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Retinol uptake by isolated retinal pigment epithelium plasma membranes
Exp. Eye Res. (1981) 32, 69-75
Retinol Uptake by Isolated Retinal Pigment Epithelium Plasma Membranes SIMONE OTTONELLO A:ND G I O V A N N I M A R A I ~ I
Institute of Molecular Biology and Institute of Ophthalmology, University of Parma, 43100 Parma, Italy (Received 19 February 1980, New York) The uptake of retinol has been investigated by incubating suspensions of isolated bovine retinal pigment epithelium plasma membranes in the presence of [aH]retinol-retinol-binding protein complex. After SDS gel electrophoresis of the labelled membranes, [aH]retinol is found to be associated with a protein band of molecular weight 16000. The binding process is saturable, specific and shows a linear dependence on the membrane concentration in the incubation mixture. A dissociation constant of the order of l0 -9 has been calculated for the retinol-receptor interaction. These observations confirm previous data obtained with isolated pigment epithelium cells and suggest the existence of an intermediate step, at the membrane level, in the transfer of retinol from its plasma carrier to the cell cytoplasm. Key words: retinol; retinol-binding protein ; retinal pigment epithelium ; plasma membranes; retinol receptor; intracellular retinol-binding protein.
1. I n t r o d u c t i o n R e t i n o l is of p r i m a r y i m p o r t a n c e Jn t h e m e t a b o l i s m of t h e eye where t h e a l d e h y d e of v i t a m i n A c o n s t i t u t e s t h e c h r o m o p h o r e of visual p i g m e n t molecules. A v a i l a b l e evidence i n d i c a t e s in t h e r e t i n a l p i g m e n t e p i t h e l i u m ( R P E ) t h e m o n o l a y e r of cells c o n s t i t u t i n g t h e b l o o d - r e t i n a l b a r r i e r b e t w e e n t h e c h o r o i d a l e x t r a v a s c u l a r space a n d t h e n e u r a l retina. R P E cells are i n v o l v e d in t h e u p t a k e o f r e t i n o l from b l o o d a n d in t h e t r a n s f e r of this molecule (or of its d e r i v a t i v e s ) to a n d from t h e p h o t o r e c e p t o r cells. V i t a m i n A circulates in t h e blood b o u n d to a specific c a r r i e r p l a s m a p r o t e i n , t h e r e t i n o l - b i n d i n g p r o t e i n ( R B P ) , a n d t h e u p t a k e o f retinol b y R P E occurs t h r o u g h t h e i n t e r a c t i o n o f R B P p r e s e n t in the choroid e x t r a v a s c u l a r fluid w i t h a specific r e c e p t o r on R P E p l a s m a m e m b r a n e s (Heller, 1975; M a r a i n i a n d Gozzoli, 1975). D u r i n g t h e i n t e r a c t i o n o f R B P w i t h its receptor, r e t i n o l is released from its c a r r i e r to some o t h e r c o m p o n e n t of t h e cell. R B P does n o t cross t h e p l a s m a m e m b r a n e a n d is r e m o v e d from its b i n d i n g place b y c o m p e t i t i v e d i s p l a c e m e n t b y o t h e r R B P molecules (Chi-Ching Chen a n d Heller, 1977). A f t e r i n c u b a t i o n of i s o l a t e d R P E cells in t h e presence o f [ a H ] r e t i n o l - R B P , Maraini, Ottonello, Gozzoli a n d Merli (1977) f o u n d t h a t in t h e p l a s m a m e m b r a n e s of t h e cells r a d i o a c t i v e retinol was a s s o c i a t e d w i t h a m e m b r a n e p r o t e i n o f m o l e c u l a r w e i g h t 14500. This p r o t e i n was different from exogenous R B P a d d e d to t h e cell suspension a n d was identified as being a n o r m a l c o n s t i t u e n t of R P E p l a s m a m e m b r a n e s showing retinol-binding properties. To c h a r a c t e r i z e f u r t h e r t h e i n t e r a c t i o n b e t w e e n r e t i n o l a n d its m e m b r a n e r e c e p t o r we t o o k a d v a n t a g e of the a v a i l a b i l i t y o f an e x p e r i m e n t a l s y s t e m in which t h e process of retinol t r a n s p o r t could n o t p r o g r e s s b e y o n d t h e m e m b r a n e level. W e therefore s t u d i e d t h e t r a n s f e r o f r e t i n o l from its carrier p l a s m a p r o t e i n to t h e m e m b r a n e b i n d i n g 9 1980 Academic Press Inc. (London) Limited
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p r o t e i n in suspensions of i s o l a t e d R P E p l a s m a m e m b r a n e s . T h e results of such a s t u d y are r e p o r t e d in t h e p r e s e n t p a p e r .
2. M a t e r i a l s a n d M e t h o d s
Preparation of pigment epithelial cells R P E cells were isolated from freshly obtained bovine eyes under normal room illumination according to the procedure described by Berman and Feeney (1976). After isolation the cells were washed three times with 0"32 M-sucrose. Examination of R P E preparations by light microscopy after the final washing demonstrated t h a t contamination by rod outer segments, choroidal remnants or red blood cells was negligible.
