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Preparation and characterization of bovine retinal pigment epithelial cell plasma membrane Laird, Dale W. 1984

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PREPARATION AND CHARACTERIZATION OF BOVINE RETINAL PIGMENT E P I T H E L I A L CELL PLASMA MEMBRANE by DALE W.LAIRD B . S c . , U n i v e r s i t y of P r i n c e Edward I s l a n d , C h a r l o t t e t o w n , P r i n c e Edward I s l a n d , 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF BIOCHEMISTRY FACULTY OF MEDICINE UNIVERSITY OF BRITISH COLUMBIA We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA O c t o b e r , 1 9 8 4 © D a l e W. L a i r d , 1984 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department/, or by his or her representatives. I t i s under s tood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. k o '/A. Department of (^^>^^d^^ The University of B r i t i s h CoMimbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date DE-6 (3/81) - i i -ABSTRACT A 7-9 fold enriched preparation of bovine r e t i n a l pigment e p i t h e l i a l c e l l plasma membrane was prepared and characterized by enzymatic analysis. SDS-polyacrylamide gel electrophoresis and transmission electron microscopy revealed a large rod outer segment contamination in the preparation due to a tight r e t i n a l pigment e p i t h e l i a l c e l l u l a r adhesion to the rod photoreceptor c e l l s . The contaminating rhodopsin was p a r t i a l l y removed by a n t i -rhodopsin immunoaffinity chromatography as determined by SDS-polyacrylamide gels stained with coomassie blue or s i l v e r . Monoclonal antibodies raised against the r e t i n a l pigment e p i t h e l i a l plasma membrane preparation cross reacted with rod outer segment preparations. A monoclonal antibody, designated Rho-5A3, was c l a s s i f i e d as an I cJ G3 kappa l i g h t chain immunoglobulin. It was shown to be s p e c i f i c for rhodopsin as determined by radioimmune labeling of bovine rod outer segment membrane proteins e l e c t r o p h o r e t i c a l l y transferred to CNBr-activated paper. Limited proteolytic digestion of rhodopsin followed by electrophoretic transfer to CNBr- activated paper l o c a l i z e d the binding s i t e of t h i s antibody to the N-terminal two-thirds of the rhodopsin molecule. Competition assays with rhodopsin polypeptides further defined the antigenic s i t e to be within the 17-39 amino-acid segment of rhodopsin. The Rho-5A3 antibody did not bind to sealed ROS discs or frozen-thawed ROS discs but did bind to Triton X-100 s o l u b i l i z e d discs indicating a detergent s o l u b i l i z a t i o n dependence for antigenic s i t e a c c e s s i b i l i t y . - i i i -C u l t u r e s o f b o v i n e r e t i n a l pigment e p i t h e l i a l c e l l s were s t a r t e d by i n i t i a l e n z y m a t i c i s o l a t i o n f o l l o w e d by r e c o v e r y i n RPMI-1640 c u l t u r e medium. The r e t i n a l p i g m e n t e p i t h e l i a l c e l l s e s t a b l i s h e d a d o u b l i n g t i m e of 52 h o u r s u n t i l t h e c e l l s r e a c h e d c u l t u r e c o n f l u e n c y . The c e l l s a l s o m a i n t a i n e d many of t h e i r i n  v i v