolated pigment epithelial cells from bovine eyes were shown to be capable of taking up retinol and retinoic acid. The uptake was dependent on the ligands being presented to the cells as complexes with serum retinol-binding protein. Neither free retinol nor retinol or retinoic acid presented as serum albumin complexes were taken up by the pigment epithelial cells. The transport of retinal and retinoic acid was a relatively rapid, temperature-dependent process. The uptake of retinol and retinoic acid was accompanied by dissociation of the ligand retinal-binding protein complex, with the ligand being taken up by the cells while the cariier protein remained outside the cell. Retinol which was taken up by the pigment epithelial cells was found in the cytosol as retinal. About a third of the transported retinol was found to be bound to a high molecular weight protein (>1.5 x 10”) while the rest was found as free retinol. These results were similar to those previously obtained by the direct addition of retinol to pigment epithelial cytosol (Heller, J. (197Q.I. Biol. Chem. 251, 2952). The retinoic acid which was taken up by the pigment epithelial cells was found as retinal-like material, partly bound to a high molecular weight cytosol protein and partly free. This retinol-like material was not retinol since it was not incorporated into rhodopsin in ciao. Fresh leucocytes and red cells from either human or bovine blood did not show an3 uptake of either retinol or retinoic acid when presented to the cells as their respective serum retinol-binding protein complexes.

Isolated pigment epithelial cells from bovine eyes were shown to be capable of taking up retinol and retinoic acid. The uptake was dependent on the ligands being presented to the cells as complexes with serum retinol-binding protein. Neither free retinol nor retinol or retinoic acid presented as serum albumin complexes were taken up by the pigment epithelial cells. The transport of retinal and retinoic acid was a relatively rapid, temperature-dependent process. The uptake of retinol and retinoic acid was accompanied by dissociation of the ligand retinal-binding protein complex, with the ligand being taken up by the cells while the cariier protein remained outside the cell.
Retinol which was taken up by the pigment epithelial cells was found in the cytosol as retinal. About a third of the transported retinol was found to be bound to a high molecular weight protein (>1.5 x 10") while the rest was found as free retinol. These results were similar to those previously obtained by the direct addition of retinol to pigment epithelial cytosol (Heller, J. (197Q.I. Biol. Chem. 251, 2952). The retinoic acid which was taken up by the pigment epithelial cells was found as retinal-like material, partly bound to a high molecular weight cytosol protein and partly free. This retinol-like material was not retinol since it was not incorporated into rhodopsin in ciao. Fresh leucocytes and red cells from either human or bovine blood did not show an3 uptake of either retinol or retinoic acid when presented to the cells as their respective serum retinol-binding protein complexes. thus, vitamin A-depleted animals which are supplied with retinoic acid thrive, although their vision is lost (6, 7). Goodman and his colleagues (8) have shown that dietary retinoic acid is carried as a complex with serum albumin and apparently is not found as a complex with serum RBP' although it is well established that retinoic acid can combine in vitro with apo-RBP to form a stable retinoic acid.RBP complex. Since retinol is normally found in serum only as a complex with RBP Cl), we were interested in investigating the uptake of retinol and retinoic acid by pigment epithelial cells.
Experiments using "'I-labeled RBP have previously indicated that the RBP molecule does not enter the pigment epithelial cells. This was shown both by in vitro binding experiments (3) and by in uiuo injection of the labeled protein followed by autoradiography (4). Previous experiments in our laboratory have shown that when retinol is added in vitro to the cytosol from pigment epithelial cells, the chromophore is bound to a high molecular weight lipoglycoprotein (9). In the present series of experiments, we report the uptake of retinol and retinoic acid by isolated pigment epithelial cells from their respective holo-RBP complexes. We also found that isolated human white and red blood cells do not show any uptake of retinol or retinoic acid under similar conditions. Additionally, it was found that retinol which was taken up by the cells was present in the cytosol as retinol bound to a high molecular weight protein.