Isolation of plasma membranes Plasma membranes were isolated by fractionating the R P E homogenate on a discontinuous sucrose graxiient as previously described (Maraini et al., 1977). After the last washing R P E was resuspended in Calcium free Krebs-Ringer solution (pH 7"4) containing 10 -3 M-EDTA, homogenized in a tight fitting Teflon-glass homogenizer at 4~ and layered on top of a discontinuous sucrose gradient. The following sucrose solutions (containing l0 -3 M-EDTA) were layered in the centrifuge tube from bottom to top : 44, 40, 36, 33, 15 %. Centrifugation time was 150 min at 27000 r / m i n in a SW 40 rotor with an L5 Spinco ultracentrifuge. Plasma membranes were collected at the interface between 15 and 33% sucrose, diluted with Krebs-Ringer and peUetted at 20000 r / m i n for 30 min. The purity of the preparation was controlled by electron microscopy of fixed samples.
[aH]Retinol uptake assay In a typical experiment plasma membranes (0"1~)-3 mg of proteins) were suspended in a calcium-free Krebs-Ringer solution. The suspension was then forced several times through a 22-gauge syringe needle and incubated at 37~ in the presence of varying amounts of [SH]retinol-RBP (10 -l~ to 10-~1 tool in 5t)-500 #l containing 5000-50000 ct/min) in a total volume of 1 ml. At various times aliquots of the incubation mixture were filtered on Millipore filters (GS-0"22 #m). Filters were then washed with a large volume (usually 10 ml) of buffer solution, air-dried, placed in scintillation vials, dissolved in Instagel and counted in a P a c k a r d Tricarb scintillation spectrometer. The background due to [3H]retinol aspecifically bound to the filter was evaluated in each experiment by running a blank in the same conditions and using this value for correction.
Electrophoresis of the labelled membranes In some experiments after incubation the membranes were diluted with Krebs-Ringer, sedimented by centrifuging for 30 min at 20000 r/rain, dissolved in 3 % SDS, 1% mercaptoethanol, 30 mM-Tris-HC1 (pH 8"9)) and subjected to eleetrophoresis on 8'5 % polyaerylamide gels according to Weber and Osborn (1969). In some control experiments the electrophoretic separation was also performed in a Tris-glycine buffer system according to Laemmli (1970). The results obtained with the two systems were superimposable. The gels were calibrated for molecular weight determinations using serum albumin (mol.wt 68000), human R B P (mol.wt 21000), Soybean trypsin inhibitor (mol.wt 20000), myoglobin (mol.wt 17000) and cytochrome c (mol.wt 12000). For radioactivity determinations the gels were sliced, dissolved in H,O, at 45~ overnight, resuspended in Instagel and counted as described above.
Other procedures Human R B P was isolated from plasma according to Peterson and Bergg~rd (1971) and purified by affinity chromatography on prealbumin coupled Sepharose 4B according to Vahlquist, Nilsson and Peterson (1971). [3H]Retinol-RBP, prepared according to Heller and Horwitz (1973), was stored in plastic vials at - 2 0 ~ and filtered on Millipore GS-0"22 # m filters immediately before use. [3H]Retinol-bovine serum albumin complex was prepared as detailed by Chi-Ching Chen and Heller (1977). Proteins were determined by the method of Lowry, Rosebrough, F a r r and Randall (1951) or, in the case of pure proteins, by measuring the absorbance at 280 nm.
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Chemicals
Proteins used as electrophoretic standards were purchased from Boehringer. [3H]Retinol was from NEN chemicals. All other reagents were of analytical grade (Merck). 3. R e s u l t s
When isolated plasma membranes of R P E cells were incubated, under the above described experimental conditions in the presence of [3H]retinol-RBP, a rapid incorporation of radioactivity in the membrane fraction was observed. This membranebound radioactivity cannot be removed by a second incubation (over a period of 10 min) of the plasma membranes in the presence of a 200-fold molar excess of unlabeled R B P before filtering. When the labelled membranes were subjected to SDS polyacrylamide gel electrophoresis and the gels were tested for radioactivity distribution, 90 % of the total gel radioactivity was found to be associated with a protein band of mol.wt 16000. This is in good agreement with the results previously obtained by incubating isolated R P E cells in the presence of [3H]retinol-RBP (Maraini et al., 1977). The mobility of this radioactivity containing band in membrane SDS gel electrophoresis was clearly higher than that of holo-RBP added to the membrane pellet immediately before detergent solubilization (Fig. 1). Control SDS gel electrophoresis of equivalent amounts of free [3H]retino] showed that the radioactivity peak had an electrophoretic mobility clearly higher than that of the tracking dye. The same was true with both phosphate and glycine buffer electrophoretic systems and
2-0[ +
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2000 c
I000
o.ob\/.
-
. . . .
]
Relative mobility FIO. 1. Staining ( ) and labelling ( Q - - Q ) profiles of SDS gel eleetrophoresis of proteins from RPE plasma membranes pre-incubated with [aH]retinol-RBP. Arrows indicate the mobility of proteins of known molecular weight: bovine serum albumin (mol.wt 68000), human holo-RBP (mol.wt 21000), myoglobin (mol.wt 17000), cytochrome c (mol.wt 12000).
72
S. O T T O N E L L O
A N D G. M A R A I N I
F 7" o
~4
P, i
0
I
I
I
....
