Maintenance of retinyl ester synthetase activity in cultured human retinal pigment epithelial cells

E.rp. E!/e //es. ( 1 9 8 7 ) 4 5 , 187-190 L E T T E R TO T I l E E D I T O R S

Maintenance of Retinyl Ester Synthetase Activity in Cultured Human Retinal Pigment Epithelial Cells Tile 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 ) c o n t a i n s h i g h levels o f r e t i n y l e s t e r s y n t h e t a s e ( R E S ) , t h e e n z y m e t h a t c o n v e r t s r e t i n o l to its e s t e r s o f l ) a l m i t a t e a n d o t h e r flirty a c i d s ; t h e h i g h e s t specific a c t i v i t y o f R E S is l)resent in t h e m i c r o s o m e s u h e e l l u l a r f r a c t i o n o f t h e R I ) E ( B e r m a n , H o r o w i t z , Segal, F i s h e r a n d F e e n e y - l ~ u r n s , 19811). T h i s e n z y m e p l a y s an i m p o r t a n t role in t h e v i t a m i n A cycle. R e t i n o l r e l e a s e d d u r i n g l i g h t a d a l ) t a t i o n is s t o r e d in t h e R P E ~.ts t h e e s t e r ( l ) o w l i n g . 1960), whi(.h is less m e m h r a n o l y t i c t h a n r e t i n o t ((Joo(tali, F i s h e r a n d L u c y , 19811). C u l t u r e d R P E l)rovides a useful s y s t e m fi>r s t u d y i n g t h e role ()f t h i s t i s s u e in v i t a m i n A m e t a b o l i s m ; v i a b l e c u l t u r e s o f p u r e R P E cells c a n be m a i n t a i n e ( l u n d e r d e f i n e d c o n d i t i o n s for m o n t h s , d u r i n g w h i c h v i t a m i n A - m e t a b o l i z i n g l~rocesses can he m o n i t o r e d . H o w e v e r , s o m e e n z y m e a c t i v i t i e s de('line r a p i d l y i~1 !{1)1~ in vitro, i n c l u d i n g R E S (Bridges, O k a , F o n g , Liou a n d A l v a r e z , 1986) a n d ll-cis-retinyl l ) a l m i t a t e h y d r o l a s e (Ishizaki, H a l e y , l)as a n d G o u r a s , 1986), a n d t h e c a p a c i t y o f i n t a c t c u l t u r e d R P E cells to esterii~v all-txa~s-retinol also de(.reases w i t h t i m e in v i t r o ( F l o o d , Bridges, A l v a r e z , B l a n e r a n d G o u r a s , 1983). A m o u n t s o f cellular retinolb i n d i n g p r o t e i n ( B l a n e r , F l o o d , P i a n t e d o s i , F a s a n o a n d G o u r a s , 1985; B r i d g e s et al., 1986) a n d c e l l u l a r r e t i n a l d e h y d e - b i n d i n g p r o t e i n ( B r i d g e s et al., 1986) also d e c l i n e w i t h t i m e in culture(l R P E . R P E cells c u l t u r e d in a m e d i u m w i t h h o r m o n e s , o t h e r a d d e d d e f i n e d c o m p o n e n t s , b o v i n e r e t i n a e x t r a c t , a~ld 1 ~¼J b o v i n e c a l f s e r u m h a v e been s