Uptake, processing and release of retinoids by cultured human retinal pigment epithelium

Exp.

Eye Res. (1990)

Uptake, JOHN

51, 717-728

Processing G. FLANNERY”t,

and Release of Retinoids Retinal Pigment Epithelium WILLIAM

by Cultured

O’DAY”“, BRUCE A. PFEFFER*, DEAN BOK””

JOSEPH

Human HORWITZ”

AND

aJules Stein Eye Institute UCLA School of Medicine, b Laboratory of Mechanisms of Ocular Diseases, Section on Pathophysiology, National Eye Institute, National Institutes of Health, Bethesda, MD, and c Department of Anatomy and Cell Biology, UCLA School of Medicine, Los Angeles, CA 90024, U.S.A. (Received

2 March

1990 and accepted

in revised form 23 May 1990)

Upon absorption of a photon, the 11-cisretinaldehyde chromophoreof rhodopsinis isomerizedand reduced to all-trans retinol (vitamin A) in the photoreceptor outer segments. whereupon it leaves the photoreceptors, and moves to the retinal pigment epithelium (RPE). To clarify the function of the RPE in the regeneration of 11 -cis retinaldehyde, we delivered all-trans retinol to monolayer cultures of human RPE. During delivery the retinol was associated with its putative natural carrier, interphotoreceptor retinoid binding protein (IRBP). IRBP has been proposed as a carrier protein involved in the exchange of retinoids between the photoreceptors and the retinal pigment epithelium. The retinoid composition of RPE cells and culture medium was analyzed by HPLC following several incubation periods. The RPE monolayer was found to process all-trans retinol into two distinct end-products: all-trans retinyl palmitate, which remained within the RPE monolayer; and 1 I-cis retinaldehyde which was released into the culture medium. These results demonstrate retinoid isomerase. retinol oxidoreductase and retinyl ester synthetase activity in human RPE cells cultured under the appropriate conditions. They show that IRBP can serve as a carrier of retinol through an aqueous medium to the RPE, and they illustrate that the visual cycle can be studied in vitro. Keg words: retinal pigment epithelium; visual cycle: retinol binding protein ; IRBP; tissue culture : retinoids : vitamin A.

1. Introduction The initial reaction of the visual cycle within the photoreceptor cell has been established for over 30 years: the isomerization of the 11-cis retinaldehyde chromophore of rhodopsin to all-trans retinaldehyde by light (Hubbard and Wald, 1952). The regeneration of 11-cis retinaldehyde required for the regeneration of rhodopsin does not occur within the photoreceptor cell, however. Instead, upon bleaching of rhodopsin to opsin and all-truns retinaldehyde, and reduction of the retinaldehyde to all-trans retinol, retinol leaves the photoreceptors (Dowling, 1960 ; Futterman, 19 63 ; Bridges, 19 76) and traverses the interphotoreceptor spaceto the retina1 pigment epithelium (RPE) where it is stored as the retinyl ester (Krinsky, 1958 ; Dowling, 1960; Hubbard and Dowling, 1962; Bridges, 1976). The course by which retinoids progress from the photoreceptors to the retinal pigment epithelium and back again in the regeneration of rhodopsin remains a fundamental question in the elucidation of the visual cycle. The importance of the RPE in the regeneration * This work was presented Meeting of the Association

in a preliminary form at the Annual for Research in Vision and Ophthalmology. May 1988 (Flannery et al.. 1988. Invest. Ophthalmol. Vis. Sci. 29 (ARVO Suppl.). 416). t For correspondence at: Jules Stein Eye Institute, 100 Stein Plaza. Los Angeles. CA 90024. U.S.A.

00144835/90/120717+12

$03.00/O

of visual pigment was demonstrated initially by Ewald and Kiihne in 1877, when neurosensory retina in contact with the RPE regenerated visual pigment, whereas retina separated from the RPE did not. More recently, the concentration of retinol within the interphotoreceptor matrix has been shown to rise following light adaptation (Saari et al., 1985), providing further evidence of a migration of retinol from the photoreceptors to the RPE. Finally, a cell fraction possessingall-trans retinol isomerizing activity, capable of restoring all-trans retinol to the 11-cis configuration, has been isolated from the RPE of frogs and cattle (Bernstein, Law and Rando, 1987; Fulton and Rando, 1987). Interphotoreceptor retinoid binding protein (IRBP), a glycoprotein found in the extracellular space surrounded by the photoreceptors, RPE and Miiller cells, has been proposed as the carrier protein involved in the transfer of retinoids between these cells (Adler and Severin, 1981; Liou et al., 1982 ; Lai et al., 1982). Recently, several studies (Rando and Bangerter, 1982 ; Fex and Johannesson, 1987, 1988: Ho et al., 1989) have demonstrated that retinoids can be spontaneously transferred between membranes through the aqueous phase, and that intercellular retinoid transfer may not require the participation of specific transport proteins. However, binding proteins such as 0 1990 Academic