J - -
I00 200 Membrene profeins (fig)
FIG. 2. [3H]Retinol uptake as a function of R P E plasma membrane concentration in the presence of a constant a m o u n t of [3H]retinol-RBP (3 pmol) in the incubation mixture. Incubation time was 5 min at 37~
also when [SH]retinol was added to unlabeled R P E plasma membranes dissolved in SDS before the electrophoresis, I n both cases the radioactivity peak had thus migrated much faster than control protein standards of 17000 daltons. The kinetics of the uptake process was investigated by incubating isolated R P E plasma membranes in the presence of [3H]retinol-RBP and withdrawing, filtering and washing aliquots of the incubation mixture at different time intervals. The uptake is a very rapid process and is virtually complete within 2 min. Its time course is superimposable to t h a t obtained by studying the uptake of retinol by isolated R P E cells (Maraini and Gozzoli, 1975; Chi-Ching Chen and Heller, 1977). As shown in Fig. 2 the uptake rate shows a linear dependence on the membrane concentration in the incubation mixture. In the presence of the usual amounts of membranes and [aH]retinol-RBP (about 200/zg of membrane proteins and 2"9 pmol of [aH]retinol-RBP in the incubation mixture) the uptake was about 5 % of total radioactivity. The specificity of [aH]retinol uptake from R B P to isolated R P E plasma membranes was then investigated. Several experiments indicate t h a t the uptake process is not influenced by the presence of different amounts of bovine serum albumin (up to 200 times molar excess with respect to [aH]retinol-RBP). In different experiments, 100-fold molar excess of unlabelled R B P totally inhibited [3H]retinol uptake. Since previous evidence indicated t h a t no uptake is observed by incubating isolated cells in the presence of free retinol (Maraini and Gozzoli, 1975 ; Rask and Peterson, 1976 ; Chi-Ching Chen and Heller, 1977), we have further tested the specificity of the uptake process by using [aH]retinol bound to bovine serum albumin. Under identical conditions as those utilized for [aH]retinol-RBP experiments, uptake of retinol from [aH]retinolbovine serum albumin complex by R P E plasma membranes showed a linear dependence from ligand concentration (up to 4 x 10 -7 M) analogous to t h a t observed by running a blank in the same conditions. On the other hand incubation of aliquots of a cell membrane suspension with increasing amounts of [aH]retinol-RBP did not show this linear dependence of uptake from ligand concentration indicating t h a t the binding of retinol to its receptor is a saturable process (Fig. 3). A plot of the ratio of bound to free ligand versus the concentration of bound ligand is linear, as reported in Fig. 4. By assuming a single binding site on the receptor, we
R E T I N O L U P T A K E IN PIGMENT E P I T H E L I U M
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i ~
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i 2
i 3
i
4
i
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./
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5 [SH]RefinoI-RBP (x 10-8 m)
I
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FIG. 3. Specific [aH]retinol uptake by isolated RPE plasma membranes incubated for 5 rain at 37~ in the presence of increasing amounts of [SH]retinol-RBP (continuous line). Each point represents the mean 4-S.D. of three experiments. Non-specificbinding to the filter was measured at different [SH]retinolRBP concentrations (dotted line). Double reciprocal plot of the data is shown in the inset.
1.5i x
~
I-0
r-1
~= o-5
o
5 I0 ESH]Retinol bound (x I0 -II M)
i
15
Fto. 4. Scatchard plot of [aH]retinol equilibrium binding data to RPE plasma membranes, Isolated membranes (50 #g of membrane proteins) were incubated for 5 rain at 37~ in the presence of different amounts of [SH]retinol-RBP. The line represents a linear least-squares fit to the data. e s t i m a t e a c o n c e n t r a t i o n of receptor molecules of 1"5 • 10 -1~ M in a n assay m i x t u r e c o n t a i n i n g 50/~g m e m b r a n e p r o t e i n s / m l . F r o m the same plot we can calculate a dissociation c o n s t a n t of the order of 5 x 10 -9. Since the molecular weight of the retinol receptor, as shown b y SDS gel electrophoresis, appears to be 16000, the receptor p r o t e i n appears to represent a b o u t 0"005 % of t o t a l p r o t e i n s of R P E p l a s m a m e m b r a n e s . Since this figure is well below the s e n s i t i v i t y of p r o t e i n d e t e c t i o n in p o l y a c r y l a m i d e gel electrophoresis, the presence of a Coomassie blue s t a i n e d b a n d c o - m i g r a t i n g with the r a d i o a c t i v i t y peak in the m e m b r a n e e l e c t r o p h e r o g r a m could be e x p l a i n e d b y the existence of non-receptor proteins of similar molecular weight.