Uptake of I"HIRetinol by Pigment Epithelial
Cells -When pigment epithelial cells were incubated with ["Hlretinol RBP complex, there was a rapid uptake by the cells of the labeled retinol ( Fig. 1). The uptake of retinol was rapid for the first 2 to 3 min and was about 80% complete in that time. The loss of I:'H]retinol in the extracellular medium corresponded to the gain of ["Hlretinol in the cells (Fig. 1). When the uptake experiment was performed by adding free I:'H]retinol (instead of ["Hlretinol RBP complex) to the pigment epithelial cells, there was no uptake of labeled material by the cells (Fig. 2). These experiments indicate that retinol RBP complex is an essential component of the uptake process by the pigment epithelial cells and that free retinol cannot be transported in this system. Effect ofAddition ofFresh Retinol .RBP -As seen in Fig. 1, the uptake of I"Hlretino1 by pigment epithelial cells leveled off after about 10 min of incubation at 37". To establish whether this leveling off was due to saturation of the pigment epithelial cells by retinol or to exhaustion of the retinol 'RBP in the medium, the pigment epithelial cells aRer 10 min of incubation were recovered by centrifugation at 170 x g for 5 min and incubated in additional retinol RBP. As shown in Fig. 2, the uptake of retinol by pigment epithelial cells following this procedure was as effective as during the first period of incubation. The addition of fresh retinol RBP to pigment epithelial cells was repeated four times, resulting in a similar uptake of retinol following each addition. Moreover, in vivo injection of ["H]retinol.RBP led, after 5 days, to a total retinol uptake by pigment epithelial cells that was about loo-fold larger than that seen after 10 min of in vitro incubation.Y On the other hand, addition of fresh pigment epithelial cells to the supernatant from the first incubation did not result in any further uptake of retinol by the fresh pigment epithelial cells (Fig. 2). These experiments indicated that the reduced uptake of retinol by the pigment epithelial cells after 10 min of incubation at 37" was due to depletion of retinol . RBP complex in the medium and not due to saturation of the pigment epithelial cells by retinol.
Since the supernatant medium after lo-min incubation with [:'H]retinol.RBP still contained about 80% of the initial 3H counts, we investigated the fate of the retinol RBP complex in the extracellular medium. When the radioactive supernatant at the termination of the incubation was subjected to gel filtration chromatography, the counts appeared as a low molecular weight (<lOOO) substance, while no radioactivity appeared at the position of retinol . RBP complex. Addition of excess protein (egg albumin) to the incubation medium did not affect the extent of RBP degradation.
The conclusion drawn from this experiment was that the ["Hlretinol. RBP in the incubation mixture was progressively broken down.

Effect
of Temperature on Retinol Uptake -As shown in Fig.  3, very little uptake was observed at 4" and there was only a slight difference between uptake at 37" and at 22". When the pigment epithelial cells were incubated for 10 min at 4" and then raised to 37", there was an increase in retinol uptake, without any lag period and with an uptake pattern similar to that shown by pigment epithelial cells that were not kept in the cold. This experiment illustrates that the cold treatment did not affect the uptake potential of the cells and that when the incubation temperature was raised back to 37", the retinol uptake seemed to be normal. In other words, the transport of per cent uptake was taken as the difference between the counts of the cellular fraction at zero time and after 60-min incubation. One hundred per cent loss was taken as the difference between the counts after 60-min incubation and at zero time in the extracellular medium which was obtained by centrifuging the incubation mixture at 170 x g for 10 min. 0, uptake by the pigment epithelial cells; 0, loss in the extracellular medium. NaI under identical conditions as those used for iodination with Y. As seen from Fig. 4 and Table I, the [3H]retinol*I-labeled RBP was as effective in the delivery of retinol to the cells as noniodinated retinol * RBP. These experiments show that there is a dissociation between retinol uptake and RBP binding by the pigment epithelial cells. Additionally, while retinol could not be displaced from the pigment epithelial cells once it was taken up, the RBP moiety could be displaced rapidly by the addition of excess retinol+RBP, providing further evidence that the retinol and the RBP are dissociated during the transport process and are then present in two distinct compartments.
Cell Specificity of Binding of Retinol.RBP and Uptake of Retinol - Fig. 5, A and B, demonstrated that there was neither binding of retinol RBP nor uptake of retinol by bovine leucocytes or red blood cells. By using retinoic acid. RBP, again we found no binding and uptake by bovine cells (Fig. 5C). Similar results were found in the human white and red blood cells.