h o w n to r e t a i n b a s a l l y localized r e c e p t o r s for s e r u m r e t i n o l - b i n d i n g l)rotein (Pfeffer, C l a r k , F l a n n e r y a n d Bok, 1981i). T'o d e t e r m i n e if t h i s m e d i u m m i g h t also p r e s e r v e o t h e r R P E l)rol)erties t h a t arc involve(l in v i t a m i n A m e t a b o l i s m , R E S a c t i v i t y w a s m e a s u r e d in R P E c u l t u r e d in t h i s m e d i u m a n d in fi'eshly is(~lated R P E . T h e r e s u l t s s h o w t h a t R P E c u l t u r e d in t h i s m e d i u m r e t a i n s u !) to 40 ~¼J o f f r e s h - t i s s u e R E 8 levels, while I{ES levels d e c l i n e r a p i d l y in R P E g r o w n in a m o r e c o n v e n t i o n a l medium. H u m a n R P E cells w e r e i s o l a t e d as l)reviously d e s c r i b e d ( E d w a r d s , 1982) fi'om a u t o p s y eyes o b t a i n e d t h r o u g h t h e N a t i o n a l I)isease l~esearch l n t e r c h a ~ l g e ( P h i l a (lell)hia, PA). Briefly, t h e a n t e r i o r s e g m e n t , v i t r e o u s , a n d r e t i n a w e r e r e m o v e d , t h e e x p o s e d R P E in t h e e y e c u I) w a s i n ( : u b a t e d w i t h 2.5 m g m1-1 tryt)sin in c a l c i u m - a n d m a g n e s i u m - f i ' e e t)alaneed salt s o l u t i o n , a n d t h e R P E was r e m o v e d by g e n t l y pipe t t i n g t h e t r y p s i n s o l u t i o n a g a i n s t t h e R P E . Cells were c e n t r i f u g e d , r e s u s l ) e n d e d in m e d i u m , a n d see(led in p l a s t i c flasks ( F a l c o n , O i n a r d , CA) a t 12-17 ~ 10 a cells cm -'2. T w o me(lia w e r e u s e d : o n e was t h e m e d i u m r o u t i n e l y u s e d in t h i s l a b o r a t o r y ( 2 0 ~ F B S ) , w h i c h c o n t a i n e d H a m ' s F - 1 0 N u t r i e n t S o l u t i o n (Gibco, G r a n d I s l a n d , N Y ) s u p p l e m e n t e ( t w i t h 30 mM a d d i t i o n a l g l u c o s e a n d 20 ~¼, ( v : v ) fetal b o v i n e s e r u m ( F B S , A r m o u r , K a n k a k e e , IL). T h e o t h e r m e d i u m , d e s i g n a t e d P M m e d i u m , c o n s i s t e d o f M e d i u m 199 (Gii)co; f o r m u l a t i o n i n c l u d e s 0"43/~M r e t i n y l a c e t a t e ) S U l ) p l e m e n t e d w i t h 10/~g ml -~ b o v i n e insulin, 5/~,g ml -I t r a n s f e r r i n , 20 nM h y d r o ( . o r t i s o n e , I n~1 trii o d o t h y r o n i n e , 0-3/~m linoleic acid, 8%t~g lnl -I b o v i n e s e r u m a l b u m i n , 0"3/Lg m1-1 p u t r c s c i n e ( S i g m a , S t Louis, MO), 1% (v :v) n e w b o r n c a l f s e r u m ( H a z l e t o n , D e n v e r , 0 0 1 4 - 4 8 3 5 / 8 7 / 0 7 0 i 87 + 04 $ 3 . 0 0 / 0