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S, OTTONELLO AND G. MARAINI 4. D i s c u s s i o n
I n the previous section we have shown t h a t the radioactive retinol is recovered bound to isolated R P E plasma membranes after incubation in the presence of [aH]retinol-RBP, thus extending previous findings obtained on isolated cells (Maraini et al., 1977). In agreement with the observations of Rask and Peterson (1976) on intestinal mucosal cells, the uptake process does not seem to depend on the existence of a functioning cell metabolism. The binding process is saturable and is inhibited by an excess of cold ligand. This indicates t h a t the observed phenomenon is a specific binding process. After SDS polyacrylamide gel electrophoresis the membrane bound retinol is recovered in a position corresponding to a molecular weight of 16000; the migration distance of this protein can be unambiguosly separated from t h a t of holo-RBP and of free [aH]retinol ran in the presence of R P E plasma membranes. These observations indicate t h a t the membrane bound radioactive retinol we are actually measuring is no more associated with the plasma carrier and is not free retinol which was removed from its plasma R B P and had independently migrated in the gel. Previous d a t a (Maraini et al., 1977) obtained using [12SI]RBP support the conclusion on the existence of a retinol-binding protein in R P E plasma membranes. The fact t h a t the a m o u n t of membrane-bound radioactivity does not decrease after incubation of the labelled membranes in the presence of excess cold holo-RBP before washing, indicates t h a t the receptor-bound [3H]retinol does not exchange with R B P - b o u n d retinol. The dissociation constant for the binding process in our experimental conditions is lower than t h a t of 2 x 10 -~ reported for retinol R B P interaction (Cogan, Kopelman, Mokady and Shinitzky, 1976), but is considerably higher than t h a t of 10 -12 found by Heller (1975) studying the interaction of plasma R B P with its membrane receptor. This further supports the conclusion t h a t we are investigating a different process. These data, together with the fact t h a t plasma R B P does not cross the cell m e m b r a n e (Heller, 1975; R a s k and Peterson, 1976), suggest the existence of an intermediate step at the membrane level in the transfer of retinol from its plasma carrier into R P E cells. This m a y be necessary to prevent both oxidative degradation of free retinol and its toxic effects on biological membranes. Intraeellular retinol-binding proteins have been identified in R P E cells (Wiggert, Bergsma and Chader, 1976; Saari, Bunt, F u t t e r m a n and Berman, 1978). As retinolbinding proteins from other tissues (Ong and Chytil, 1978), the soluble retinol receptor of R P E cells has a molecular weight of about 17000 and a dissociation constant for retinol of the order of 10 -8. These figures are very similar to those which we have Obtained for the membrane retinol receptor. The possibility therefore exists t h a t these two receptors are indeed the same protein which could be functioning in two different states (membrane bound a n d / o r soluble). The ability of isolated membranes to specifically take up retinol from its plasma carrier seems to rule out the possibility t h a t the existence of a retinol receptor functioning in the plasma membranes of R P E is simply due to cytosol contamination. Preliminary experiments carried out in this laboratory indicate t h a t by incubating [SH]retinol-labelled membranes with cold R P E cytosol, radioactivity can be specifically transferred to the soluble phase. With further experiments it will perhaps be possible to ascertain whether the radioactivity transfer which we observe represents the exchange of membrane-bound vitamin A or the exchange of the retinol-receptor complex.
RETINOL UPTAKE IN PIGMENT EPITHELIUM
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ACKNOWLEDGMENTS This work was supported by grants from the Ministero della Pubblica Istruzione and the Consiglio Nazionale delle Ricerche, Roma. We thank Drs G. L. Rossi, E. Bignetti and A. Merli for critically reading and discussing the manuscript. REFERENCES Berman, E. R. and Feeney, L. (1976). Clean start for retinal pigment epithelium. Invest. Ophthalmol. 15, 238-40. Chi-Ching Chen and Heller, J. (1977). Uptake of retinot and retinoic acid from serum retinol-binding protein by retinal pigment epithelial cells. J. Biol. Chem. 252, 5216-21. Cogan, U., Koperlman, M., Mokady, S. and Shinitzky, M. (1976). Binding affinities of retinol and related compounds to retinol-binding proteins. Eur. J. Biochem. 65, 71-8. Heller, J. (1975). Interaction of plasma retinol-binding protein with its receptor. J. Biol. Chem. 250, 3613-19. Heller, J. and Horwitz, J. (1973). Conformational changes following interaction between retinol isomers and human retinol-binding protein and between retinol-binding protein and prealbumin. J. Biol. Chem. 248, 6308-16. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of phage T4. Nature (London) 227, 680-85. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951 ). Protein measurements with the folin phenol reagent. J. Biol. Chem. 193, 265-75. Maraini, G. and Gozzoli, F. (1975). Binding of retinol to isolated retinal pigment epithelium in the presence and absence of retinol-binding protein. Invest. Ophthalmol. 14, 785-7. Maraini, G., Ottonello, S., Gozzoli, F. and Merli, A. (1977). Identification of a membrane protein binding the retinol in retinal pigment epithelium. Nature (London) 265, 68-9. Ong, D. E. and Chytil, F. (1978). Cellular retinol-binding protein from rat liver. J. Biol. Chem. 253, 828-32. Peterson, P. A. and Bergggtrd, I . J . (1971). Isolation and properties of a human retinoltransporting protein. J. Biol. Chem. 246, 25-33. Rask, L. and Peterson, P. A. (1976). In vitro uptake of vitamin A from the retinol:binding plasma protein to mucosal epithelial cells from the monkey's small intestine. J. Biol. Chem. 251, 6360-66. Saari, J. C., Bunt, A. M., Futterman, S. and Berman, E. R. (1977). Localization of cellular retinol-binding protein in bovine retina and retinal pigment epithelium, with a consideration of the pigment epithelium isolation technique. Invest. Ophthalmol. Vis. Sci. 16, 797-806. Vahlquist, A., Nilsson, S. F. and Peterson, P. A. (1971). Isolation of the human retinol-binding protein by affinity chromatography. Eur. J. Biochem. 20, 160-68. Weber, K. and Osborn, M. (1969}. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 4406-12. Wiggert, B. O., Bergsma, D. R. and Chader, G. J. (1976). Retinol receptors of the retina and pigment epithelium: further characterization and species variation. Exp. Eye Res. 22, 411-18.
