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Localization of retinoid-binding proteins in developing rat retina
E p. Eye Re,~. ( 1985), 41,299-304
L o c a li ~ a t l o" n
of Retinoid-binding Proteins Rat Retina*
A~IY J. EISENFELD, AN~; H . B U N T - M ! L A M
in Developing
AND JOH~
C. S A A R I
!
Depart
ent of Ophthalmol , ~eattle,
iver~ity of 8hi 98195 U.S.A.
(Received 8 October 1984 and accepted 6
ton ;School of
dicine,
1 9 8 5 , New Y;grk)
Antibodies to cellular retino|-binding prot-ein (CRBP) and cellular retinal-binding pro~in (CRALBP) were obtained m rabbits immunized with tigens purified m bovine ~ t i n a . Antigens were localized on frozen sections of rat retina using indirect FITC immunofluor~2scence. In tile retinal pigment epithelium of rats from postnatal day i (the day of birth) to postnatal day 32, specific staining with anti-CRBP was restric~d to the cytoplasm; the nuclei were unstained. In the neurosensory retina, Muller cell end t were stained with anti-CRBP at all s examined. No CRBP reactivity w found in the pigment epithelium of the ciliary body at any a ~ examined. On postnatal d 14 and 32, in addition Muller cell end t and radial pro es, o fine lamin in the inner plexiform I r re faintly positive l%r CRBP. Anti-CRALBP stained the cytopla~um of the R P E and Muller cells in the adult rat. In developing rat retina, in addition to Muller cells and the ~etinal pigment epithelium, the ciliary body pigment epithelium and the o u ~ r epithelium of the iris w e ~ stained with anti-CRALBP in the first postnatal w ~ k . The intensity of staining of the ciliary body pigment epithelium dec~e d g ually from postnatal day 8 until postnatal d 14, at which point a clear line of demarcation w ~ tbund be e~n the sitively stained ~tinal pigment epithelium and the a nt, lightly stMned, pigment epithelial cells of the ciliary body. y wo • retina; retinoid-binding pro~ins; development; cellular retinal-binding protein; cellular mtinol-binding protein.
1. I n t r o d u c t i o n C e l l u l a r r e t i n o l - b i n d i n g p r o t e i n ( C R B P ) , f i r s t x~epor~d b y B a s h o r , T o f t a n d C h y t i l (1973), is a n i n t r a c e l l u l a r p r o ~ i n p r e s e n t in ~ a n y t i s s u e s o f t h e b o d y . I t s p r e s e n c e in a n u m b e r o f e p i t h e l i a l cell t y p e s t h a t d e p e n d o n v i t a m i n A f o r n o r m a l di rentiation a n d m e t a b o l i s m h a s led t o t h e s u g g e s t i o n t h a t C R B P m a y f u n c t i o n as a t r a n s c y t o plasmic carrier that accepts tinol fro~ serum tinol-binding protein (RBP) and t r a n s p o r t s it t a r g e t cell n u c l e i f o r i n t e r a c t i o n w i t h t h e g e n o m e ( T a k a s e , O n g a n d C h y t i l , 1979). D e l i v e r y o f r e t i n o l t o t h e t a ~ e t e p i t h e l i u m p r o b a b l y i n v o l v e s r e c e p t o r s in t h e cell s u r f a c e m e m b r a n e ( H e l l e r , 1 9 7 5 ; B o k a n d H e l l e r , i 9 7 6 ; R;ask a n d P e t ~ r s o n , 1976). U n l i k e C R B P t h a t is w i d e l y d i s t r i b u t e d , c e l l u l a r tinal-binding pro~in (CRALBP), w h i c h b i n d s 11-ci,s-retina! a n d I l-cis- t i n o l , is r e s t r i c t e d i n l o c a l i z a t i o n t o t h e tinal p i g e n t e p i t h e l i u m ( R ; P E ) a n d r e t i n a ( F u t t e r m a n a n d S a a r i , 1977). B e c a u s e o f t h i s r e s t r i c t e d l o c a l i z a t i o n a n d t h e 11-cis t ~ r m o f i t s e n d o g e n o u s r e t i n o i d s , it h a s b e e n s u g g e s t e d t h a t C R A L B P m a y b e i n v o l v e d in t h e v i s u a l c y c l e , p e r h a p s f o r r e g e n e r a t i o n o f b l e a c h e d r h o d o p s i n ( F u t t e r m a n , S a a r i a n d B l a i r 1977).
* This s~udy was supposed in part by USPHS Research G r a n ~ EY-05563, EY-07013, EY-01730, EY-0!311 and EY-02317, and in p a ~ by an unrestricted grant from Research to Prevent Blindness, Inc. Dr Bunt-Milam is a William and Ma D" Greve In~rnational Research Scholar of R e , a r c h to P ~ v e n t Blindness, Incorporated. 14-4835/85/0
299 + 0 6 ~ 3 .
/0
(~) i985 Academic P ~ s s Inc. (London)Limited
300
A.J. E I S E N F E L I) E T A L.