Press Limited

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J. G. FLANNERY

IRBP may serve to direct and facilitate this movement, and protect the retinoids during transfer. The primary evidence for the role of IRBP in retinoid transfer, to date. has been circumstantial, namely, the restriction of this protein to the interphotoreceptor space (Adler and Martin, 1982 ; Pfeffer et al., 198 3 ; Bunt-Milam and Saari, 1983), the detection of endogenous retinoids bound to the protein upon its isolation, and light/dark differences in recovered retinoids bound to the protein (Wiggert et al., 1979 ; Adler and Martin, 1982: Liou et al., 1982; Saari et al., 1985 ; Adler, Evans and Stafford, 198 5 : Livrea et al., 1987). The assumption that a carrier is required for retinoids to traverse the extracellular space is based upon the chemical properties of the retinoids, primarily their limited solubility in aqueous solution, and their susceptibility to oxidative damage, rather than on evidence for a functional retinoid binding protein. In this report, we investigated the ability of cultured RPE cells to internalize all-truns retinol delivered by IRBP and to incorporate this retinoid in the reactions of the visual cycle. Cultured RPE, although a model system with excellent potential for the study of retinoid processing by a specific cell type, has not been used extensively for this purpose.

2. Materials

and Methods

isolation and Culture of RPE Cells Cultures of human RPE cells were established from eyes of fetuses ranging in gestational age from 18 to 24 weeks. Primary cultures were started with explants of RPE obtained by dissecting sheets of RPE from the choroid in Ca*+- and Mg2+-free Hank’s balanced salt solution (HBSS). We used the Konigsberg variant without bicarbonate (Konigsberg, 1963), omitting Ca’+ and Mg*’ when specified. RPE sheets were washed three times in fresh HBSS with sedimentation between washes (100 g for 2 min). Thereafter, they were transferred to loo-mm culture dishes containing the low Ca”+ growth medium outlined in Table I. Reducing the Ca2+ concentration has been shown to promote the disassembly of epithelial junctions (Martinez-Palomo et al., 1980; Fujimoto and Ogawa, 1982) and cell substratum adhesion (Takeichi and Okada, 19 72). The cells are released into the medium as proliferation continues at confluence, following which they could be concentrated by centrifugation (100 g for 2 min) and used to initiate subcultures (Pfeffer et al., 1986). These secondary cultures were seeded at a density of 1.2 x lo5 cells crnmZ in ZOO-mm plastic dishes containing the normal [Caz+] growth medium described in Table II. This Ca2+ concentration promoted the formation of cell-to-cell junctional complexes in the developing monolayer. Initially, the serum concentration in the seeding medium was raised to 10% to promote attachment of cells to the substrate. At the first subsequent change of medium, the serum con-

ET AL.

centration was reduced to 1% and maintained at that level thereafter. Because of the low serum concentration in the medium, trace nutrients growth factors and hormones were included to augment serum components that are active in the growth and maintenance of cells in culture (Table II: Pfeffer et al.. 1986). Secondary cultures were maintained until they were confluent, well-melanized monolayers. Primary and passaged cultures were maintained in a tissue culture incubator in a 9 5 y0 air/ 5 y0 CO, atmosphere at 37°C. Culture medium was changed every 3-4 days and cultures were maintained for 2-i months prior to use. The total number of RPE cells was estimated to be 2.4 x 10’ cells per loo-mm dish by counting similar cultures with an ocular micrometer (Mircheff et al., 1990. Preparation of Apo and Holo-IRBP IRBP was purified from the retinas of 100-200 recently enucleated bovine eyes by a modification of the concanavalin A affinity procedures of Adler and Klucznik (1982). IRBP obtained by this procedure was further purified by anion exchange chromatography using a 50 x 5 mm Mono-Q column (Pharmacia. Uppsala, Sweden), and a linear O-O.5 M NaCl gradient containing 10 mM Tris, 2 mM EDTA. 100 mM X-Dmethyl mannoside at pH 7.5. The flow rate was 0.5 ml min-I. Protein peaks were detected by absorbance at 280 nm. SDS-polyacrylamide gel electrophoresis demonstrated a single band with an apparent M, of 140000. The immunologic identity of the IRBP was confirmed by Western blot analysis yielding a single reactive band with affinity purified rabbit antibovine IRBP. Apo-IRBP was prepared by washing the purified protein with KHBSS in a Centricon filtration unit (Amicon Corp., Lexington, MA) to remove residual endogenous retinoids from the protein. HoloIRBP was reconstituted by addition of HPLC-purified all-tmns retinol to the apo-IRBP. All-truns retinol (Sigma, St Louis. MO) was further purified by isolation of the all-truns retinol peak from a preparative scale separation of retinol isomers using the same HPLC methods utilized for analytical separations. All-trans retinol was collected with a fraction collector, evaporated to dryness under nitrogen and redissolved in a minimum volume ( < 20 /II) of ethanol. Analytical and preparative separation of the retinol and retinal isomers was performed with either a 5 ,MM spherical silica LiChrosorb RT-Si-60 column (4.6 x 2 50 mm: Merck, Darmstadt, Germany) or a 8-,HM irregular silica Dynamax column (4.6 x 2 50 mm ; Rainin, Inc.. Emeryville, CA) using a Varian 5500 HPLC. The Dynamax column provided a significantly longer retention of the retinoids than the LiChrosorb column, and was used primarily to maximize the separation of the retinaldehyde isomers and the

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RPE

TABLE I Lo/Ca” medium for initiating human RPE cultures. Medium was CEM 2000 without CaCl, (Scott Laboratories, Fiskeville, RI) with CaCl, added to raise the Ca2+ level to O-05 mM. The following ingredients were added Ingredient 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Amount 1-l