o c h a r a c t e r i s t i c s s u c h a s a h i g h d e g r e e of p i g m e n t a t i o n and an abundance of m i c r o v i l l i . C e l l s u r f a c e g l y c o p r o t e i n s l a b e l e d w i t h FITC-Con A, FITC-WGA, and FITC-RCA showed dense and random s u r f a c e l a b e l i n g p a t t e r n s . F l u o r e s c e n t l a b e l s were i n d u c e d t o r e d i s t r i b u t e t o c e n t r a l s p o t s and c l e a r f r o m t h e c e l l s u r f a c e by i n c u b a t i n g t h e l a b e l e d c e l l s i n b u f f e r f o r 60 m i n u t e s a t 37°C. T r e a t m e n t by t h e a p p r o p r i a t e s a c c h a r i d e i n h i b i t o r s i n d i c a t e d t h a t t h e l a b e l e d s i t e s had u n dergone e n d o c y t o s i s by t h e c e l l . C o n t i n u o u s l a b e l i n g e x p e r i m e n t s i n d i c a t e d t h a t r e d i s t r i b u t i o n and i n t e r n a l i z a t i o n i s c o n s t a n t l y o c c u r i n g so t h a t p r e v i o u s l y u n l a b e l e d r e c e p t o r s become a c c e s s i b l e f o r l a b e l i n g . As a r e s u l t a d e n s e p a t t e r n of l a b e l on t h e c e l l s u r f a c e was m a i n t a i n e d . The p r o t e i n a c t i n , w i t h a p p a r e n t M =46,000, was d e t e c t e d w i t h r a b b i t a n t i - a c t i n a n t i s e r a l a b e l i n g of t h e RPE p l a s m a membrane p r e p a r a t i o n p r o t e i n s e l e c t r o p h o r e t i c a l l y t r a n s f e r r e d t o n i t r o c e l l u l o s e p a p e r . I m m u n o f l u o r e s c e n t l a b e l i n g u s i n g t h e r a b b i t a n t i - a c t i n a n t i s e r a c o n f i r m e d b i o c h e m i c a l s t u d i e s t h a t a c t i n was a major component of t h e b o v i n e RPE c e l l . The a c t i v a t i o n of a c t i n f i l a m e n t s may p l a y an i m p o r t a n t r o l e i n t h e p h a g o c y t o s i s of b o v i n e r o d o u t e r segments by r e t i n a l p i gment e p i t h e l i a l c e l l s i n t i s s u e c u l t u r e . - i v -S c a n n i n g e l e c t r o n m i c r o s c o p y and t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y have shown t h a t 2 week o l d b o v i n e r e t i n a l pigment e p i t h e l i a l c e l l s i n v i t r o c a n be i n d u c e d t o r e c o g n i z e , a t t a c h , and e n g u l f d a r k a d a p t e d s e a l e d r o d o u t e r segments. In summary a m o n o c l o n a l a n t i b o d y was r a i s e d a g a i n s t a b o v i n e r e t i n a l pigment e p i t h e l i a l c e l l p l a s m a membrane p r e p a r a t i o n , however, t h e a n t i b o d y r a i s e d p r o v e d t o be s p e c i f i c f o r r h o d o p s i n , a c o n t a m i n a t i n g ROS p r o t e i n f o u n d i n t h e p r e p a r a t i o n . The m o n o c l o n a l a n t i b o d y , d e s i g n a t e d Rho-5A3, was f u l l y c h a r a c t e r i z e d and i t s a n t i g e n i c s i t e d e t e r m i n e d . F i n a l l y , b o v i n e r e t i n a l p i g m e n t e p i t h e l i a l c e l l s grown i n t i s s u e c u l t u r e a c t e d as a model s y s t e m f o r s t u d y i n g c e l l s u r f a c e components and r o d o u t e r segment p h a g o c y t o s i s a t t h e l e v e l s of f l u o r e s c e n c e , s c a n n i n g e l e c t r o n m i c r o s c o p y , and t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y . - v -ACKNOWLEDGEMENTS I would l i k e to thank Dr. Robert Molday for his patience and guidance in the supervision of thi s work. I would also l i k e to extend my admiration and gratitude to Dr. Dave Hicks for his help with a l l electron microscopic techniques and his donation of the Lowicryl sections used. F i n a l l y , I would l i k e to thank Don Mackenzie, Simon Wong, and Laurie Molday for their excellent theore t i c a l and technical advice. - v i -L I S T OF ABBREVIATIONS BSA b o v i n e serum a l b u m i n CHAPS 3 - [ ( 3 - c h o l a m i d o p r o p y l ) d i m e t h y l a m m o n i o ] - 1 - p r o p a n e s u l f o n a t e Con A c o n c a n a v a l i n A DMSO d i m e t h y l s u l f o x i d e EDTA e t h y l e n e d i a m i n e t e t r a a c e t a t e FCS f e t a l c a l f serum F I T C f l u o r e s c e i n i s o t h i o c y a n a t e HAT h y p o x a n t h i n e , a m i n o p t e r i n , t h y m i d i n e IMDM I s c o v e ' s M o d i f i e d D u l b e c c o ' s Medium Ig i m m u n o g l o b u l i n M r m o l e c u l a r w e i g h t PBS p h o s p h a t e - b u f f e r e d s a l i n e PEG p o l y e t h y l e n e g l y c o l PMSF p h e n y l m e t h y l s u l f o n y l f l u o r i d e RIA radioimmune a s s a y RCA R i c i n i u s communis a g g l u t i n i n ROS r o d o u t e r segment RPE r e t i n a l p i gment e p i t h e l i a l SDS sodium d o d e c y l s u l f a t e SEM s c a n n i n g e l e c t r o n m i c r o s c o p y TCA t r i c h l o r o a c e t i c a c i d •TEM t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y T r i s t r i s ( h y d r o x y m e t h y l ) aminomethane WGA wheat germ a g g l u t i n i n - v i i -TABLE OF CONTENTS PAGE ABSTRACT i i ACKNOWLEDGEMENTS v LIST OF ABBREVIATIONS v i TABLE OF CONTENTS v i i LIST OF TABLES x i LIST OF FIGURES x i i INTRODUCTION 1 . Retina 1 2. Photoreceptor c e l l s 1 3. Location of the r e t i n a l pigment e p i t h e l i u m 4 4. Function of the r e t i n a l pigment e p i t h e l i u m 4 5. Pathology of the r e t i n a l pigment e p i t h e l i u m 5 6. Process of phagocytosis 8 7. The c y t o s k e l e t a l system i n phagocytosis 9 8. Energy source of phagocytosis 11 9. Latency p e r i o d i n phagocytosis 11 10. Shedding or "pinching o f f " of ROS d i s c packets.. 12 11. RPE c e l l s i n t i s s u e c u l t u r e 13 12. E f f e c t of aging on RPE c e l l morphology and enzyme a c t i v i t y 14 13. L e c t i n c e l l surface l a b e l s 15 14. Immunological approaches to studying membranes.. 16 15. Thesis i n v e s t i g a t i o n 17 - v i i i -EXPERIMENTAL PROCEDURES 1 . M a t e r i a l s 19 2. P r o t e i n a s s a y s 20 3. I s o l a t i o n o f b o v i n e RPE c e l l s 20 4. P r e p a r a t o n of b o v i n e RPE c e l l p l a s m a membrane... 