C R B P has been localized to t|le R P E in t h e bovine (Saari, B u n t - M i l a m , B r e d b e r g a n d G a r w i n , 1984) and to t h e R P E and inner (vitread) p o r t i o n s of t h e Muller (.ells in t h e r a t r e t i n a (Bok, 0rig a n d Chytil, 1984). C R A L B P has been localized to the R P E a n d all p o r t i o n s of retinal Muller cells in several species ( B u n t - M i l a m a n d Saari, 1983). In this r e p o r t , we describe the imn!un
2.
aterials and
ethods
Sprague-Dawley rats (Tyler Labs, Bellevue, WA, U.S.A.) were maintained ira a 12 hr light/12 hr dark environment. Rats from postnatal d 1 (PI or the das, of birth). P2, I~4, P6, PS, P I0, P I4 and P32 were enucleated under ether aneatl~esia between I "00 and 2"30 p.m. The cornea was slit, the lens and vitreous removed and the glol)e was immersed in 4 °/oj depolymerized paraformaldehyde in 0"13 ~ phosphate I)u r (pH 7"4). After 6 hr in fixative at 4 °C, the eyes were transferred to 3 0 % sucrose and stored at 4 °C overnight. r immunofluo~scence, 20 p m sections were cut with a c ~ o s t a t at -°C. The ctions were m o u n ~ d on chrome alum-gelatin coated slides and air-dried overnight at room ~ m p e r a t u r e . Anti-CRALBP was prepared in rabbits against bovine CI~.ALBP as previously described (Bunt-Milam and Saari, i983). Anti-CRBP was prepared in rabbits by injecting S * et performic acid oxidized bovine C R B P as previously described (caarl a|., 1084) 6 Sera samples were ~ s ~ d ~ r the anti-CRBP activity using an ELISA ass with non-per~rmic acid oxidized C R B P (Saari et al., 1984). ]mmunofluorescence techniques were performed as previously described (Bunt-Milam and Saari, 198'3; Saari et al., 1984). Control experiments were conducted fi)r all ages, including substitution of the specific primary antibody with nonimmune antiserum t)r witli specific antibody that had been absorbed witll 10-25/~g//ll purified a n t i ~ n ~ r 4-6 hr.
3. R e s u l t s Sections t r e a t e d with n o n i m m u n e or a n t i g e n - d b s o r b e d a n t i b o d i e s were negative. s h o w i n g only a pale y e l l o w / g r e e n autofluorescence t h a t was m o s t p r o n o u n c e d ii~ e r y t h r o c y t e s a n d p h o t o ceptor o u t e r s e g m e n t s [Figs l(c), 2(a). 3(c)]. T h e R P E s t a i n e d positively for C R B P a n d C R A L B P a t all ages e x a m i n e d (PI to P32). R P E c y t o p l a s m was heavily labeled [Fig. l (a),(b)] a n d pale images of R P E nuclei were resolved w i t h i n tile intensely s t a i n e d c y t o p l a s m . S t a i n i n g of the Muller cell e n d f e e t was v a r i a b l e with a n t i - C R B P for all ages e x a ined [Figs 1 (b), 2(t))]. More c o n s i s t e n t l y , a n t i - C R B P p r o d u c e d f a i n t s t a i n i n g of two fine l a m i n a e ill the inner p l e x i f o r m l a y e r on P14 and P 3 2 in a d d i t i o n to radial processes of" tile Muller cells [~ g . 2(b)]. On P I , in a d d i t i o n to the R P E , a n t i - C I ~ A L B P also s t a i n e d tile c i l i a ~ l)ody p i g m e n t e d e p i t h e l i u m (CPE) [Fig. 3(a)] a n d t h e o u t e r e p i t h e l i u m of t h e iris [Fig. 3(b)]. T h e ciliary b o d y n o n p i g m e n t e d e p i t h e l i u m a n d the inner e p i t h e l i u m of t h e iris were n o t labeled. This s t a i n i n g p a t t e r n p e r s i s ~ d until P8, after which the s t a i n i n g g r a d u a l l y di inished until P14, w h e n the C P E a n d iris e p i t h e l i u m were very faintly s t a i n e d with a n t i - C R A L B P . B y P14, t h e r e was a clear line of d e m a r c a t i o n a t the o r a s e r r a t a be een t h e h e a v i l y stained R P E a n d t h e lightly s t a i n e d C P E , as seen in the a d u l t [:Fig. l(a)]. T h e r e was no a n t i - C R B P s t a i n i n g of the choroid, ciliary b o d y or iris a t a n y of t h e ages e x a m i n e d [Fig. 1(b)]. On P I t h e r e was some a n t i - C R A L B P s t a i n i n g of radial processes in t h e retina, especially n e a r the inner limiting m e m b r a n e , where the s t a i n e d processes resembled
RETINOII)-llINI)IN(]
PROTEINS
I N R A T I¢~ET1NA
30.W
F1o. I. Indirect immunofluoreseenee of a P32 rat retina, 118 × . (a) Treated with anti-CRALBP. The retinal pigment epithelium (RPE) is heavily labeled, There is faint staining of the ciliary body pigmented el)ithelium (CB). Muller cell radial processes are labeled and can be seen in the o u ~ r limiting membrane (*), inner nuclear layer (INL) and inner plexiform layer (IPL). Muller cell endfeet are heavily labeled (arrowheads). (b) Treated with anti-CRllP. The retinal pigment epithelium (RPE) is heavily stained. There is no staining of the ciliary t)ody epithelium (CB). Muller cell endfeet (arrowheads) are labeled. (e) 'l'reat~ed with nonimmune antibody.