Selenious acid (Na salt) Hydrocortisone Calf serum (heat-inactivated) Linoleic acid (complexed with albumin Insulin (bovine) Transferrin Putrescine-HCl L-glutamine Triiodothyronine Bovine retina extract*

1.75 $ug 10.0 /lg 1.0% (v/v) 84.0 yg 5.0 mg 5.0 mg 0.3 mg 292.0 mg 6.5 ng 1.0% (v/v)

* Bovine retina extract was prepared by placing 12 freshly-dissected bovine retinas in 100 ml of Ca2+ and Mg*‘-free Konigsberg’s-Hank’s balanced salt solution (KHBSS). sonicating briefly at 4°C and sting in the dark for 2 hr at 4°C. The suspension was centrifuged and aliquots of the supematant were stored at - 80°C.

TABLE

II

Normal mediumfor maintenanceof human RPE cultures. The basalmediumwas a 7 : 1 mixture of CEM 2000 (Scott Laboratories)and MEM (Eagle). It contained 1.5-1.8 mM Ca”+from CaCl,. Thefollowing ingredientswere added Ingredient 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Amount lk’

ZnSO,.7H,O CuSO,.5H,O MnCl, .4H,O Seleniousacid (Na salt) Hydrocortisone Calf serum(heat-inactivated) Linoleic acid (complexedwith albumin) Insulin Transferrin Putrescine-HCl L-ascorbicacid L-glutamine Triiodothyronine

14. Bovine retina extract

72.0 ,ug 125.0 ng 50.0 ng 1.75 /lug 10.0,ug 1.0% (v/v) 84.0 pug 5.0 mg 5.0 mg 0.3 mg 22.5 mg 292.0 mg 6.5 ng 0.5% (v/v)

Heat inactivated calf serum was obtained from Hazelton Biologics. Inc. (Lenexa, KS). Selenious acid and putrescine were from Collaborative Research Inc. (Bedford, MA). All other organic reagents (except for bovine retina extract) were from Sigma Chemical Co. (St Louis, MO).

internal standard (Fig. 5). The absorbance was monitored at 340 nm. Retinoids were separated by isocratic elution in 2 y. dioxane in n-hexane at a flow rate of 1 ml min-‘. Normal phase separations of retinyl esters were made with a LiChrosorb RT-Si-60 column in a solvent consisting of 0.2 y. methyl-tert-butyl ether in hexane at a flow rate of 1.0 ml min-‘. All extraction and purification procedures were carried out under non-isomerizing safelight illumination.

Preparation of lRBP/Retinol SupplementedCulture Media In a typical incubation, monolayers of human RPE were incubated in the dark with culture medium containing 1.38 ,UM IRBP (7 nmol IRBP/5 ml

medium). This is approx. 4% of the IRBP concentration of the human interphotoreceptor matrix, which has been estimated at 3 7 ,UM [5400 rig/l ,~l (Fong et al., 1984)]. IRBP was complexed with 2.77 moles of all-bans retinol per mole of protein by combining 1.0 mg of purified apo-IRBP with 5.4 pg of all-trans retinol (19 nmol) in 1 ml of Eagle’s balanced salt solution (EBSS) without phenol red (GIBCO). To this was added 4 ml of a 1: 1 mixture of CME/Eagle’s MEM resulting in a final retinol concentration of 3.8 ,UM. The absorbance spectra of the IRBP-retinol in EBSSwas rapidly scanned in a spectrophotometer to determine the precise IRBP/retinol ratio (A,,,/A,,,). The maximum spectral ratios (A,,JA,,,) obtained for the binding of all-trans retinol IRBP were 1.0. The osmolarity was measured by a vapor pressure osmometer (Wescor, Inc. Logan, UT), and adjusted to

720

J. G. FLANNERY

FIG. 1. Confluent monolayer

isotonicity with the normal culture medium (325 m0sm) by addition of NaCl if necessary. The quantity of retinol administered to the RPE culture was chosen with reference to estimates of the total rhodopsin content per human eye of 3.94 nmol (Crescitelli and Dartnall, 1953 ; Plantner, Barbour and Kean, 1988), and the volume of the subretinal space of the human eye [0.02 ml (Adler and Severin, 198 l)]. A 100% bleach of the retina could result in a 200 ,UM maximal concentration of retinol in the interphotoreceptor matrix (IPM). However, the loading of retinol associatedwith IRBP in vivo has been estimated to be in the range of 3-30s (Adler and Evans, 1983), suggesting that micromolar concentrations of retinol in the IPM during bleaching represent physiological levels. The typical human eye is estimated to contain 4.2-6.1 x lo6 RPE cells over a surface of approx. 2000 mm2 (Hogan, Alvarado and Weddell, 19 71; Farber et al., 19 8 5 ). Our RPE monolayers cultured in loo-mm dishes contained approx. 3.6-5.2 times as many RPE cells as a single intact eye, ranging from 1.4 x 10’ to 3.2 x lo7 cells covering an area of 5800 mm2. The cell density of these preparations is very similar to that of the in vivo human RPE, in the range of 2500-4000 cells mm-‘. The 3.8 ,UM retinol concentration in the culture medium at the start of an incubation represents approx. 4% of the retinoid recovered from the IPM in vivo, or a bleach of 2% of the rhodopsin in the retina. Extraction of Retinoids All experiments were performed in darkness or dim red Light. All steps of the extraction procedures were

of human

ET AL.