22 5. Enzyme a s s a y s A. 5 ± n u c l e o t i d a s e 23 B. N a + K + ATPase 24 C. NADPH c y t o c h r o m e c r e d u c t a s e 24 D. S u c c i n a t e c y t o c h r o m e c r e d u c t a s e 25 6. S D S - g e l e l e c t r o p h o r e s i s and g e l t r a n s f e r 25 7. P r e p a r a t i o n of g o a t a n t i - m o u s e I g and g o a t a n t i -r a b b i t I g a n t i b o d y r e a g e n t s 27 8. P o l y p e p t i d e d e t e c t i o n by a n t i b o d y 27 9. S i l v e r s t a i n t e c h n i q u e 28 10. M o n o c l o n a l a n t i b o d y t e c h n i q u e s A. I m m u n i z a t i o n o f BALB/C mice 28 B. P r e p a r a t i o n o f t i s s u e c u l t u r e myeloma c e l l s 28 C. P r o d u c t i o n of h y b r i d o m a c e l l s 29 D. S t a n d a r d RIA 31 E . Hybridoma c l o n i n g . . 31 F. S t o r a g e of v i a b l e h y b r i d o m a c e l l s 32 G. S o l i d - P h a s e c o m p e t i t i o n a s s a y s 32 H. L o w i c r y l t h i n s e c t i o n l a b e l i n g 33 11. A d u l t b o v i n e RPE c e l l s i n t i s s u e c u l t u r e A. C u l t u r i n g of b o v i n e RPE c e l l s 34 B. F l u o r e s c e n t d e t e c t i o n of a c t i n i n RPE c e l l s 34 - i x -C. F l u o r e s c e n t l e c t i n l a b e l e d b o v i n e RPE c e l l s 35 D. D i s c o n t i n u o u s f l u o r e s c e n t l e c t i n l a b e l i n g . . 36 E. C o n t i n u o u s f l u o r e s c e n t l e c t i n l a b e l i n g 36 12. P h a g o c y t o s i s o f b o v i n e ROS by b o v i n e RPE c e l l s A. P r e p a r a t i o n o f s e a l e d d a r k a d a p t e d ROS 37 B. P h a g o c y t o s i s o f ROS by a d u l t b o v i n e RPE c e l l s i n v i t r o . . 37 C. P r e p a r a t i o n o f samples f o r TEM 38 D. P r e p a r a t i o n of samp l e s f o r SEM 38 SECTION 1 ANALYSIS OF BOVINE RPE PLASMA MEMBRANE PREPARATIONS RESULTS 1. P r e p a r a t i o n of b o v i n e RPE plasma membrane 40 2. D e t e c t i o n o f a c t i n i n t h e RPE p l a s m a membrane p r e p a r a t i o n 46 3. TEM o b s e r v a t i o n o f s u b c e l l u l a r f r a c t i o n a t i o n . . . . 46 DISCUSSION 49 SECTION 2 THE PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES RESULTS 1. P r o d u c t i o n o f m o n o c l o n a l a n t i b o d i e s 52 2. C h a r a c t e r i z a t i o n o f Rho-5A3 a n t i b o d y 52 3. S u b c l a s s i f i c a t i o n o f Rho-5A3 a n t i b o d y 56 4. I d e n t i f i c a t i o n o f Rho-5A3 a n t i g e n o f b o v i n e ROS membrane 56 5. C o m p e t i t i v e i n h i b i t i o n of Rho-5A3 a n t i b o d y 59 - x -6. A n a l y s i s o f Rho-5A3 a n t i b o d y b i n d i n g t o r h o d o p s i n p e p t i d e s 59 7. L o w i c r y l t h i n s e c t i o n l a b e l i n g 62 DISCUSSION 65 SECTION 3 ANALYSIS OF BOVINE RPE CELLS IN VITRO RESULTS 1. I s o l a t i o n o f b o v i n e RPE c e l l s 70 2. The m o r p h o l o g y o f b o v i n e RPE c e l l s i n v i t r o 70 3. F l u o r e s c e n t d e t e c t i o n of a c t i n 74 4. D i s c o n t i n u o u s l e c t i n l a b e l i n g of b o v i n e RPE 74 5. C o n t i n u o u s f l u o r e s c e n t l e c t i n l a b e l i n g of b o v i n e RPE c e l l s i n v i t r o 78 6. P r o b i n g RPE c u l t u r e d c e l l s w i t h Rho-1D4 84 7. P h a g o c y t o s i s of ROS by b o v i n e RPE c e l l s i n v i t r o 84 DISCUSSION 91 CONCLUSIONS 95 BIBLIOGRAPHY 97 - x i -L I S T OF TABLES TABLE PAGE 1. D i s t r i b u t i o n of e n z y m a t i c m a r k e r s i n v a r i o u s f r a c t i o n s r e c o v e r e d d u r i n g membrane i s o l a t i o n . . . 