Fro. 2. lndilvet immunofluorescence of a I)32 rat retina. 142x. ( a ) T r e a t e d with nonirnmune antibody. (b) Treated with anti-CRBP. The retinal pigment epithelium (RPE) is heavily labeled. There m staining of Muller cell end t (M) and ~ i n t staining of two fine laminae (arrowheads)in the inner plexifi3rm l ~ e r . *, autofluoreseent blood vessels.
Muller cell endfeet (Fig. 4). The staining of Muller radial processes beca e more intense during the first week, and by P l 0 positively stained Muller solnata were present in the inner nuclear layer. By PI4, Muller cell radial processes and ramifications in the plexiform layers were heavily stained spanning the retina f r o the inner to outer limiting mernbranes [Fig. l(a)].
302
A.,I. E I S E N F E I ,
I) E T AI,.
F1o. 3. Indirect immunofluoreseenee of P6 r a t retin~ treated with anti-CR, A L B P . (a,h ~ 1 4 2 × ; e ~-56 x ) (a) Ora serrata anti ciliary body. Tile retinal pigment epithelium ( R P E ) and ciliary body p i g m e n ~ d epithelium (CB) are labeled. The inner epithelium of the iris (arrowhead) is unlabeled. There is labeling of pro awes at the vitrea[ surface of the retina (arrow). NB, neurobl ts; *, autofluorescent red blood cells' sehoroid. (b)Ciliary i~ody and iris. Tim ciliary body pigmented epithelium (CB)and outer epithelium of the iris (arrowhead) are labeled. The unl)igmented ciliary body pigment epithelium and the inner epithelium of the iris ( * ) a negative. LC, lens capsule. (e) 1)6 rat retina treated with nonimmune antibody.
FIo. 4. I n d i ~ c t immunofluorescence of the inner p o t i o n of a rat retina, 296 x . ( a ) T r e a t e d with nonimmune antibody. (b) T r e a ~ d with a n t i - C R A L B P . N o ~ +_-he faint ~taining of processes in the neurob|as¢ic layer (NB) and the more intense staining of processes near the vitreal su~Stce of the retina. V, v i t ~ o u s .
R E T I N O I D - B I N D I N G PROTEINS IN RAT RETINA
303
4. D i s c u s s i o n
A n t i - C R B P was localized in tile R P E cytoplasm of th e developing rat eye a t all ages examined. This is consistent with t h e e v i d e n c e t h a t R P E cells have receptors for R B P on the basolateral p l a s m a l e m m a (Bok and Heller, 1976; Bok, 1982). Staining with a n t i - C R B P was p r o m i n e n t at all times in the R P E b u t was a b s e n t from the CPE, even though this layer of epithelium also has sdrface ~eceptors for retinol-binding protein (Bok and Heller, 1976). Differing a m o u n t s of CP~BP in contiguous cells of the same differentiated tissue have also been found in the e p i d i d y m i s by P o r t e r et al. (1983), who suggested t h a t one m a j o r function of CI~BP, and t h u s retinol, m i g h t be m a i n t e n a n c e of the high rate o f g e n o m i c expression t h a t occurs in certain differentiated cells with increased need for retinol. CR, B ~ ! I I ) , a n o t h e r cellular retinol-binding protein, is found in the small intestine of the rat. Crow an d Ong (1985) found a b r u p t t e r m i n a t i o n of labeled epithelium a t the gastroduodenal an d ileocecal junctions. Age-related changes in the level and localization of C R B P h a v e been d o c u m e n t e d in the liver, where the a m o u n t of CI:tBP increases sharply at birth and declines after 21 days to lower levels characteristic of the a d u l t (Ong a n d Chytil, 1976; K ato , K a t o and Goodman, 1985). The present s t u d y d e , t r a i n e d tl~at C R A L B P is expressed d u r i n g the first postnatal week in both the R P E and CPE, two epithelia t h a t have a c o m m o n embryologic origin from the o u t e r layer ot" the optic cup. The C P E staining decreases from P8 to P14, after which the C P E stained very faintly with a n t i - C R A L B P . The decline of a n t i - C R A L B P staining during this second p o s tn atal week coincides t e m p o r a l l y with the m a t u r a t i o n of photoreceptor o u t e r s e g m e n t s in the retina (Weidman and K u w a b a r a , 1968). This transitory expression of C R A L B P in the C P E during the first postnatal week was unexpected and its significance remains to be de t e rmi ne d. A n t i - C R A L B P was also localized in Muller cell radial processes in the early post na t a l retina. In the a d u l t r a t retina, the Muller cell basal endfeet, s o m a t a and fine processes t h a t ramify in the plexiform and nuclear layers are heavily stained with anti-CP~ALBP. This staining p a t t e r n is the same as t h a t seen previously in the retina of the a d u l t cow, m onke y, and h u m a n (Bunt-Milam an d Saari, 1983). F a i n t an d variable staining for C R B P was also found in Muller radial processes and endfeet. Unlike a n t i - C R A L B P staining, the label was always restricted to the inner r e t i n a ; Muller s o m a t a and processes in the o u ter plexiform and o u t e r nuclear layers were consistently negative with a n t i - C R B P . Such differences in staining intensity m a y reflect differences in the absolute a m o u n t s of the two antigens present in the retina. I n the bovine retina, the ratio of C R A L B P to C R B P is 7 : 1 (Saari et al., 1984). Localization of C R A L B P to Muller cells raised the u n e x p e c t e d possibility t h a t this glial cell might be involved in the metabolism of v i t a m i n A (Bunt-Milam and Saari, 1983). The report by Bok et al. (1984) and the results reported here s t r e n g t h e n t h a t hypothesis, since it now appears t h a t at least two cellular retinoid-binding proteins, C R A L B P and C R B P , m a y be present in Muller cells. I t will be i m p o r t a n t to confirm these results with methods i n d e p e n d e n t of the specificity of th e antibodies and to elucidate the specific role played by the Muller cell in retinoid metabolism. ACKNOWDEDGMENTS The authors thank Faridah l)ahlan, lngrid Klock, and Gl~g Garwin for technical assistance, Brad Clifton, Ron Jones and Pat Siedlak for photographic help, and Julie Seng for secretarial assistance.