Retinol Uptake by Isolated Retinal Pigment Epithelium Plasma Membranes SIMONE OTTONELLO A:ND G I O V A N N I M A R A I ~ I
Institute of Molecular Biology and Institute of Ophthalmology, University of Parma, 43100 Parma, Italy (Received 19 February 1980, New York) The uptake of retinol has been investigated by incubating suspensions of isolated bovine retinal pigment epithelium plasma membranes in the presence of [aH]retinol-retinol-binding protein complex. After SDS gel electrophoresis of the labelled membranes, [aH]retinol is found to be associated with a protein band of molecular weight 16000. The binding process is saturable, specific and shows a linear dependence on the membrane concentration in the incubation mixture. A dissociation constant of the order of l0 -9 has been calculated for the retinol-receptor interaction. These observations confirm previous data obtained with isolated pigment epithelium cells and suggest the existence of an intermediate step, at the membrane level, in the transfer of retinol from its plasma carrier to the cell cytoplasm. Key words: retinol; retinol-binding protein ; retinal pigment epithelium ; plasma membranes; retinol receptor; intracellular retinol-binding protein.
1. I n t r o d u c t i o n R e t i n o l is of p r i m a r y i m p o r t a n c e Jn t h e m e t a b o l i s m of t h e eye where t h e a l d e h y d e of v i t a m i n A c o n s t i t u t e s t h e c h r o m o p h o r e of visual p i g m e n t molecules. A v a i l a b l e evidence i n d i c a t e s in t h e r e t i n a l p i g m e n t e p i t h e l i u m ( R P E ) t h e m o n o l a y e r of cells c o n s t i t u t i n g t h e b l o o d - r e t i n a l b a r r i e r b e t w e e n t h e c h o r o i d a l e x t r a v a s c u l a r space a n d t h e n e u r a l retina. R P E cells are i n v o l v e d in t h e u p t a k e o f r e t i n o l from b l o o d a n d in t h e t r a n s f e r of this molecule (or of its d e r i v a t i v e s ) to a n d from t h e p h o t o r e c e p t o r cells. V i t a m i n A circulates in t h e blood b o u n d to a specific c a r r i e r p l a s m a p r o t e i n , t h e r e t i n o l - b i n d i n g p r o t e i n ( R B P ) , a n d t h e u p t a k e o f retinol b y R P E occurs t h r o u g h t h e i n t e r a c t i o n o f R B P p r e s e n t in the choroid e x t r a v a s c u l a r fluid w i t h a specific r e c e p t o r on R P E p l a s m a m e m b r a n e s (Heller, 1975; M a r a i n i a n d Gozzoli, 1975). D u r i n g t h e i n t e r a c t i o n o f R B P w i t h its receptor, r e t i n o l is released from its c a r r i e r to some o t h e r c o m p o n e n t of t h e cell. R B P does n o t cross t h e p l a s m a m e m b r a n e a n d is r e m o v e d from its b i n d i n g place b y c o m p e t i t i v e d i s p l a c e m e n t b y o t h e r R B P molecules (Chi-Ching Chen a n d Heller, 1977). A f t e r i n c u b a t i o n of i s o l a t e d R P E cells in t h e presence o f [ a H ] r e t i n o l - R B P , Maraini, Ottonello, Gozzoli a n d Merli (1977) f o u n d t h a t in t h e p l a s m a m e m b r a n e s of t h e cells r a d i o a c t i v e retinol was a s s o c i a t e d w i t h a m e m b r a n e p r o t e i n o f m o l e c u l a r w e i g h t 14500. This p r o t e i n was different from exogenous R B P a d d e d to t h e cell suspension a n d was identified as being a n o r m a l c o n s t i t u e n t of R P E p l a s m a m e m b r a n e s showing retinol-binding properties. To c h a r a c t e r i z e f u r t h e r t h e i n t e r a c t i o n b e t w e e n r e t i n o l a n d its m e m b r a n e r e c e p t o r we t o o k a d v a n t a g e of the a v a i l a b i l i t y o f an e x p e r i m e n t a l s y s t e m in which t h e process of retinol t r a n s p o r t could n o t p r o g r e s s b e y o n d t h e m e m b r a n e level. W e therefore s t u d i e d t h e t r a n s f e r o f r e t i n o l from its carrier p l a s m a p r o t e i n to t h e m e m b r a n e b i n d i n g 9 1980 Academic Press Inc. (London) Limited
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p r o t e i n in suspensions of i s o l a t e d R P E p l a s m a m e m b r a n e s . T h e results of such a s t u d y are r e p o r t e d in t h e p r e s e n t p a p e r .
2. M a t e r i a l s a n d M e t h o d s
Preparation of pigment epithelial cells R P E cells were isolated from freshly obtained bovine eyes under normal room illumination according to the procedure described by Berman and Feeney (1976). After isolation the cells were washed three times with 0"32 M-sucrose. Examination of R P E preparations by light microscopy after the final washing demonstrated t h a t contamination by rod outer segments, choroidal remnants or red blood cells was negligible.