Uptake of Retinoic Acid from Retinoic Acid .RBP by Pigment Epithelial Cells -The uptake of retinoic acid by incubation of retinoic acid. RBP with pigment epithelial cells is shown in Figs. 5 and 6. The extent of retinoic acid uptake was similar to that of retinol uptake. This striking similarity was also observed in the binding studies of retinol . '2sI-labeled RBP and retinoic acid. I25 I-labeled RBP to pigment epithelial cells.3 Uptake of Retinol and Retinoic Acid from Bovine Serum Albumin Complex -Both retinol and retinoic acid bind in vitro to serum albumin forming a relatively stable complex. Goodman and his colleagues (8) have recently shown that unlike retinol, which is normally carried in plasma as a complex with RBP, retinoic acid seems to be complexed with serum albumin. To check the specificity of RBP in the transport of retinol and retinoic acid into pigment epithelial cells, we tested the ability of pigment epithelial cells to take up either compound when presented to the cells as a complex with bovine serum albumin. Neither retinol nor retinoic acid were taken up by pigment epithelial cells when presented to the cells as a complex with bovine serum albumin (Fig. 6). To show that there was no inhibition by bovine serum albumin of the transport mechanism, we have added either retinal. or retinoic acid. RBP complex and the normal, prompt uptake of either retinol or retinoic acid was observed (Fig. 6). The conclusion drawn from these experiments was that RBP is an obligatory carrier protein for uptake by pigment epithelial cells and that retinoic acid (which is normally carried by serum albumin) cannot be utilized by the pigment epithelial cells.
Gel Filtration Chromatography of Cytosol after Retinol or Retinoic Acid Uptake-When the cytosol prepared from pigment epithelial cells that have been loaded with [SH]retinol (supplied as [3H]retinol.RBP) was examined, it was found that radioactivity was present in this fraction. To establish the nature of the radioactive compound, the labeled cytosol was subjected to gel filtration chromatography.
As seen in Fig. 7A, about a third of the radioactivity was bound to a protein which appeared in the column's void volume, while the rest of the label appeared as a low molecular weight (4000) material. These results were similar to those observed in a previous study (9) in which free retinol was added to cytosol from isolated pigment epithelial cells.
When this experiment was repeated after 13H]retinoic acid uptake by the pigment epithelial cells, the chromatographic pattern of the cytosol was similar to that obtained after retinol I"HJretinoic acid uptake was found in the cytosol and, in both cases, about a third of the radioactivity was bound to a high molecular weight protein.

Identification
of Cytosol-labeled Material after Retinol or Retinoic Acid Uptake -Thin layer chromatography of the extracted cytosol retinol protein complex showed that about 95%' of the recovered counts were found in a spot corresponding to retinol. No radioactivity was found on the chromatogram in spots corresponding to retinoic acid, retinal, or retinyl esters. Total recovery of radioactivity from the plate was 75% of the counts in the cytosol. Similar results were obtained when the low molecular weight material found after gel filtration chromatography ( Fig. 7A) was extracted and subjected to thin layer chromatography.
When the pigment epithelial cytosol obtained from cells that had taken up ["Hlretinoic acid (from the RBP complex) was examined in the same way, 98% of the radioactivity in the chromophore protein complex (Fig. 7B) was due to retinol-like material as judged by thin layer chromatography.
The low molecular weight radioactive fraction from the gel filtration chromatography (Fig. 7B) was also found to be retinol-like. To exclude the possibility that this reduction of ["Hlretinoic acid happened during the extraction of the cytosol and the thin layer chromatography, equal amounts of labeled cytosol were mixed with I sH]retinoic acid. RBP, extracted with organic solvents, and subjected to thin layer chromatography as above. Forty-four per cent of the counts were found in the retinol fraction and 54% in the retinoic acid fraction. It is thus clear that retinoic acid was not reduced to retinol during extraction and thin layer chromatography.