304
A.J. EISENFEI,1) ET AL. REFE
RENCI,~S
Bashor, M. M., Taft, D. O. and Chyti[, F. (1973). In vitro bmt~,nv, :£ re~:uc! i~) rat tissue components. Proc. Natl. Acad. Sci. U.S.A. 70, 3483-7. Bok, D. (1982). Autoradiographic studies on the polarity of plasma mcmJ.~rane receptors in retinal pigment epithelialcells.|n The Structure of the E~/e (Ed. Hollyfield,J. G.). P. 247. Else mr Biomedical, N e w York. Bok, D. and Heller, J. (1976). Transport of retinal from the hlood to the retina: an autoradiographic study of the pigment epithelial cell surface receptor for plasma ' O retinal-binding protein. Exp. Eye Res. 22, 395-40,. Bok, D., Ong, D. E. and Chytil, F. (1984). ]mmunoeytoehemieal [oealization of retinalbinding proteins in the retina. Invest, Ophthalmol. Vis. Sci. 25, 877-83. Bunt-Milam, A. H. and Saari, J. C. (1983). Immunocytochemical localizationof two retinoidbinding proteins in veri~hrate retina. J. Cell Biol. 97, 703-12. Bunt-Milam, A. H., Eisenfeld, A. J. and Saari,J. C. (I984). Immunoeytochemieal localization of retinoid-binding proteins in adult and developing retinas, ln~est. Opbthalmol. l'is. 8ci. Suppl. 25, 276. Crow, J. A. and Ong, 1). E. (! 985). Cell specific immunohistochemical localization of a cellular retinal-Binding protein (type two) in the small intestine of the rat. Prec. Natl. Acttd. Sci. U.S.A. 82, 4707-II. Futterman, S. and Saari, J. C. (1977). Occurrence of 1i-cis-retinal-binding protein restricted to the retina. Invest. Ophthal~ol. Vis. Sci. 16, 768-71. Futterman, S., Saari, J.C. and Blair, S. (1977). Occurrence of a binding protein fi~r l l-cis-retinal in the retina. J. Biol. Chem. 252, 3267-71. Heller, J. (1975). Interaction of plasma retinal-binding protein with its receptor. J. Biol. Ch,em. 250, 3613-19. Ong, 1). E. (1984). A novel retinol-tfinding protein from rat: l)urification and partial characterization. J. Biol. Chen~. 259, 1476-82. Ong, D. E. and Chytil, FI (1976).-Changes in the levels of cellular retinal- and retinoic acid-binding proteins of liver and lung during perinatal development of rat. Proc. Natl. Acad. Sci. U.S.A. 73, 3976-8. Porter, S.B., Fraker, L. l)., Chytil, F. and Ong, I). E. (1983). Localization of cellular retinal-binding protein in several rat tissues. Proc. Natl. Acad. Sci. U.S.A. 80, 6586-90. Rask, L. and Peterson, P. A. (l (.)76). In vitro uptake of vitamin A from retinal-binding plasma protein to mucosal epithelial cells from the monkey's small intestine. J. Biol. Chem. 251, 6360-6. Saari, J. C., Bunt-Milam, A. H., Bredberg, D. L. and Garwin, G. G. (1984). Properties and immunocytoehemical localization of three retinoid-binding proteins fi'om bovine retina. Vision Res. 24, 1595-603. Takase, S., Ong, D. E. and Chytil, F. (1979). Cellular retinal-binding protein alh)ws specific interaction of retinal with the nucleus in vitro. Proc. Natl. Acad. ,Nci. U.S.A. 76. 2204-8. Weidman, T. A. and Kuwabara, T. (1968). Postnatal development of the rat retina. Arch. Ophthalmol. 79, 470-84.