41 2. C o m p a r i s o n of Rho-5A3 m o n o c l o n a l a n t i b o d y b i n d i n g t o T r i t o n X-100 and SDS s o l u b i l i z e d r o d o u t e r segments 55 3. I d e n t i f i c a t i o n o f Rho~5A3 m o n o c l o n a l a n t i b o d y I g s u b t y p e 57 - x i i -LIST OF FIGURES FIGURE PAGE 1. Diagram t o i l l u s t r a t e the l a y e r s of the r e t i n a 2 2. Diagram of a rod p h o t o r e c e p t o r c e l l 3 3. Coomassie b l u e and s i l v e r s t a i n e d S D S - p o l y a c r y l a m i d e g e l s of v a r i o u s membrane f r a c t i o n s 44 4. A RPE c e l l homogenate f r a c t i o n a t i o n p r o f i l e of a t30-50% (w/w) c o n t i n u o u s s u c r o s e g r a d i e n t 45 5. D e t e c t i o n of a c t i n i n r o d o u t e r segments and bovine RPE plasma membrane p r e p a r a t i o n by SDS-poly-a c r y l a m i d e g e l e l e t r o p h o r e s i s and immunoblot 47 6. T r a n s m i s s i o n e l e c t r o n m i c r o g r a p h s of b o v i n e RPE c e l l plasma membrane p r e p a r a t i o n s 48 7. A n t i b o d y t i t r a t i o n c u r v e f o r b l o o d from a BALB/C mouse immunized w i t h bovine RPE plasma membranes .... 53 8. T i t r a t i o n c u r v e of Rho-5A3 hybridoma c u l t u r e f l u i d a g a i n s t s o l u b i l i z e d r o d o u t e r segments 54 9. A n a l y s i s of p o l y p e p t i d e s from u n t r e a t e d and p r o t e a s e d i g e s t e d rod o u t e r segments by monoclonal a n t i b o d y b i n d i n g 58 10. I n h i b i t i o n of Rho~5A3 a n t i b o d y b i n d i n g t o s o l u b i l i z e d rod o u t e r segments by r h o d o p s i n 60 11. I n h i b i t i o n of Rho-5A3 a n t i b o d y b i n d i n g t o s o l u b i l i z e d ROS d i s k s by fr o z e n / t h a w e d , s e a l e d , and s o l u b i l i z e d ROS d i s k s ' . . 61 - x i i i -12. Inhibition of Rho~5A3 antibody binding to s o l u b i l i z e d rod outer segments by rhodopsin polypeptides 63 13. Transmission electron micrograph of a Rho~5A3 antibody gold dextran labeled rod photoreceptor c e l l embedded in Lowicryl resin 64 14. A diagrammatic model of rhodopsin 66 15. Bovine r e t i n a l pigment e p i t h e l i a l c e l l s in v i t r o 71 16. Scanning electron micrograph of a 10 day old bovine r e t i n a l pigment e p i t h e l i a l c e l l 72 17. Scanning electron micrograph of a 10 day old bovine r e t i n a l pigment e p i t h e l i a l c e l l 73 18. Scanning electron micrograph of a 10 day old bovine r e t i n a l pigment e p i t h e l i a l c e l l grown on glass 75 19. Fluorescent micrographs of 2 week old bovine RPE c e l l s incubated with rabbit a n t i - a c t i n antisera 76 20. Fluorescent micrographs of unfixed RPE c e l l s labeled with FITC-Con A 79 21. Fluorescent micrographs of unfixed RPE c e l l s labeled with FITC-WGA 80 22. Fluorescent micrographs of unfixed RPE c e l l s labeled with FITC-RCA 81 23. ' Fluorescent micrographs of unfixed RPE c e l l s labeled continuously with FITC-Con A, FITC-WGA, and FITC-RCA. 82 24. Rho-1D4 monoclonal antibody binding to bovine RPE c e l l s in tissue culture 85 -xiv-25. S c a n n i n g e l e c t r o n m i c r o g r a p h o f a 2 week o l d b o v i n e RPE c e l l i n c u b a t e d f o r 5 h w i t h s e a l e d r o d o u t