fetal RPE cells. x 660

carried out on ice at 5°C unless noted otherwise. A single loo-mm culture dish of confluent RPE was used for each experiment. Culture medium was removed and the cells were rinsed with three lo-ml washes with HBSS. To analyze for retinoids present in the cells prior to incubation in all-trans retinol-IRBP, a strip of RPE measuring approximately 11 x 83 mm was aseptically removed with a Teflon policeman. The cells were transferred to a polypropylene microcentrifuge tube with 0.5 ml HBSS and stored at - 80 “C until extracted. A 05-ml sample of culture medium with added all-trans retinol-IRBP was also frozen for later extraction. The remainder of the experimental medium (4.5 ml) was then added to the culture dish which was incubated in darkness for l-14 hr. Following incubation, the culture medium was removed and a l.O-ml aliquot was removed and stored at -80°C until extraction. The RPE monolayer was rinsed three times with HBSS (10 ml per rinse). The cells were then removed by scraping and transferred to a microcentrifuge tube containing 0.5 ml HBSS. Quantitative data in this report were derived from samples of RPE and media extracted in parallel according to the method of Suzuki et al. (1986) with modification. Cells and media from additional experiments were extracted according to the method of Bernstein, Law and Rando ( 198 7) yielding qualitatively similar results, but with smaller yields of all retinoid classes. A total of 15 experiments were conducted, of which the last four were used for the quantitative analyses. Modifications in our use of the method of Suzuki et al. (1986) are described below. RPE cells were homogenized in an iced microcentrifuge tube for 2 min using a polypropylene pestle

RETINOID

PROCESSING

BY CULTURED I I-as

HUMAN

721

ratlnaldshyde ali-trons

I

RPE

rstinaidehyde all-tram

retinol

T

0.002

n (A)

Normal-phose HPLC standards

(B)

Ohr

(C)

Ihr

J&k + I I-cis

(D)

au

retinoldthyde

II-cis

retinaldahyde

I I-cis

retinaldshyde

6hr

t (C)

14hr

-t

0

5

IO

15

20

25

30 Time

35

40

45

50

55

60

(min)

FIG. 2. Normal phaseHPLCprofilesof RPE cell extracts. A, Absorbanceprofile showingelution times of verified retinoid standards.B, Control strip of RPEceilsremovedprior to incubation (0 hr) with IRBP-all-trunsretinol, showingno endogenous retinol or retinaldehydeisomers.C, RPEcell monolayerfollowing 1 hr incubation of IRBP-all-trunsretinol. A minor peakof 1lcis retinaldehydeis observed.D, Six-hour incubation with IRBP-all-transretinol. Elution profile isvery similarto that obtained following 1-hr incubation. E, Fourteen-hourincubation with IRBP-all-transretinol. Profile is not significantly different than that obtainedfollowing a 1-hr incubation. Scaleon all HPLCrecords:0.002 aufs (absorbanceunits full scale).

and centrifuged at 600 g for 10 min at 5°C. The supernatant was divided equally between two microcentrifuge tubes and 250 ,~l of 6 M formaldehyde in phosphate buffer, pH 7.5, was added to each. The tubes were incubated for 2 min at room temperature and 300 ,~l dichloromethane was then added and shaken vigorously. The extracts were incubated an additional 10 min and 600 ,ul hexane (hexane-BHT, with butylated hydroxytoluene, 0.1 mg ml-l) was added, the tubes shaken well and centrifuged at 13000 g for 10 min at 5°C. The dichloromethanehexane layer was then transferred to a fresh microcentrifuge tube and evaporated in a stream of dry nitrogen gas. The extracted retinoid was promptly redissolved in 50 ~1 hexane-BHT for HPLC analysis. This procedure provided two identical aliquots, to allow for analysis of retinyl esters (in duplicate), and for isomers of retinol and retinaldehyde (in duplicate). Samples of culture medium were extracted in a similar manner. The extractions were performed in glass-stoppered tubes followed by centrifugation in 15ml Corex tubes. AI1 extractions of RPE and media were performed to provide two identical aliquots in hexane-BHT for HPLC analysis of isomers of retinol, retinaldehyde and retinyl esters. Control experiments

were performed in which the entire population of RPE cells covering a loo-mm culture dish were extracted without prior administration of retinoid (two experiments). Control samples (5 ml) of normal culture medium which had not been used to feed cells were also extracted. Experimental extracts were quantitated by comparing their integrated peak areas and retention times to those obtained from known quantities of verified retinoid standards (Hoffman La-Roche, Nutley, NJ, and Sigma, Inc., St Louis, MO). Retinoid standards were chromatographed immediately prior and subsequent to each experimental injection. In order to control for the possible misidentification of the retinal isomers, due to changes in elution time, an 11-cis retinaldehyde internal standard was added to alternate sample extracts prior to their chromatography. All extracts were analyzed in duplicate by two different HPLC column/solvent systems to separately quantify retinol and retinaldehyde isomers and retinyl esters. The proportion of the retinol which was internalized by the monolayer was determined by comparison of the quantity of retinoids extracted from the control aliquots of media containing IRBP-all-truns retinol with the amount of retinol and retinaldehyde isomers

722

J. G. FLANNERY

(A)

Normal-phase retinyl e;ter standard

(B)

Ohr

(C)

Ihr

(D)

ET

AL.