L o c a li ~ a t l o" n
of Retinoid-binding Proteins Rat Retina*
A~IY J. EISENFELD, AN~; H . B U N T - M ! L A M
in Developing
AND JOH~
C. S A A R I
!
Depart
ent of Ophthalmol , ~eattle,
iver~ity of 8hi 98195 U.S.A.
(Received 8 October 1984 and accepted 6
ton ;School of
dicine,
1 9 8 5 , New Y;grk)
Antibodies to cellular retino|-binding prot-ein (CRBP) and cellular retinal-binding pro~in (CRALBP) were obtained m rabbits immunized with tigens purified m bovine ~ t i n a . Antigens were localized on frozen sections of rat retina using indirect FITC immunofluor~2scence. In tile retinal pigment epithelium of rats from postnatal day i (the day of birth) to postnatal day 32, specific staining with anti-CRBP was restric~d to the cytoplasm; the nuclei were unstained. In the neurosensory retina, Muller cell end t were stained with anti-CRBP at all s examined. No CRBP reactivity w found in the pigment epithelium of the ciliary body at any a ~ examined. On postnatal d 14 and 32, in addition Muller cell end t and radial pro es, o fine lamin in the inner plexiform I r re faintly positive l%r CRBP. Anti-CRALBP stained the cytopla~um of the R P E and Muller cells in the adult rat. In developing rat retina, in addition to Muller cells and the ~etinal pigment epithelium, the ciliary body pigment epithelium and the o u ~ r epithelium of the iris w e ~ stained with anti-CRALBP in the first postnatal w ~ k . The intensity of staining of the ciliary body pigment epithelium dec~e d g ually from postnatal day 8 until postnatal d 14, at which point a clear line of demarcation w ~ tbund be e~n the sitively stained ~tinal pigment epithelium and the a nt, lightly stMned, pigment epithelial cells of the ciliary body. y wo • retina; retinoid-binding pro~ins; development; cellular retinal-binding protein; cellular mtinol-binding protein.
1. I n t r o d u c t i o n C e l l u l a r r e t i n o l - b i n d i n g p r o t e i n ( C R B P ) , f i r s t x~epor~d b y B a s h o r , T o f t a n d C h y t i l (1973), is a n i n t r a c e l l u l a r p r o ~ i n p r e s e n t in ~ a n y t i s s u e s o f t h e b o d y . I t s p r e s e n c e in a n u m b e r o f e p i t h e l i a l cell t y p e s t h a t d e p e n d o n v i t a m i n A f o r n o r m a l di rentiation a n d m e t a b o l i s m h a s led t o t h e s u g g e s t i o n t h a t C R B P m a y f u n c t i o n as a t r a n s c y t o plasmic carrier that accepts tinol fro~ serum tinol-binding protein (RBP) and t r a n s p o r t s it t a r g e t cell n u c l e i f o r i n t e r a c t i o n w i t h t h e g e n o m e ( T a k a s e , O n g a n d C h y t i l , 1979). D e l i v e r y o f r e t i n o l t o t h e t a ~ e t e p i t h e l i u m p r o b a b l y i n v o l v e s r e c e p t o r s in t h e cell s u r f a c e m e m b r a n e ( H e l l e r , 1 9 7 5 ; B o k a n d H e l l e r , i 9 7 6 ; R;ask a n d P e t ~ r s o n , 1976). U n l i k e C R B P t h a t is w i d e l y d i s t r i b u t e d , c e l l u l a r tinal-binding pro~in (CRALBP), w h i c h b i n d s 11-ci,s-retina! a n d I l-cis- t i n o l , is r e s t r i c t e d i n l o c a l i z a t i o n t o t h e tinal p i g e n t e p i t h e l i u m ( R ; P E ) a n d r e t i n a ( F u t t e r m a n a n d S a a r i , 1977). B e c a u s e o f t h i s r e s t r i c t e d l o c a l i z a t i o n a n d t h e 11-cis t ~ r m o f i t s e n d o g e n o u s r e t i n o i d s , it h a s b e e n s u g g e s t e d t h a t C R A L B P m a y b e i n v o l v e d in t h e v i s u a l c y c l e , p e r h a p s f o r r e g e n e r a t i o n o f b l e a c h e d r h o d o p s i n ( F u t t e r m a n , S a a r i a n d B l a i r 1977).
* This s~udy was supposed in part by USPHS Research G r a n ~ EY-05563, EY-07013, EY-01730, EY-0!311 and EY-02317, and in p a ~ by an unrestricted grant from Research to Prevent Blindness, Inc. Dr Bunt-Milam is a William and Ma D" Greve In~rnational Research Scholar of R e , a r c h to P ~ v e n t Blindness, Incorporated. 14-4835/85/0
299 + 0 6 ~ 3 .
/0
(~) i985 Academic P ~ s s Inc. (London)Limited
300
A.J. E I S E N F E L I) E T A L.