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PA), a n d 1 % (v : v) b o v i n e r e t i n a e x t r a c t ; this is similar to tile IllCdiuln tised by ]'ft,fl't'l" et al. (1986) for tile initiation of p r i m a r y c u l t u r e s of h u m a n R P E e x c e p t t h a t t h e final CaCI~ e o n c e n t r a t i o n in the p r e s e n t e x p e r i m e n t s was 1'8 m~1. N o a n t i b i o t i c s w e r e a d d e d to t h e media. 'File r e t i n a e x t r a c t was tile s u p e r n a t a n t o b t a i n e d by h o m o g e n i z i n g six b o v i n e r e t i n a s in 50 ml E a r l e ' s b a l a n e e d salt solution and eentrifllging the h o m o g e n a t e at lO000t)g for" I hr at 4°C (B.A. Pfi'ffer. pcrs. c o m m . ) . T h r e e sots of R P E c u l t u r e s were used; one sot was p r e p a r e d from each eye ~)f a n o r m a l m a l e age(I 64 years, a n d t h e t h i r d set w a s l)relmre(l from (.ells pooled fi'om both eyes of a n o r m a l m a l e aged 66 years. All c u l t u r e s were i n i t i a t e d in PM m e d i u m . One c u l t u r e of each set was m a i n t a i n e d in PM m e d i u m for' 22 tlas's, while a scoop.l(! c u l t u r e ot' e a c h set was s w i t c h e d from PM m e d i u m to 2 0 % F B S a t 9 (.lays in vitro a n d m a i n t a i n e d an a d d i t i o n a l 13 clays in t h e l a t t e r me(tiunl. All c u l t u r e s w e r e t h e n rinsed live t i m e s in t)'15 M NaCl, h o m o g e n i z e d in 0-25 M s u c r o s e - 0"Ol M T r i s - a c e t a t e (pH 7"0). a n d c e n t r i f u g e d a t 14(~000g for i hr. T h e pellets (l)artieulate fi'actions) w e r e r c s u s p e n d e d 1)y brief s o n i c a t i o n in 0"1 m~z d i t h i o t h r e i t o l - 0"01 ~1 T r i s - H C l (pH 7"0) (Saari a n d B r e d b e r g , 1982), assayetl for l)rotein ( L o w r y , R o s e b r o u g h , F a r r and R a n d a l l , 195I), a n d stored at --80°C. R E S a c t i v i t y was a s s a y e d e s s e n t i a l l y as p r e v i o u s l y d e s c r i b e d ( B e r m a n et al., 1980; Saari a n d B r e d b e r g , 1(,)82). R e a c t i o n m i x t u r e s (0"2 ml final v o l u m e ) c o n t a i n e d e n z y m e ( e q u i v a l e n t to 15/tg of p a r t i c u l a t e fi'action l)rotein t)'om c u l t u r e d cells or 0 " 6 - 1 5 / t g fi'om freshly isolated R P E ) , 0"1 M T r i s - H C I (pH 8"2), a n d !/tCi of I:~H lall-tran.s-retinol as s u b s ( r a t e ( A m e r s h a m . Chicago, IL, 53 Ci mmo1-1, or New E n g l a n d Nuclear', Boston, MA, 20 Ci retool-l). S a m p l e s were incubatetl tbr 15 rain in a 31 t., s h a k e r w a t e r b a t h . T h e reac.tion was t e r m i n a t e d by a d d i n g 0",5 ml of ice-cold e t h a n o l t h a t c'ontained 0".5 n m o l each of all-lrans-retinol a n d all-trans-retinyl p a l m i t a t e (Sigma) to serve as m a r k e r c o m p o u n d s fbr H P L C . F o r a n a l y s i s the m i x t u r e was e x t r a c t e d twice at r o o m t,e m l ) e r a t u r e with 1 ml of h e x a n e each t i m e (Saari a n d B r e d b e r g , 1982). T h e pooled h e x a n e e x t r a c t s were e w q ) o r a t e d to 5001d, h a l f o f which was a n a l y s e d for r e t i n o i d s by n o r m a l - p h a s e H P L C on a B e c k m a n S y s t e m 344 i n s t r u m e n t with all Ultrasl)here silica c o l u m n (5-/zm particle size) e l u t e d at 1 ml rain -1 with a step g r a d i e n t o f d i o x a n e in h e x a n e (2 % d i o x a n e for 13 rain followed 1)y 8 % dioxal~e fbr 12 rain) ( a d a p t e d from Bridges a n d Alvarez, 1982). M a r k e r r e t i n o i d s w e r e d e t e c t e d by al)sorban('~.e a t 340 n m and corI:csponding r a d i o a c t i v e r c t i n o i d s w e r e m e a s u r e d by scintillation c o u n t i n g of 1 ml fi-actions. R e t i n y l p a h n i t a t c was e l u t e d a t 3"6 rain a n d retinol at 22 rain. R E S specific a c t i v i t y was e x p r e s s e d as t h e p e r c e n t a g e of [aH]retinot esterified per /~g p a r t i c u l a t e fraction protein d u r i n g t h e 15 rain i n c u b a t i o n . P e r c e n t a g e esterification was c a l c u l a t e d as (cl)ml~ v x 1 0 0 ) - (cpniRp q-eI)mi~on), w h e r e cl)m is c o u n t s per rain for t h e r a d i o a c t i v i t y t h a t m i g r a t e d w i t h t h e r e t i n y l p a h n i t a t e ( R P ) or retinol ( R O H ) m a r k e r s . L i n e a r i t y of t h e r e a c t i o n w i t h a m o u n t of p a r t i c u l a t e fi'action p r o t e i n was m e a s u r e d with r e p r e s e n t a t i v e fi'esh tissue samples. All results w e r e based on a m o u n t s of s a m p l e t h a t g a v e r e a c t i o n r a t e s in t h e r a n g e 0-1-30 % esterification. T h e r a t e o f vea(;tion as a f u n c t i o n o f t o t a l p a r t i c u l a t e fraction p r o t e i n was linear w h e n esterification (tld nut e x c e e d 2 5 - 3 0 % of total [aH]retinol; this a c t i v i t y was e q u i v a l e n t to 10-25 tzg of p a r t i c u l a t e protein from c u l t u r e d cells or 5 / t g of fi'esh tissue p a r t i c u l a t e p r o t e i n . R e p r o d u c i b i l i t y was w i t h i n 20 % of t h e m e a n for d u p l i c a t e assays of r e p r e s e n t a t i v e samples. Boiling of t h e p a r t i c u l a t e fraction for 1 rain c o m p l e t e l y a b o l i s h e d esterification a c t i v i t y . 4