The conclusion drawn from this series of experiments was that retinoic acid uptake (from RBP complex) by pigment epithelial cell is followed by the reduction of the acid. This retinol-like material is not vitamin A itself since it is not incorporated into rhodopsin in uir?o.z In contrast, retinol (provided as retinol . RBP complex) is rapidly incorporated as the chromophore of rhodopsin following injection into the living frog.2

DISCUSSION
The polyene lipid retinol is transported in plasma as a complex with a specific protein. the retinol-binding protein.
We have previously shown that pigment epithelial cells in bovine (3) and rat (13) possess a specific cell surface receptor for RBP. This receptor is found only on one side of the cell, the side facing the systemic (choroidal) blood supply. In the present study we have utilized pigment epithelial cells isolated from bovine eyes to investigate the transport of retinol to pigment epithelial cells.
The experiments reported in this paper show that retinol is taken up by pigment epithelial cells only if presented as a retinol.RBP complex, while neither free retinol nor retinol bovine serum albumin complex were sequestered by the cells.
The uptake of retinol (from retinol RBP complex) was rapid, temperature-dependent, and did not show saturation at the level employed in this study. The reduced uptake of retinol after a few minutes of incubation was due to the breakdown of retinol .RBP complex and was not due to saturation of the cells by retinol (Fig. 2). Since the amount of retinol taken up by the cells was 5-to 6-fold larger than the number of cell surface RBP receptors (as determined by binding studies) (Table I), we conclude that there was a rapid exchange at the receptor site of retinol RBP molecules with apo-RBP molecules (molecules that have already delivered their retinol complement to the cell). In this connection, it is interesting to note that previous binding studies have indicated that apo-RBP is less firmly bound to the RBP receptor site than are retinol RBP molecules (3). Maraini and Gozzoli (14) have shown that RBP is essential for retinol "binding" by pigment epithelium. Rask and Peterson (15) have also shown that retinol is taken up in vitro by intestinal mucosal epithelial cells when supplied as the retinol RBP complex.
Previous work by Dowling and Wald (6) has shown that while dietary retinoic acid can maintain vitamin A-deficient animals in good health, it did not supply the chromophoric group (retinol or retinal) for the visual pigments and the animals became blind. Goodman and his colleagues have shown that dietary retinoic acid is normally bound to serum albumin and not to RBP (8). The experiments reported in the present study show that when retinoic acid is used to form a complex with RBP, it is taken up by pigment epithelial cells as effectively as retinol (Fig. 6), at least during the incubation periods which were employed in this study. In a recent study, we have found' that although retinoic acid is taken up by pigment epithelial cells following an in uiuo injection of retinoic acid. RBP complex, the internalized retinoic acid is not incorporated into rhodopsin. In contrast. retinol (derived from retinol RBP) is effectively incorporated as the chromophore (retinal) of rhodopsin." It is interesting to note that neither white nor red blood cells show any uptake of retinol or retinoic acid from their respective RBP complexes. Whether this is due to the absence of RBP receptors on the surface of these cells or to the RBP receptor being at so low a concentration as to be undetected is not known at the present time.
Following the in vitro uptake of either retinol or retinoic Uptake of Retinol and Retinoic Acid 5221 acid by pigment epithelial cells, the chromophores were found Bok and Dr. Michael 0. Hall have helped in suggestions and in the cytosol partly free and partly as a complex with a high discussions. Dr. Joseph Horwitz was most helpful in the critical reading of this manuscript. molecular weight protein. In previous experiments in which retinol was added directly to cytosol from bovine pigment epithelial cells, it was also found that the chromophore was bound to a high molecular weight lipoglycoprotein (9). In other words, retinol which was internalized by intact pigment epithelial cells from retinal. RBP in the medium was bound to a similar high molecular weight protein as is free retinol added to isolated cytosol from pigment epithelial cells. Whether these two intracellular cytosol retinol-binding proteins are actually one and the same remains to be established.
We have previously shown that photoreceptor cells do not have a receptor for RBP (3, 4). Consequently the pigment epithelial cells would seem to be the only avenue for entry of retinol from the blood. The retinol (or retinal) which is found in the photoreceptor would thus come from retinol taken up by the pigment epithelial cells. The mode of retinol transport between the pigment epithelium and the photoreceptors remains to be studied.