C R B P has been localized to t|le R P E in t h e bovine (Saari, B u n t - M i l a m , B r e d b e r g a n d G a r w i n , 1984) and to t h e R P E and inner (vitread) p o r t i o n s of t h e Muller (.ells in t h e r a t r e t i n a (Bok, 0rig a n d Chytil, 1984). C R A L B P has been localized to the R P E a n d all p o r t i o n s of retinal Muller cells in several species ( B u n t - M i l a m a n d Saari, 1983). In this r e p o r t , we describe the imn!un
2.
aterials and
ethods
Sprague-Dawley rats (Tyler Labs, Bellevue, WA, U.S.A.) were maintained ira a 12 hr light/12 hr dark environment. Rats from postnatal d 1 (PI or the das, of birth). P2, I~4, P6, PS, P I0, P I4 and P32 were enucleated under ether aneatl~esia between I "00 and 2"30 p.m. The cornea was slit, the lens and vitreous removed and the glol)e was immersed in 4 °/oj depolymerized paraformaldehyde in 0"13 ~ phosphate I)u r (pH 7"4). After 6 hr in fixative at 4 °C, the eyes were transferred to 3 0 % sucrose and stored at 4 °C overnight. r immunofluo~scence, 20 p m sections were cut with a c ~ o s t a t at -°C. The ctions were m o u n ~ d on chrome alum-gelatin coated slides and air-dried overnight at room ~ m p e r a t u r e . Anti-CRALBP was prepared in rabbits against bovine CI~.ALBP as previously described (Bunt-Milam and Saari, i983). Anti-CRBP was prepared in rabbits by injecting S * et performic acid oxidized bovine C R B P as previously described (caarl a|., 1084) 6 Sera samples were ~ s ~ d ~ r the anti-CRBP activity using an ELISA ass with non-per~rmic acid oxidized C R B P (Saari et al., 1984). ]mmunofluorescence techniques were performed as previously described (Bunt-Milam and Saari, 198'3; Saari et al., 1984). Control experiments were conducted fi)r all ages, including substitution of the specific primary antibody with nonimmune antiserum t)r witli specific antibody that had been absorbed witll 10-25/~g//ll purified a n t i ~ n ~ r 4-6 hr.
3. R e s u l t s Sections t r e a t e d with n o n i m m u n e or a n t i g e n - d b s o r b e d a n t i b o d i e s were negative. s h o w i n g only a pale y e l l o w / g r e e n autofluorescence t h a t was m o s t p r o n o u n c e d ii~ e r y t h r o c y t e s a n d p h o t o ceptor o u t e r s e g m e n t s [Figs l(c), 2(a). 3(c)]. T h e R P E s t a i n e d positively for C R B P a n d C R A L B P a t all ages e x a m i n e d (PI to P32). R P E c y t o p l a s m was heavily labeled [Fig. l (a),(b)] a n d pale images of R P E nuclei were resolved w i t h i n tile intensely s t a i n e d c y t o p l a s m . S t a i n i n g of the Muller cell e n d f e e t was v a r i a b l e with a n t i - C R B P for all ages e x a ined [Figs 1 (b), 2(t))]. More c o n s i s t e n t l y , a n t i - C R B P p r o d u c e d f a i n t s t a i n i n g of two fine l a m i n a e ill the inner p l e x i f o r m l a y e r on P14 and P 3 2 in a d d i t i o n to radial processes of" tile Muller cells [~ g . 2(b)]. On P I , in a d d i t i o n to the R P E , a n t i - C I ~ A L B P also s t a i n e d tile c i l i a ~ l)ody p i g m e n t e d e p i t h e l i u m (CPE) [Fig. 3(a)] a n d t h e o u t e r e p i t h e l i u m of t h e iris [Fig. 3(b)]. T h e ciliary b o d y n o n p i g m e n t e d e p i t h e l i u m a n d the inner e p i t h e l i u m of t h e iris were n o t labeled. This s t a i n i n g p a t t e r n p e r s i s ~ d until P8, after which the s t a i n i n g g r a d u a l l y di inished until P14, w h e n the C P E a n d iris e p i t h e l i u m were very faintly s t a i n e d with a n t i - C R A L B P . B y P14, t h e r e was a clear line of d e m a r c a t i o n a t the o r a s e r r a t a be een t h e h e a v i l y stained R P E a n d t h e lightly s t a i n e d C P E , as seen in the a d u l t [:Fig. l(a)]. T h e r e was no a n t i - C R B P s t a i n i n g of the choroid, ciliary b o d y or iris a t a n y of t h e ages e x a m i n e d [Fig. 1(b)]. On P I t h e r e was some a n t i - C R A L B P s t a i n i n g of radial processes in t h e retina, especially n e a r the inner limiting m e m b r a n e , where the s t a i n e d processes resembled
RETINOII)-llINI)IN(]
PROTEINS
I N R A T I¢~ET1NA
30.W
F1o. I. Indirect immunofluoreseenee of a P32 rat retina, 118 × . (a) Treated with anti-CRALBP. The retinal pigment epithelium (RPE) is heavily labeled, There is faint staining of the ciliary body pigmented el)ithelium (CB). Muller cell radial processes are labeled and can be seen in the o u ~ r limiting membrane (*), inner nuclear layer (INL) and inner plexiform layer (IPL). Muller cell endfeet are heavily labeled (arrowheads). (b) Treated with anti-CRllP. The retinal pigment epithelium (RPE) is heavily stained. There is no staining of the ciliary t)ody epithelium (CB). Muller cell endfeet (arrowheads) are labeled. (e) 'l'reat~ed with nonimmune antibody.