a

•

~O

R E T I N Y L ESTER SVNTHETASE IN C U L T U R E D RPE

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189

I~ Fresh "13ssue [ ] Cultured in PM [] Cultured in 20°1=FBS

2

er" 0

Left Eye Donor 1

Right Eye PooledEyes Donor 1 Donor 2

Mean

Fro. i. Retinyl ester synthetase activity in cultured and freshly isolated human retinal pigment epithelium, laH]all-tra~.~-retinoi was incubated with atiquots of the particulate fraction from freshly isolated R P E or R P E cultured in the indicated me(lia. Retinoids were extracted with hexane and analysed by high-pressure liquid c h r o m a t o g r a p h y as described in the text. Retinyl ester synthetase activity is expressed as the percentage esterification of [aHjretinol per ttg particulate fraction protein. Vertical lines on the bars for mean values indicate one standard deviation.

H u m a n R P E cells cultured for 22 (lays ill PM medium retained levels of R E S a c t i v i t y corresponding to 1-2-2"2% esterification of [aHIall-tran,'-retinol per Fg l)rotein (Fig. 1). This compares with 5 % esterification per Fg t'or freshly isolated R P E from the same eyes. Cultures t h a t were switched to 20 % F B S medium for the last 13 (lays in vitro retained R E S a c t i v i t y of only 0"1-0"4% esterification per Fg. Similar results were obtained with R P E cultured from an additional donor in which cells were maintained for 25 d a y s either in PM medium or in Medium 199 supplemented with 2 0 % fetal bovine serum (data not shown). Thus, I ( P E m a i n t a i n e d in PM medium retained about 25-42 % of R E S levels present in fi'eshly isolated R P E , while R P E maintained in 20 % F B S retained only 2-8 % of fresh-tissue R E S levels. The retention of relatively high R E S levels b y R P E cultured in PM medium shows t h a t PM medium is superior to other previously used media for the ill vitro m a i n t e n a n c e of this e n z y m e activity. I t remains to be determined if other properties of the R P E involved in v i t a m i n A metabolism, such as retinoid-binding proteins or vitamin-A-metabolizing enzymes in addition to R E S , are retained in R P E grown in PM medium. Since the retention of R E S by R P E can now be m a n i p u l a t e d in vitro, it should be possible to determine which components of PM medium p l a y a role in the m a i n t e n a n c e of R E S activity. Compared with media t h a t do not m a i n t a i n R E S levels, unique components of PM medium include retina extract, triiodothyronine, hydroeortisone, linoleic acid, putrescine, and low levels of newborn calf serum (Pfeffer et al., 1986). Media not capable of m a i n t a i n i n g R E S in cultured R P E include media with 20 % fetal bovine serum as the only source of hormones and growth factors (control medium in this st,u d y ; Bridges et al., 1986) and a serum-free medium supplemented with insulin, transferrin, epithelial growth factor, follicle-stimulating hormone, retinoie acid, and selenium (Bridges et al., 1986). F u r t h e r work should indicate whether a n y of the components unique to PM medium play a role in controlling the levels of retinyl ester s y n t h e t a s e a c t i v i t y in the R P E .