-

-

6hr

0.016 au

(E)

14hr

-

0 5

IO 15 20

25 30 35 40 45 50 55 60 Time

(min)

FIG. 3. Normal phaseHPLCprofile of retinyl estersextracted from RPEmonolayer. A, Absorbanceprofile showingelution timesof verified retinyl ester standards.B, RPEcontrol strip extracted prior to incubation of monolayerwith IRBP-all-truns retinol complex.A broadpeak at the minimum level of detectionis observedat the elution time of all-trunsretinyl palmitate. C, One-hourincubation with all-tram retinol-IRBP complex.A smallpeakof all-trunsretinyl palmitateis observed.D, Six-hour incubation with all-transretinol-IRBP complex.A prominent peakcorrespondingto all-trans retinyl palmitate is observed at 35 min, and a minor peak of 1l-cis retinyl palmitate at 25 min. E, Fourteen-hour incubation with all-tram retinol-IRBP complex.At eightfold attenuation, major peaksof all-trunsand 11-cis retinyl palmitate are observed.Scalefor HPLCrecords: A-D, 0.002 aufs.Scalefor record: 0.016 aufs.

and retinyl esters recovered from the monolayer and the medium following incubation with the IRBP-alltrans retinol.

3. Results Morphological/Appearanceof Monolayer-s

The appearance of RPE cells used in this study closely resembled the appearance of RPE cells in vivo. Fetal human RPE cells were grown for a minimum of 1 month, by which time they contained many melanosomes and had attained a hexagonal, cobblestone appearance (Fig. 1). They developed a marked

apical and basal morphologic polarity, with prominent apical microvilli, and well-developed basal infoldings as determined by electron microscopy, a configuration characteristic of RPE in vivo. The polarity of the RPE monolayer is invariably established with the basolateral surface in contact with the plastic support, and therefore the culture medium primarily interacts with the apical membrane. In order to model the return of

all-trans retinol from the photoreceptors, we ,restricted the retinol to the apical surface of the epithelial monolayer by using cells attached to an impermeant plastic support and bound the retinol to IRBP, the proposed retinoid carrier protein of the interphotoreceptor space.

RETINOID

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HUMAN

I I-cis f F ; z E

(6)

rstinaldehyde all-irons

retinal

*

B 2 3 .-8 ‘cL

0~0020u

Ohr II-cis

(C)

retinoldehyde

all-trots

,’

723

RPE

retinoldehyde

Ihr 1I-CIS retinoldehyde

(D)

6hr

(E)

14hr

all-irons

ratinol \

h

*

II-cisrstlnaldehyde

0 5 IO

15 20 25

30 35 40 Time

45 50

55 60

(min)

FIG. 4. Normal phaseHPLCrecordsfrom culture mediumextracts. A, Absorbanceprofile showingelution timesof verified retinoid standards.B, Control aliquot (0.5 ml) of culture mediumremovedimmediately(0 hr) following addition of IRBP-alltram retinol. A peak at 55 min correspondingto all-tram retinol is observed.C, Culture medium (5.0 ml) following a I-hr incubation of IRBP-all-transretinol with RPEmonolayer.A small,quantity of 11-cisretinaldehydeiseluted at 6 min. A peak of all-trunsretinol is observedat 55 min. D, Six-hour incubation of IRBP-all-tram retinol with the RPEmonolayer.A prominent peakof 1Ids retinaldehydeis observed,and the peakof all-transretinol is significantly reduced.E, Fourteen-hourincubation of IRBP-all-tram retinol with the RPEmonolayer.A major peakof 11-cisretinaldehydeisobserved.The peakof all-transretinol is no longer evident. Scaleon all HPLCrecords: 0.002 aufs.

Analysis of Retinol and Retinaldehyde Isomers Within RPE Cell Extracts The HPLC profile of the cell extract at 0 hr [Fig. 2(B)] shows that no retinol and retinaldehyde isomers were detectable in the 1 x 10 cm strip of cells which was removed prior to the addition of IRBP-retinol. These cells served as a control for endogenous retinoids within the cultured cells prior to experimental incubations. In control experiments in which the entire population of RPE cells covering a loo-mm culture dish were extracted without prior administration of retinoid (two experiments), endogenous levels of retinol and retinaldehyde were also found to be below the level of detection by our methods. Samples (5 ml) of normal culture medium which had not been used to feed cells were also extracted and found to contain such low levels of retinoids that they were unmeasurable. The low level of endogenous retinoids allowed us to follow retinoid processing without the necessity of using radiolabels to distinguish newly synthesized compounds.