Fro. 2. lndilvet immunofluorescence of a I)32 rat retina. 142x. ( a ) T r e a t e d with nonirnmune antibody. (b) Treated with anti-CRBP. The retinal pigment epithelium (RPE) is heavily labeled. There m staining of Muller cell end t (M) and ~ i n t staining of two fine laminae (arrowheads)in the inner plexifi3rm l ~ e r . *, autofluoreseent blood vessels.
Muller cell endfeet (Fig. 4). The staining of Muller radial processes beca e more intense during the first week, and by P l 0 positively stained Muller solnata were present in the inner nuclear layer. By PI4, Muller cell radial processes and ramifications in the plexiform layers were heavily stained spanning the retina f r o the inner to outer limiting mernbranes [Fig. l(a)].
302
A.,I. E I S E N F E I ,
I) E T AI,.
F1o. 3. Indirect immunofluoreseenee of P6 r a t retin~ treated with anti-CR, A L B P . (a,h ~ 1 4 2 × ; e ~-56 x ) (a) Ora serrata anti ciliary body. Tile retinal pigment epithelium ( R P E ) and ciliary body p i g m e n ~ d epithelium (CB) are labeled. The inner epithelium of the iris (arrowhead) is unlabeled. There is labeling of pro awes at the vitrea[ surface of the retina (arrow). NB, neurobl ts; *, autofluorescent red blood cells' sehoroid. (b)Ciliary i~ody and iris. Tim ciliary body pigmented epithelium (CB)and outer epithelium of the iris (arrowhead) are labeled. The unl)igmented ciliary body pigment epithelium and the inner epithelium of the iris ( * ) a negative. LC, lens capsule. (e) 1)6 rat retina treated with nonimmune antibody.
FIo. 4. I n d i ~ c t immunofluorescence of the inner p o t i o n of a rat retina, 296 x . ( a ) T r e a t e d with nonimmune antibody. (b) T r e a ~ d with a n t i - C R A L B P . N o ~ +_-he faint ~taining of processes in the neurob|as¢ic layer (NB) and the more intense staining of processes near the vitreal su~Stce of the retina. V, v i t ~ o u s .
R E T I N O I D - B I N D I N G PROTEINS IN RAT RETINA
303
4. D i s c u s s i o n
A n t i - C R B P was localized in tile R P E cytoplasm of th e developing rat eye a t all ages examined. This is consistent with t h e e v i d e n c e t h a t R P E cells have receptors for R B P on the basolateral p l a s m a l e m m a (Bok and Heller, 1976; Bok, 1982). Staining with a n t i - C R B P was p r o m i n e n t at all times in the R P E b u t was a b s e n t from the CPE, even though this layer of epithelium also has sdrface ~eceptors for retinol-binding protein (Bok and Heller, 1976). Differing a m o u n t s of CP~BP in contiguous cells of the same differentiated tissue have also been found in the e p i d i d y m i s by P o r t e r et al. (1983), who suggested t h a t one m a j o r function of CI~BP, and t h u s retinol, m i g h t be m a i n t e n a n c e of the high rate o f g e n o m i c expression t h a t occurs in certain differentiated cells with increased need for retinol. CR, B ~ ! I I ) , a n o t h e r cellular retinol-binding protein, is found in the small intestine of the rat. Crow an d Ong (1985) found a b r u p t t e r m i n a t i o n of labeled epithelium a t the gastroduodenal an d ileocecal junctions. Age-related changes in the level and localization of C R B P h a v e been d o c u m e n t e d in the liver, where the a m o u n t of CI:tBP increases sharply at birth and declines after 21 days to lower levels characteristic of the a d u l t (Ong a n d Chytil, 1976; K ato , K a t o and Goodman, 1985). The present s t u d y d e , t r a i n e d tl~at C R A L B P is expressed d u r i n g the first postnatal week in both the R P E and CPE, two epithelia t h a t have a c o m m o n embryologic origin from the o u t e r layer ot" the optic cup. The C P E staining decreases from P8 to P14, after which the C P E stained very faintly with a n t i - C R A L B P . The decline of a n t i - C R A L B P staining during this second p o s tn atal week coincides t e m p o r a l l y with the m a t u r a t i o n of photoreceptor o u t e r s e g m e n t s in the retina (Weidman and K u w a b a r a , 1968). This transitory expression of C R A L B P in the C P E during the first postnatal week was unexpected and its significance remains to be de t e rmi ne d. A n t i - C R A L B P was also localized in Muller cell radial processes in the early post na t a l retina. In the a d u l t r a t retina, the Muller cell basal endfeet, s o m a t a and fine processes t h a t ramify in the plexiform and nuclear layers are heavily stained with anti-CP~ALBP. This staining p a t t e r n is the same as t h a t seen previously in the retina of the a d u l t cow, m onke y, and h u m a n (Bunt-Milam an d Saari, 1983). F a i n t an d variable staining for C R B P was also found in Muller radial processes and endfeet. Unlike a n t i - C R A L B P staining, the label was always restricted to the inner r e t i n a ; Muller s o m a t a and processes in the o u ter plexiform and o u t e r nuclear layers were consistently negative with a n t i - C R B P . Such differences in staining intensity m a y reflect differences in the absolute a m o u n t s of the two antigens present in the retina. I n the bovine retina, the ratio of C R A L B P to C R B P is 7 : 1 (Saari et al., 1984). Localization of C R A L B P to Muller cells raised the u n e x p e c t e d possibility t h a t this glial cell might be involved in the metabolism of v i t a m i n A (Bunt-Milam and Saari, 1983). The report by Bok et al. (1984) and the results reported here s t r e n g t h e n t h a t hypothesis, since it now appears t h a t at least two cellular retinoid-binding proteins, C R A L B P and C R B P , m a y be present in Muller cells. I t will be i m p o r t a n t to confirm these results with methods i n d e p e n d e n t of the specificity of th e antibodies and to elucidate the specific role played by the Muller cell in retinoid metabolism. ACKNOWDEDGMENTS The authors thank Faridah l)ahlan, lngrid Klock, and Gl~g Garwin for technical assistance, Brad Clifton, Ron Jones and Pat Siedlak for photographic help, and Julie Seng for secretarial assistance.