e r segments 86 26. T r a n s m i s s i o n e l e c t r o n m i c r o g r a p h s o f b o v i n e RPE c e l l s i n c u b a t e d w i t h d a r k a d a p t e d s e a l e d r o d o u t e r segments f o r 5 h i n v i t r o 88 27. T r a n s m i s s i o n e l e c t r o n m i c r o g r a p h of a n o t h e r b o v i n e RPE c e l l i n c u b a t e d w i t h d a r k a d a p t e d s e a l e d r o d o u t e r segments f o r 5 h i n v i t r o 89 - 1 -INTRODUCTION RETINA The r e t i n a has been e x t e n s i v e l y s t u d i e d o v e r t h e y e a r s as i t r e p r e s e n t s a h i g h l y complex e x t e n s i o n o f t h e n e r v o u s s y s t e m . D e v e l o p m e n t a l l y and f u n c t i o n a l l y , t h e r e t i n a i s an i s o l a t e d p a r t of t h e c e n t r a l n e r v o u s s y s t e m t o w h i c h i t r e m a i n s c o n n e c t e d by t h e o p t i c n e r v e . The v i s u a l r e t i n a c o m p r i s e s a t h i n n e r v o u s l a y e r l i n i n g t h e p o s t e r i o r p a r t of t h e e y e b a l l . In e m b r y o n i c d e v e l o p m e n t , t h e o p t i c v e s i c l e becomes t r a n s f o r m e d i n t o a t w o - l a y e r e d o p t i c cup, th e o u t e r l a y e r f o r m i n g t h e pigment e p i t h e l i u m , and t h e i n n e r l a y e r becoming t h e n e u r a l r e t i n a . L i g h t p a s s e s t h r o u g h t h e n e u r a l r e t i n a l a y e r s c o n s i s t i n g of t h e o p t i c n e r v e f i b e r l a y e r , g a n g l i o n c e l l l a y e r , i n n e r p l e x i f o r m l a y e r , i n n e r n u c l e a r l a y e r , o u t e r p l e x i f o r m l a y e r , o u t e r n u c l e a r l a y e r and p h o t o r e c e p t o r c e l l s , r e s p e c t i v e l y ( F i g . 1 ) . PHOTORECEPTOR CELLS V e r t e b r a t e p h o t o r e c e p t o r c e l l s c o n s i s t o f m o d i f i e d n e u r o n s c a l l e d r o d c e l l s and cone c e l l s . The cone c e l l s a r e r e s p o n s i b l e f o r c o l o r v i s i o n w h i l e t h e more numerous r o d c e l l s a r e r e s p o n s i b l e f o r b l a c k and w h i t e v i s i o n . The b o v i n e r o d c e l l s a r e s l e n d e r c e l l s a p p r o x i m a t e l y 60 um l o n g w i t h an a v e r a g e w i d t h of a b o u t 1.5 t o 2 um. The r o d c e l l i s d i v i d e d i n t o a r o d o u t e r segment p o r t i o n and a r o d i n n e r segment c o n n e c t e d by a t h i n c i l i u m ( F i g . 2 ) . The r o d o u t e r segment c o n s i s t s of s t a c k s o f d i s c - 2 -F i g u r e 1. D i a g r a m t o i l l u s t r a t e t h e l a y e r s of t h e r e t i n a . ( 1 . ) Pigment e p i t h e l i a l . (2.) L a y e r s o f r o d s and c o n e s . (3.) E x t e r n a l l i m i t i n g membrane. (4.) O u t e r n u c l e a r l a y e r . (5.) O u t e r p l e x i f o r m l a y e r . (6.) I n n e r n u c l e a r l a y e r . (7.) I n n e r p l e x i f o r m l a y e r . (8.) G a n g l i o n c e l l l a y e r . (9.) O p t i c f i b e r l a y e r . (10.) I n t e r n a l l i m i t i n g membrane. The arrow i n d i c a t e s t h e d i r e c t i o n of l i g h t . D i a g r a m t a k e n from r e f e r e n c e 2. -3-Outer segment - / Cilium i Inner segment Outer rod fiber Cell body Inner