Following 1 hr incubation of IRBP-all-trans retinol with RPE monolayers [Fig. 2(C)], a small peak of ll-

cis retinaldehyde was observed. After 6 and 14 hr of incubation with IRBP-all-trans retinol [Figs 2(D) and (E)] the elution profile was comparable to that obtained following 1 hr incubation, without significant gain or loss of 11-cis retinaldehyde in the RPE. Analysis of Retinyl Esters in RPE Cell Extracts The retinyl ester content of the RPE cell extracts was examined with an alternate HPLC protocol which separates cis and trans retinyl esters. Figure 3(A) shows elution ties of verified retinyl ester standards. Identical to our procedure for examining the retinol and retinal isomers, a control strip of RPE was analyzed prior to incubation of the monolayer with IRBP-alltrans retinol complex in order to establish the baseline level of retinyl esters present in the RPE monolayer [Fig. 3(B)]. In all experiments, this level was at or below our detection threshold. In control experiments in which the entire cell complement of 100~mm culture dishes were extracted without prior administration of retinoid (two experiments), endogenous levels of retinyl esters were also found to be at the minimum level of detection by our methods. A small

724

J. G. FLANNERY

(A)

Retinaldehyde standards

[B)

Apical before

medium incubation

ET AL.

x

II-cis-ret~noldahyda )L

(C)

Aplcal after

medium incubation

y II-cis-ret~naldehyde

c

( D)

I I-cis retinaldehyde standard

II-cis-retinoldehyda I

(C )

Apical after I I-cis

medium incubation retinaldehyde

t -I-IllIll

0

2

4

6

8

IO

I

I

I

I

l

I

I

I2

I4

I6

I8

20

22

24

Time

(min)

FIG. 5. Normal phase HPLC records from culture medium extracts. A, Absorbance profile showing elution times of verified retinoid standards. B, Control allquot (0.5 ml) removed immediately (0 hr) following addition of IRBP-all-truns retinol. C, Culture medium (1.0 ml) following a 6-hr incubation of IRBP-all-tram retinol with RPE monolayer. At 15 min a peak of 1 lcis retinaldehyde is eluted. D, Absorbance profile of authentic 1 I-cis retinaldehyde standard. E, Culture medium (I.0 ml) following a 6-hr incubation of IRBP-all-tram retinol with RPE monolayer, supplemented with internal standard chromatographed in (D) above. Scale on all HPLC records: 0.002 aufs. peak of all-truns retinyl palmitate was observed following 1 hr incubation with all-trans retinol-IRBP complex [Fig. 3(C)]. The amount of all-tram retinyl palmitate increased significantly with 6 hr of incubation, and a small amount of ll-cis retinyl palmitate was also detectable [Fig. 3(D)]. After 14 hr of incubation [Fig. 3(E)] a major peak corresponding to all-tram retinyl palmitate was observed, and the quantity of 1 1-cis retinyl palmitate also increased. The predominant retinoid observed in all cell extracts was the all-truns stereoisomer of retinyl palmitate, with a smaller proportion of 11-ciS retinyl palmitate. The quantity of retinyl ester observed was typically 50-100 times greater than that of retinol or retinaldehyde isomers within the RPE cell extracts at a corresponding incubation period.

Analysis of Retinol and Retinaldehyde lsomers in Culture Media The control aliquot of culture medium (0.5 ml). which was removed before incubation with the RPE monolayer, showed a single peak of all-truns retinol. This suggests that association of the all-tram retinol with the IRBP molecule alone, or with other components of the culture medium did not cause isomerization of the retinol or its oxidation to the aldehyde [Fig. 4(B)]. At the end of each incubation, the entire volume of culture medium (5 ml) was removed and analyzed by HPLC. Extracts of culture medium following a 1-hr incubation of IRBP-all-trans retinol with RPE monolayers disclosed 11-cis and all-truns retinaldehyde to be

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minor components [Fig. 4(C)]. The quantity of 11-cis retinaldehyde was significantly increased following 6 hr of incubation [Fig. 4(D)] with little change in the quantity of all-trans retinaldehyde, and a substantial decreasein the quantity of all-trans retinol. Following a 14-hr incubation [Fig. 4(E)] the 11-cis retinaldehyde was predominant in the medium extract, with a minor peak of all-trans retinaldehyde, and an unmeasurable quantity of all-trans retinol. Useof internal Standardsin ldentijkation of Retinoid homers

no1 in medium inal in medium

Due to the potential problem of misidentification of the geometric isomers of retinaldehyde and retinol in normal phase HPLC separations, we used the internal standard technique routinely in our analysis of retinoid isomers. Figure 5 is an example of our results, using this method. This separation was performed using five sequential sampleinjections with the Rainin column (as described in Materials and Methods) to maximize the separation of the retinaldehyde isomers. Figure S(A) shows the initial separation of a standard mixture of retinaldehyde isomers, and Fig. 5(B) is the absorbance profile of the second injection, a sample of culture medium which was not incubated with RPE cells. All-trans retinol is not seen in this figure, as its elution occurs at a much greater retention time. -For injections 3 and 5, an extract from media following delivery of all-trans retinol-IRBP to RPE cells for a 6-hr incubation period was divided into two equal samples. Figure 5(C) is the absorbance profile obtained from the first half of this media extract. The profile is similar to that obtained in Fig. 4(D) with the exception that the retention times are extended by the use of the Rainin column. Figure 5(D) represents the fourth injection, the elution of an authentic ll-cis retinaldehyde standard. For the final injection, the remaining half of the media sample was combined with a sample of 1 lcis retinaldehyde identical to that used for the fourth injection and chromatographed. Figure 5(E) shows the co-elution of the 11-cis retinaldehyde internal standard with the predominant peak of the media sample, supporting its identification as 1 I-cis retinaldehyde.