Isolation of plasma membranes Plasma membranes were isolated by fractionating the R P E homogenate on a discontinuous sucrose graxiient as previously described (Maraini et al., 1977). After the last washing R P E was resuspended in Calcium free Krebs-Ringer solution (pH 7"4) containing 10 -3 M-EDTA, homogenized in a tight fitting Teflon-glass homogenizer at 4~ and layered on top of a discontinuous sucrose gradient. The following sucrose solutions (containing l0 -3 M-EDTA) were layered in the centrifuge tube from bottom to top : 44, 40, 36, 33, 15 %. Centrifugation time was 150 min at 27000 r / m i n in a SW 40 rotor with an L5 Spinco ultracentrifuge. Plasma membranes were collected at the interface between 15 and 33% sucrose, diluted with Krebs-Ringer and peUetted at 20000 r / m i n for 30 min. The purity of the preparation was controlled by electron microscopy of fixed samples.
[aH]Retinol uptake assay In a typical experiment plasma membranes (0"1~)-3 mg of proteins) were suspended in a calcium-free Krebs-Ringer solution. The suspension was then forced several times through a 22-gauge syringe needle and incubated at 37~ in the presence of varying amounts of [SH]retinol-RBP (10 -l~ to 10-~1 tool in 5t)-500 #l containing 5000-50000 ct/min) in a total volume of 1 ml. At various times aliquots of the incubation mixture were filtered on Millipore filters (GS-0"22 #m). Filters were then washed with a large volume (usually 10 ml) of buffer solution, air-dried, placed in scintillation vials, dissolved in Instagel and counted in a P a c k a r d Tricarb scintillation spectrometer. The background due to [3H]retinol aspecifically bound to the filter was evaluated in each experiment by running a blank in the same conditions and using this value for correction.
Electrophoresis of the labelled membranes In some experiments after incubation the membranes were diluted with Krebs-Ringer, sedimented by centrifuging for 30 min at 20000 r/rain, dissolved in 3 % SDS, 1% mercaptoethanol, 30 mM-Tris-HC1 (pH 8"9)) and subjected to eleetrophoresis on 8'5 % polyaerylamide gels according to Weber and Osborn (1969). In some control experiments the electrophoretic separation was also performed in a Tris-glycine buffer system according to Laemmli (1970). The results obtained with the two systems were superimposable. The gels were calibrated for molecular weight determinations using serum albumin (mol.wt 68000), human R B P (mol.wt 21000), Soybean trypsin inhibitor (mol.wt 20000), myoglobin (mol.wt 17000) and cytochrome c (mol.wt 12000). For radioactivity determinations the gels were sliced, dissolved in H,O, at 45~ overnight, resuspended in Instagel and counted as described above.
Other procedures Human R B P was isolated from plasma according to Peterson and Bergg~rd (1971) and purified by affinity chromatography on prealbumin coupled Sepharose 4B according to Vahlquist, Nilsson and Peterson (1971). [3H]Retinol-RBP, prepared according to Heller and Horwitz (1973), was stored in plastic vials at - 2 0 ~ and filtered on Millipore GS-0"22 # m filters immediately before use. [3H]Retinol-bovine serum albumin complex was prepared as detailed by Chi-Ching Chen and Heller (1977). Proteins were determined by the method of Lowry, Rosebrough, F a r r and Randall (1951) or, in the case of pure proteins, by measuring the absorbance at 280 nm.
R E T I N O L U P T A K E IN P I G M E N T E P I T H E L I U M
71
Chemicals
Proteins used as electrophoretic standards were purchased from Boehringer. [3H]Retinol was from NEN chemicals. All other reagents were of analytical grade (Merck). 3. R e s u l t s
When isolated plasma membranes of R P E cells were incubated, under the above described experimental conditions in the presence of [3H]retinol-RBP, a rapid incorporation of radioactivity in the membrane fraction was observed. This membranebound radioactivity cannot be removed by a second incubation (over a period of 10 min) of the plasma membranes in the presence of a 200-fold molar excess of unlabeled R B P before filtering. When the labelled membranes were subjected to SDS polyacrylamide gel electrophoresis and the gels were tested for radioactivity distribution, 90 % of the total gel radioactivity was found to be associated with a protein band of mol.wt 16000. This is in good agreement with the results previously obtained by incubating isolated R P E cells in the presence of [3H]retinol-RBP (Maraini et al., 1977). The mobility of this radioactivity containing band in membrane SDS gel electrophoresis was clearly higher than that of holo-RBP added to the membrane pellet immediately before detergent solubilization (Fig. 1). Control SDS gel electrophoresis of equivalent amounts of free [3H]retino] showed that the radioactivity peak had an electrophoretic mobility clearly higher than that of the tracking dye. The same was true with both phosphate and glycine buffer electrophoretic systems and
2-0[ +
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g
m
2000 c
I000
o.ob\/.
-
. . . .
]
Relative mobility FIO. 1. Staining ( ) and labelling ( Q - - Q ) profiles of SDS gel eleetrophoresis of proteins from RPE plasma membranes pre-incubated with [aH]retinol-RBP. Arrows indicate the mobility of proteins of known molecular weight: bovine serum albumin (mol.wt 68000), human holo-RBP (mol.wt 21000), myoglobin (mol.wt 17000), cytochrome c (mol.wt 12000).
72
S. O T T O N E L L O
A N D G. M A R A I N I
F 7" o
~4
P, i
0
I
I
I
....