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R.B. E D W A R D S ET AL. ACKNOWLEDGMENT

This research was s u p p o r t e d by National I n s t i t u t e s of Health grants EY02028 (RBE) and EY04368 (AJA), Specialized Research Center Grant EY02014, a n d by the Retinitis P i g m e n t o s a F o u n d a t i o n F i g h t i n g Blindness, Baltimore, MD.

Berman-Gund Laboratory for the Study of Retinal Degenerations, Harvard Medical School, Massachusetts Eye and Ear Infirmary and *Eye Research Institute of Retina Foundation, 20 Staniford Street, Boston, M A 02114, U.S.A.

R o s s B. EDWARDS

*ALICE J. ADLER AND *ROMMA E. SOVTHWlCK

(Received 6 February 1987 and accepted in revised f o r m 10 March 1987) REFERENCES B e r m a n , E. R., Horowitz, J., Segal, N., Fisher, S. and F e e n e y - B u r n s , L. (1980). E n z y m a t i c esterification of v i t a m i n A in t h e p i g m e n t epithelium o f bovine retina. Biochem. Biophys. Acta 630, 36-46. Blaner, W. S., Flood, M. T., Piantedosi, R., Fasano, M. K. a n d Gouras, P. (1985). Cellular retinol-binding protein levels in fresh and cultured h u m a n retinal p i g m e n t epithelial cells. Invest. Ophthalmol. Vis. Sci. 26(Suppl.), 16. Bridges, C. D. B. and Alvarez, R. A. (1982). M e a s u r e m e n t of t h e v i t a m i n A cycle. Met]is. Enzymol. 81, 463-85. Bridges, C. D. B., Oka, M. S., Fong, S.-L., Liou, G. I. and Alvarez, R. A. (1986). Retinoidbinding proteins and retinol esterifieation in cultured retinal p i g m e n t epithelium cells. Neurochem. Int. 8, 527-34. Dowling, J . E . (1960). Chemistry of visual a d a p t a t i o n in the rat. Nature (London) 188, 1 14-8. Edwards, R. B. (1982). Culture of m a m m a l i a n retinal p i g m e n t epithelium and neural retina. Meths Enzyrnol. 81, 39-43. Flood, M. T., Bridges, C. D. B., Alvax:ez, R. A., Blaner, W. S. a n d Gouras, P. (1983). Vitamin A utilization in h u m a n retinal p i g m e n t epithelial cells in vitro. Invest. Ophthalmol. Iris. Sci. 24, 1227-35. Goodall, A. H., Fisher, D. a n d Lucy, J. A. (1980). Cell fusion, ha~molysis a n d mitochondrial swelling induced by retinol a n d derivatives. Biochim. Biophys. Acta 595, 9-14. Ishizaki, H., Haley, J., Das, S . R . and Gouras, P. (1986). T h e activity a n d subcellular distribution of 1 l-cis-retinyl p a l m i t a t e hydrolase in fresh a n d cultured h u m a n retinal epithelium. Invest. Ophthalnwl. Vis. Sci. 27(Suppl.), 295. Lowry, O . H . , i4osebrough, N . J . , Farr, A . L . a n d Randall, R . J . (1951). Protein m e a s u r e m e n t with the Folin phenol reagent. J. Biol. Chem. 193, 265-75. Pfeffer, B. A., Clark, V. M., F l a n n e r y , J. G. and Bok, D. (1986). Membrane receptors for retinol-binding protein in cultured h u m a n retinal p i g m e n t epithelium. Invest. Ophthalmol. Vis. ,Sci. 27, 1031-40. Saari, J . C . and Bredberg, D . L . (1982). E n z y m a t i c reduction of ll-cis-retinal b o u n d to cellular retinal-binding protein. Biochem. Biophys. Acta 716, 266-72.

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