rod fiber Rod spherule F i g u r e 2. Diagram of a r o d p h o t o r e c e p t o r c e l l a s seen by t h e e l e c t r o n m i c r o s c o p e . Note t h e l a r g e p o r t i o n of o u t e r segment membrane. D i a g r a m t a k e n from r e f e r e n c e 2. -4-membrane. The i n n e r segment c o n t a i n s a l l t h e c y t o p l a s m i c o r g a n e l l e s and m e t a b o l i c m a c h i n e r y w h i l e 90% o f t h e o u t e r segment membrane i s t h e p h o t o p i g m e n t r h o d o p s i n . The t i p of t h e o u t e r segment i s embedded i n t h e pigment e p i t h e l i u m . LOCATION OF THE RETINAL PIGMENT EPITHELIUM The c u b o i d a l m o n o s t r a t i f i e d r e t i n a l p i g m e n t e p i t h e l i u m (RPE) o r i g i n a t i n g from t h e e x t e r n a l l a y e r of t h e o p t i c v e s i c l e , r e p r e s e n t s t h e o u t e r most l a y e r of t h e v i s u a l r e t i n a . The RPE c o v e r s t h e whole p o s t e r i o r p a r t of t h e e y e b a l l a s f a r as t h e o r a s e r r a t a and i s p r e d o m i n a n t l y d a r k - c o l o r e d i n most s p e c i e s owing t o t h e m e l a n i n pigment i n i t s c e l l s . The b a s a l f a c e o f t h e pigment e p i t h e l i u m r e s t s on a b a s a l l a m i n a w h i c h r e p r e s e n t s t h e i n n e r l a y e r of B r u c h ' s membrane. T h i s s t r u c t u r e f u n c t i o n s i n t h e a d h e s i o n o f t h e pigment e p i t h e l i u m t o t h e c h o r o i d . The a p i c a l s u r f a c e of t h e r e t i n a l p i gment e p i t h e l i u m h a s m i c r o v i l l i w h i c h i n t e r d i g i t a t e t h e o u t e r segments of t h e p h o t o r e c e p t o r c e l l ( 1 ) . THE FUNCTION OF THE RETINAL PIGMENT EPITHELIUM RPE c e l l s have been shown t o be i n v o l v e d i n t h e c o n t i n u o u s r e n e w a l o f membranes i n t h e r o d o u t e r segments ( 1 ) . The p r o t e i n s y n t h e s i z e d i n t h e i n n e r segment p a s s t h r o u g h t h e c o n n e c t i n g c i l i u m t o form new d i s c s a t t h e b a s e of t h e r o d o u t e r segment. At t h e same t i m e , g r o u p s of d i s c s a r e s h e d a t t h e apex o f t h e o u t e r segment and e n g u l f e d i n t h e c y t o p l a s m o f t h e p i g m e n t e p i t h e l i u m t o form phagosomes. F i n a l l y , t h e phagosomes a r e d e g r a d e d i n t h e s e RPE c e l l s . - 5 -I t i s now e s t a b l i s h e d t h a t d u r i n g l i g h t a d a p t a t i o n o f t h e eye , t h e r e t i n a l d e h y d e l i b e r a t e d from v i s u a l pigment d u r i n g b l e a c h i n g i n t h e o u t e r segment i s r e d u c e d t o r e t i n o l and m i g r a t e s i n t o t h e pigment e p i t h e l i u m ( 1 ) . The r e v e r s e p r o c e s s t a k e s p l a c e d u r i n g d a r k a d a p t a t i o n . Thus t h e c e l l s o f t h e pigment e p i t h e l i u m p l a y a v i t a l r o l e i n t h e v i s u a l p r o c e s s . M o r e o v e r t h e pigment e p i t h e l i u m p r e v e n t s r e f l e c t i o n by a b s o r b i n g l i g h t a nd i s e s s e n t i a l f o r t h e f o r m a t i o n o f r h o d o p s i n by s t o r i n g and r e l e a s i n g v i t a m i n A ( 2 ) . T