J - -
I00 200 Membrene profeins (fig)
FIG. 2. [3H]Retinol uptake as a function of R P E plasma membrane concentration in the presence of a constant a m o u n t of [3H]retinol-RBP (3 pmol) in the incubation mixture. Incubation time was 5 min at 37~
also when [SH]retinol was added to unlabeled R P E plasma membranes dissolved in SDS before the electrophoresis, I n both cases the radioactivity peak had thus migrated much faster than control protein standards of 17000 daltons. The kinetics of the uptake process was investigated by incubating isolated R P E plasma membranes in the presence of [3H]retinol-RBP and withdrawing, filtering and washing aliquots of the incubation mixture at different time intervals. The uptake is a very rapid process and is virtually complete within 2 min. Its time course is superimposable to t h a t obtained by studying the uptake of retinol by isolated R P E cells (Maraini and Gozzoli, 1975; Chi-Ching Chen and Heller, 1977). As shown in Fig. 2 the uptake rate shows a linear dependence on the membrane concentration in the incubation mixture. In the presence of the usual amounts of membranes and [aH]retinol-RBP (about 200/zg of membrane proteins and 2"9 pmol of [aH]retinol-RBP in the incubation mixture) the uptake was about 5 % of total radioactivity. The specificity of [aH]retinol uptake from R B P to isolated R P E plasma membranes was then investigated. Several experiments indicate t h a t the uptake process is not influenced by the presence of different amounts of bovine serum albumin (up to 200 times molar excess with respect to [aH]retinol-RBP). In different experiments, 100-fold molar excess of unlabelled R B P totally inhibited [3H]retinol uptake. Since previous evidence indicated t h a t no uptake is observed by incubating isolated cells in the presence of free retinol (Maraini and Gozzoli, 1975 ; Rask and Peterson, 1976 ; Chi-Ching Chen and Heller, 1977), we have further tested the specificity of the uptake process by using [aH]retinol bound to bovine serum albumin. Under identical conditions as those utilized for [aH]retinol-RBP experiments, uptake of retinol from [aH]retinolbovine serum albumin complex by R P E plasma membranes showed a linear dependence from ligand concentration (up to 4 x 10 -7 M) analogous to t h a t observed by running a blank in the same conditions. On the other hand incubation of aliquots of a cell membrane suspension with increasing amounts of [aH]retinol-RBP did not show this linear dependence of uptake from ligand concentration indicating t h a t the binding of retinol to its receptor is a saturable process (Fig. 3). A plot of the ratio of bound to free ligand versus the concentration of bound ligand is linear, as reported in Fig. 4. By assuming a single binding site on the receptor, we
R E T I N O L U P T A K E IN PIGMENT E P I T H E L I U M
73
0.5
v
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o
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/
i ~
,a- /
I
i 2
i 3
i
4
i
/
0
./
l I
__
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5 [SH]RefinoI-RBP (x 10-8 m)
I
4
FIG. 3. Specific [aH]retinol uptake by isolated RPE plasma membranes incubated for 5 rain at 37~ in the presence of increasing amounts of [SH]retinol-RBP (continuous line). Each point represents the mean 4-S.D. of three experiments. Non-specificbinding to the filter was measured at different [SH]retinolRBP concentrations (dotted line). Double reciprocal plot of the data is shown in the inset.
1.5i x
~
I-0
r-1
~= o-5
o
5 I0 ESH]Retinol bound (x I0 -II M)
i
15
Fto. 4. Scatchard plot of [aH]retinol equilibrium binding data to RPE plasma membranes, Isolated membranes (50 #g of membrane proteins) were incubated for 5 rain at 37~ in the presence of different amounts of [SH]retinol-RBP. The line represents a linear least-squares fit to the data. e s t i m a t e a c o n c e n t r a t i o n of receptor molecules of 1"5 • 10 -1~ M in a n assay m i x t u r e c o n t a i n i n g 50/~g m e m b r a n e p r o t e i n s / m l . F r o m the same plot we can calculate a dissociation c o n s t a n t of the order of 5 x 10 -9. Since the molecular weight of the retinol receptor, as shown b y SDS gel electrophoresis, appears to be 16000, the receptor p r o t e i n appears to represent a b o u t 0"005 % of t o t a l p r o t e i n s of R P E p l a s m a m e m b r a n e s . Since this figure is well below the s e n s i t i v i t y of p r o t e i n d e t e c t i o n in p o l y a c r y l a m i d e gel electrophoresis, the presence of a Coomassie blue s t a i n e d b a n d c o - m i g r a t i n g with the r a d i o a c t i v i t y peak in the m e m b r a n e e l e c t r o p h e r o g r a m could be e x p l a i n e d b y the existence of non-receptor proteins of similar molecular weight.