Il-cis Time (hr)

retinal in RPE

14

FIG. 6. Processingof ail-tmnsretinol by RPEmonolayers supplementedwith 3.8 pM retinoi and 1.38 pM IRBP as a function of incubation time. The appearance of 11-cis retinaldehydein the culture medium is concomitant with the decreasein all-truns retinol in the medium, and the accumulation of retinyl palmitate within the RPE monolayer. The quantity of 11-cis retinaldehyde within the RPE cell extracts, remained at the baseline level of detection in all samples. Values plotted are means of the last four incubations.

Quantitative Analysis of Retinoid Processingby RPE Seventeen nanomoles of all-trans retinol were added to the medium at the outset of all incubations. The portion of this all-trans retinol recovered from the culture media decreasedrapidly with incubation time [Fig. 6(A)]. Quantitatively examining the last four incubations, the retinyl esters were observed to accumulate steadily in the RPE, reaching a mean of 1.2 nmol at 6 hr and 2.6 nmol after I4 hr of incubation [Fig. 6(B) Table III). The quantity of 11-cis retinaldehyde within the RPE was slightly above the detection threshold in cell extracts from 1-hr incubations (0.011 nmol), and remained at very low levels at all times, in all of the incubations [Fig. 6(D)]. The amount of 1 1-cis retinaldehyde which was recovered from the apical medium increased from a mean of 0.15 nmol at 1 hr, to 0.2 nmol at 6 hr, to 1.7 nmol in the 14-hr incubations [Fig. 6(C)]. Taken together, the

TABLE III

Delivery of retinoids to RPE monolayer nmol (mean+ 1 s.D.)

Time (hr) 0 1 6 14 ND, Not detected.

Retinol in medium 17.367k4.693 2.500 k 0.269 0.397kO.136 0.392 kO.273

1 1-cis retinaldehyde in medium

1 1-cis retinaldehyde in RPE

ND 0.151 kO.182 0,208 + 0.058 1.707+0.780

ND 0~011+0~005 0.025kO.023 0.056f0.048

Retinyl palmitate in RPE _.-. -.ND 0~124~0~059 0~512~0~059 2.64~0.251

726

recovery of retinoids from the initial 17.4 nmol of alltram retinol was 33.5%. 3. Discussion Our results demonstrate that cultured human retinal pigment epithelial cells are capable of internalizing all-trans retinol from IRBP, esterifying it, synthesizing 11-cis retinaldehyde and releasing it into the medium. This is significant since the ll-cis isomer of retinaldehyde is the form of the rhodopsin chromophore required for its regeneration from opsin. Since mammalian photoreceptors do not contain a retinoid isomerase, and their retinol oxidoreductase is stereospecific for the all-tram configuration (Lion et al., 1975) ; the isomerization and oxidation reactions required for conversion of all-trans retinol to 11-cis retinaldehyde must occur in the RPE. In our experiments, two predominant species were produced by the RPE from the all-tram retinol substrate ; all-tram retinyl palmitate and 1 1-cis retinaldehyde. The majority of retinoid processing by the RPE involved the synthesis of the ester, all-trans retinyl palmitate, with a smaller proportion of ll-cis retinyl palmitate. The ester form serves as a stable, storage conformation for retinoids within the RPE cell by allowing retinoids to accumulate within the epithelium without membranolytic effects. Retinyl esters may also serve as an energy store for the isomerization reactions (Deigner et al.. 1989). Additionally, the RPE has been shown to possess distinct enzymatic activities for the hydrolysis of retinyl esters back to 1 l-cis and all-trans retinol (Blaner et al., 1987). At all times examined, the quantity of retinol and retinaldehyde isomers retained by the RPE and not released into the medium remains small (approx. 1%) in comparison to the amount of retinyl palmitate which accumulated in the epithelium, and was comparable to the ratio of retinaldehyde to retinyl ester found within RPE celIs in vivo (Bridges, 1984). Rather than being stored in the RPE, 11-cis retinaldehyde was released into the culture medium. In incubations over 6 hr, as the all-tram retinol supplied by IRBP was diminished, the 11-cis isomer of retinaldehyde predominated over all other non-esteriiled (retinol and retinaldehyde) isomers in extracts from the RPE cell and culture media. The production of this 11 -cis from all-tram retinoids is energy-requiring, and biologically significant, as 1 1-cis retinoids account for only 0.1 y0 of the mixture at chemical equilibrium (Rando and Chang, 1983). These results are in agrement with recent in vitro studies on RPE cell homogenates from Ram pipiens (Bernstein et al., 1987), and the membrane fraction of bovine RPE (Fulton and Rando, 1987), from which it was concluded that the formation of 1 1-cis retinoids is the result of the action of an isomerizing enzyme. The predominance of two of the several possible retinoid species of the visual cycle within our culture system,

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ET AL.