304
A.J. EISENFEI,1) ET AL. REFE
RENCI,~S
Bashor, M. M., Taft, D. O. and Chyti[, F. (1973). In vitro bmt~,nv, :£ re~:uc! i~) rat tissue components. Proc. Natl. Acad. Sci. U.S.A. 70, 3483-7. Bok, D. (1982). Autoradiographic studies on the polarity of plasma mcmJ.~rane receptors in retinal pigment epithelialcells.|n The Structure of the E~/e (Ed. Hollyfield,J. G.). P. 247. Else mr Biomedical, N e w York. Bok, D. and Heller, J. (1976). Transport of retinal from the hlood to the retina: an autoradiographic study of the pigment epithelial cell surface receptor for plasma ' O retinal-binding protein. Exp. Eye Res. 22, 395-40,. Bok, D., Ong, D. E. and Chytil, F. (1984). ]mmunoeytoehemieal [oealization of retinalbinding proteins in the retina. Invest, Ophthalmol. Vis. Sci. 25, 877-83. Bunt-Milam, A. H. and Saari, J. C. (1983). Immunocytochemical localizationof two retinoidbinding proteins in veri~hrate retina. J. Cell Biol. 97, 703-12. Bunt-Milam, A. H., Eisenfeld, A. J. and Saari,J. C. (I984). Immunoeytochemieal localization of retinoid-binding proteins in adult and developing retinas, ln~est. Opbthalmol. l'is. 8ci. Suppl. 25, 276. Crow, J. A. and Ong, 1). E. (! 985). Cell specific immunohistochemical localization of a cellular retinal-Binding protein (type two) in the small intestine of the rat. Prec. Natl. Acttd. Sci. U.S.A. 82, 4707-II. Futterman, S. and Saari, J. C. (1977). Occurrence of 1i-cis-retinal-binding protein restricted to the retina. Invest. Ophthal~ol. Vis. Sci. 16, 768-71. Futterman, S., Saari, J.C. and Blair, S. (1977). Occurrence of a binding protein fi~r l l-cis-retinal in the retina. J. Biol. Chem. 252, 3267-71. Heller, J. (1975). Interaction of plasma retinal-binding protein with its receptor. J. Biol. Ch,em. 250, 3613-19. Ong, 1). E. (1984). A novel retinol-tfinding protein from rat: l)urification and partial characterization. J. Biol. Chen~. 259, 1476-82. Ong, D. E. and Chytil, FI (1976).-Changes in the levels of cellular retinal- and retinoic acid-binding proteins of liver and lung during perinatal development of rat. Proc. Natl. Acad. Sci. U.S.A. 73, 3976-8. Porter, S.B., Fraker, L. l)., Chytil, F. and Ong, I). E. (1983). Localization of cellular retinal-binding protein in several rat tissues. Proc. Natl. Acad. Sci. U.S.A. 80, 6586-90. Rask, L. and Peterson, P. A. (l (.)76). In vitro uptake of vitamin A from retinal-binding plasma protein to mucosal epithelial cells from the monkey's small intestine. J. Biol. Chem. 251, 6360-6. Saari, J. C., Bunt-Milam, A. H., Bredberg, D. L. and Garwin, G. G. (1984). Properties and immunocytoehemical localization of three retinoid-binding proteins fi'om bovine retina. Vision Res. 24, 1595-603. Takase, S., Ong, D. E. and Chytil, F. (1979). Cellular retinal-binding protein alh)ws specific interaction of retinal with the nucleus in vitro. Proc. Natl. Acad. ,Nci. U.S.A. 76. 2204-8. Weidman, T. A. and Kuwabara, T. (1968). Postnatal development of the rat retina. Arch. Ophthalmol. 79, 470-84.