74
S, OTTONELLO AND G. MARAINI 4. D i s c u s s i o n
I n the previous section we have shown t h a t the radioactive retinol is recovered bound to isolated R P E plasma membranes after incubation in the presence of [aH]retinol-RBP, thus extending previous findings obtained on isolated cells (Maraini et al., 1977). In agreement with the observations of Rask and Peterson (1976) on intestinal mucosal cells, the uptake process does not seem to depend on the existence of a functioning cell metabolism. The binding process is saturable and is inhibited by an excess of cold ligand. This indicates t h a t the observed phenomenon is a specific binding process. After SDS polyacrylamide gel electrophoresis the membrane bound retinol is recovered in a position corresponding to a molecular weight of 16000; the migration distance of this protein can be unambiguosly separated from t h a t of holo-RBP and of free [aH]retinol ran in the presence of R P E plasma membranes. These observations indicate t h a t the membrane bound radioactive retinol we are actually measuring is no more associated with the plasma carrier and is not free retinol which was removed from its plasma R B P and had independently migrated in the gel. Previous d a t a (Maraini et al., 1977) obtained using [12SI]RBP support the conclusion on the existence of a retinol-binding protein in R P E plasma membranes. The fact t h a t the a m o u n t of membrane-bound radioactivity does not decrease after incubation of the labelled membranes in the presence of excess cold holo-RBP before washing, indicates t h a t the receptor-bound [3H]retinol does not exchange with R B P - b o u n d retinol. The dissociation constant for the binding process in our experimental conditions is lower than t h a t of 2 x 10 -~ reported for retinol R B P interaction (Cogan, Kopelman, Mokady and Shinitzky, 1976), but is considerably higher than t h a t of 10 -12 found by Heller (1975) studying the interaction of plasma R B P with its membrane receptor. This further supports the conclusion t h a t we are investigating a different process. These data, together with the fact t h a t plasma R B P does not cross the cell m e m b r a n e (Heller, 1975; R a s k and Peterson, 1976), suggest the existence of an intermediate step at the membrane level in the transfer of retinol from its plasma carrier into R P E cells. This m a y be necessary to prevent both oxidative degradation of free retinol and its toxic effects on biological membranes. Intraeellular retinol-binding proteins have been identified in R P E cells (Wiggert, Bergsma and Chader, 1976; Saari, Bunt, F u t t e r m a n and Berman, 1978). As retinolbinding proteins from other tissues (Ong and Chytil, 1978), the soluble retinol receptor of R P E cells has a molecular weight of about 17000 and a dissociation constant for retinol of the order of 10 -8. These figures are very similar to those which we have Obtained for the membrane retinol receptor. The possibility therefore exists t h a t these two receptors are indeed the same protein which could be functioning in two different states (membrane bound a n d / o r soluble). The ability of isolated membranes to specifically take up retinol from its plasma carrier seems to rule out the possibility t h a t the existence of a retinol receptor functioning in the plasma membranes of R P E is simply due to cytosol contamination. Preliminary experiments carried out in this laboratory indicate t h a t by incubating [SH]retinol-labelled membranes with cold R P E cytosol, radioactivity can be specifically transferred to the soluble phase. With further experiments it will perhaps be possible to ascertain whether the radioactivity transfer which we observe represents the exchange of membrane-bound vitamin A or the exchange of the retinol-receptor complex.
RETINOL UPTAKE IN PIGMENT EPITHELIUM
75
ACKNOWLEDGMENTS This work was supported by grants from the Ministero della Pubblica Istruzione and the Consiglio Nazionale delle Ricerche, Roma. We thank Drs G. L. Rossi, E. Bignetti and A. Merli for critically reading and discussing the manuscript. REFERENCES Berman, E. R. and Feeney, L. (1976). Clean start for retinal pigment epithelium. Invest. Ophthalmol. 15, 238-40. Chi-Ching Chen and Heller, J. (1977). Uptake of retinot and retinoic acid from serum retinol-binding protein by retinal pigment epithelial cells. J. Biol. Chem. 252, 5216-21. Cogan, U., Koperlman, M., Mokady, S. and Shinitzky, M. (1976). Binding affinities of retinol and related compounds to retinol-binding proteins. Eur. J. Biochem. 65, 71-8. Heller, J. (1975). Interaction of plasma retinol-binding protein with its receptor. J. Biol. Chem. 250, 3613-19. Heller, J. and Horwitz, J. (1973). Conformational changes following interaction between retinol isomers and human retinol-binding protein and between retinol-binding protein and prealbumin. J. Biol. Chem. 248, 6308-16. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of phage T4. Nature (London) 227, 680-85. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951 ). Protein measurements with the folin phenol reagent. J. Biol. Chem. 193, 265-75. Maraini, G. and Gozzoli, F. (1975). Binding of retinol to isolated retinal pigment epithelium in the presence and absence of retinol-binding protein. Invest. Ophthalmol. 14, 785-7. Maraini, G., Ottonello, S., Gozzoli, F. and Merli, A. (1977). Identification of a membrane protein binding the retinol in retinal pigment epithelium. Nature (London) 265, 68-9. Ong, D. E. and Chytil, F. (1978). Cellular retinol-binding protein from rat liver. J. Biol. Chem. 253, 828-32. Peterson, P. A. and Bergggtrd, I . J . (1971). Isolation and properties of a human retinoltransporting protein. J. Biol. Chem. 246, 25-33. Rask, L. and Peterson, P. A. (1976). In vitro uptake of vitamin A from the retinol:binding plasma protein to mucosal epithelial cells from the monkey's small intestine. J. Biol. Chem. 251, 6360-66. Saari, J. C., Bunt, A. M., Futterman, S. and Berman, E. R. (1977). Localization of cellular retinol-binding protein in bovine retina and retinal pigment epithelium, with a consideration of the pigment epithelium isolation technique. Invest. Ophthalmol. Vis. Sci. 16, 797-806. Vahlquist, A., Nilsson, S. F. and Peterson, P. A. (1971). Isolation of the human retinol-binding protein by affinity chromatography. Eur. J. Biochem. 20, 160-68. Weber, K. and Osborn, M. (1969}. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 4406-12. Wiggert, B. O., Bergsma, D. R. and Chader, G. J. (1976). Retinol receptors of the retina and pigment epithelium: further characterization and species variation. Exp. Eye Res. 22, 411-18.