1 1-cis retinaldehyde and all-tram retinyl palmitate, is in agreement with the findings of Trehan, Canada. and Rando (1990), which suggestthat all-trans retinyl palmitate is the ultimate substrate for the isomerase. The predominance of all-trans over 11-cis retinyl palmitate in the pool of retinyl esterswithin the RPE is in agreement with the findings of Okajima et al. (1989) and suggests that the post-isomerization storage of ll-cis retinyl palmitate is not highly favoured in these cells in vitro. We found the accumulation of all-truns retinyl palmitate in the cultured RPE to proceed at a rate of approximately 03 nmol hr- 1 in a 100-mm culture plate, a significantly greater rate than the formation of retinol and retinaldehyde isomers. This finding correlates with the observation of greater activity for the ester synthetase relative to that of the isomerase in homogenates of bovine RPE (Fulton and Rando. 1987). Our intact ceII system differs, however, in that the primary non-esterified retinoid produced is 1 1-cis retinaldehyde, whereas the 11-cis isomer of retinol was produced by RPE cell homogenates. This difference suggeststhat the retinol oxidoreductase activity of the RPE remains viable in the cultured RPE, and is distinct from the isomeraseand ester synthetase activities. The esterification of all-trans retinol delivered via the non-specific carrier protein bovine serum albumin (BSA) has been previously demonstrated in both frog and human RPE cells in vitro (Flood et al.. 1983; Fang, Bridges and Alvarez, 198 3 ; Das and Gouras. 1988). In more recent experiments, Okajima et al. (1989) have demonstrated that the rate of esterification of all-trans retinol was higher when delivered by the natural carrier protein, IRBP. Although we did not make these comparisons in our experiments, we found the processing of the internalized retinol not to be limited to esterification. but to include the production of a significant amount of 11-cis retinaldehyde which was released into the culture medium. The specificity of delivery of retinol to the RPE by IRBP is apparently not comparable to that of the serum RBP-receptor interaction demonstrated on the basal RPE membrane in vivo (Bok and Heller, 1976. Heller and Bok. 1976) and in vitro (Pfeffer et al.. 1986). To date, no receptor for the IRBP molecule has been demonstrated on the RPE. Hollyfleld et al. (19 8 5) have demonstrated the internalization of colloidal gold-labeled IRBP within the RPE from the apical membrane, but this response appears to involve nonspecific uptake and is probably not receptormediated. Since the colloidal gold-labeled molecule is particulate, it is not known whether one can correlate these results with processesthat involve internalization of the free molecule, and therefore may or may not indicate the route by which retinoids bound to IRBP enter the RPE. On the other hand, the protection of retinoids provided by IRBP in the culture medium was significant in light of the fact that about 3 3“/” of the all-trans retinol supplied during the 14-hr in-

RETINOID

PROCESSING

BY CULTURED

HUMAN

727

RPE

cubation was recovered as retinyl ester, retinol or retinaldehyde. This finding is surprising in light of the relatively loose binding between IRBP and all-trans retinol (apparent dissociation constant : 1 x 10m6M, Alder et al., 19 8 5) and the vulnerability of retinoids to degradation in an aqueous environment. Our finding of negligible levels of retinoids in cultured RPE cells prior to incubation with retinol has a precedent in the results of Flood et al. (1983), who reported an 80% decreasein total vitamin A per RPE cell within 2-4 days in vitro. This depletion of retinoid stores has been more recently attributed to the hydrolysis of retinyl esters in responseto the presence of serum albumin in their culture medium (Das and Couras, 1988). However, the cells used in our study were cultured for 2-7 months, and the rate at which retinoids were lost from the cells is not known. The lossof endogenous vitamin A from the cultured cells may not be indicative of a dedifferentiated state, since the concentration of retinoids available in our culture media for replenishment of endogenous retinoid stores was very low. Heat inactivation of the 1% calf serum and addition of dilute aqueous bovine retina extract supplements the cells with very low levels of retinoids, which are probably just sufficient to meet the metabolic requirements of the epithelium. without providing adequate quantities for storage. Our results suggestthe presenceof functional retinyl ester synthetase, retinoid isomerase, and retinol oxidoreductase activity in human RPE cells in culture. Additionally, our observations demonstrate the delivery of all-truns retinol to the apical surface of RPE by IRBP. Although the mechanism of delivery is not known, it is clear that IRBP provides considerable protection of retinoids in light of the recovery of 3 3.5 % of the retinoid provided to the cells over a 14-hr period. Thus, although IRBP may not play a role in the targeted delivery of retinoids, it could serve as an intercellular buffer for these labile compounds (Ho et al., 1989). In this context, IRBP could serve as a binding protein not only for all-trans retinol but also for 11-cis retinaldehyde releasedby the RPE. Alternatively, 11-cis retinaldehyde may exit the RPE bound to some undiscovered protein. The use of cultured RPE stands to simplify greatly the chemical ‘dissection ’ of the visual cycle, since contamination from components of the neurosensory retina and choroid is eliminated. This approach should increase our understanding of the cell biology of the visual cycle.

Acknowledgments Supported by National Eye Institute grants EY0044, EY05624, EY00331, EY3897, ResearchTo Prevent Blindness, Inc., and a center grant from the Retinitis Pigmentosa Foundation, Fighting Blindness, Baltimore. D.B. is the Dolly Green Professor of Ophthalmology at UCLA and a Research to Prevent Blindness SeniorScientific Investigator. The authors would like to thank Dr Gary Landers for assistance with the cell extraction procedures, and Dr Paul

Bernsteinfor critical reading of the manuscript.We would alsolike to thank Dr Peter F. Sorter of Hoffman-La Roche, Nutley. NJ, for the generousgift of retinoid standards.

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