h e r e e x i s t s i n t h e RPE c e l l s a v e r y s t r o n g c e l l u l a r p o l a r i t y . The s c l e r a l f a c e of t h e c e l l , w i t h i t s b a s a l i n f o l d i n g s g i v i n g r i s e t o c o a t e d v e s i c l e s and t h e n e i g h b o u r i n g m i t o c h o n d r i a , r e p r e s e n t s a s i t e of a b s o r p t i o n and a c t i v e t r a n s p o r t i n t h e v a s c u l a r r e t i n a of mammals. The a p i c a l s u r f a c e , w i t h i t s p i g m e n t e d p r o c e s s e s i n i n t i m a t e c o n t a c t w i t h p h o t o r e c e p t o r o u t e r segments, i n d i c a t e s t h e c o m p l e m e n t a r i t y o f t h e pigment e p i t h e l i u m and t h e v i s u a l c e l l s of" t h e r e t i n a , s i n c e e x c h a n g e s a r e i m p o r t a n t and c o n t i n u o u s between t h e s e two l a y e r s . PATHOLOGY OF RETINAL PIGMENT EPITHELIUM The p a t h o l o g y o f t h e RPE c a n be s e p a r a t e d i n t o two c l a s s e s : d y s f u n c t i o n o f p o l a r i z a t i o n and p i g m e n t a t i o n of t h e c e l l ; and d y s f u n c t i o n of p h a g o c y t o s i s ( 1 ) . In t h i s p a p e r a t t e n t i o n i s p a i d more t o t h e l a t t e r . S e v e r a l r e t i n a l p a t h o l o g i e s a r e t h e r e s u l t of a d e f i c i e n c y i n t h e p h a g o c y t o s i s f u n c t i o n , w h i c h can be e x p r e s s e d a t d i f f e r e n t s t a g e s of t h e p h a g o c y t i c p r o c e s s . The m a n i f e s t a t i o n of p i g m e n t r e t i n o p a t h i e s of t h e r e t i n i t i s p i g m e n t o s a t y p e -6-i n v o l v e s d e r r a n g e m e n t o f t h e f i r s t s t e p of p h a g o c y t o s i s ; t h a t i s , t h e r e c o g n i t i o n and a d s o r p t i o n of r o d o u t e r segment m a t e r i a l by t h e a p i c a l p r o c e s s e s . The r a t C h i m e r a s a r e p a r t i c u l a r l y i n f o r m a t i v e a s t h e r d y gene i s a c t i n g e x t r i n s i c a l l y t o t h e p h o t o r e c e p t o r c e l l , but a l s o t h a t t h e pigment e p i t h e l i a l c e l l i s t h e a c t u a l s i t e o f t h e gene a c t i o n ( 3 ) . I n h e r i t e d r e t i n a l d e g e n e r a t i o n i n t h e R o y a l C o l l e g e of S u r g e o n s (RCS) r a t has been shown t o be a r e s u l t o f a s t r u c t u r a l or f u n c t i o n a l d e f e c t i n t h e r e t i n a l pigment e p i t h e l i a l c e l l s ( 3 , 4 , 5 ) . T h i s i m p o r t a n t d i s e a s e d r a t l i n e has been u s e d e x t e n s i v e l y i n s t u d y i n g t h e mechanism o f p h a g o c y t o s i s . I n i t i a l l y , i n t h e RCS r a t , normal p h o t o r e c e p t o r s h e d d i n g o c c u r s d u r i n g which t h e r e i s a c o n c o m i t a n t a c c u m u l a t i o n o f l a m e l l a r b o d i e s i n t h e e x t r a c e l l u l a r s p a c e between t h e p h o t o r e c e p t o r o u t e r segments and t h e RPE. T h i s a c c u m u l a t i o n o f l a m e l l a r m a t e r i a l i s a c c o m p a n i e d by p r o g r e s s i v e d e t e r i o r a t i o n of i n d i v i d u a l p h o t o r e c e p t o r c e l l s and a c o n s e q u e n t d e c l i n e i n r e t i n a l s e n s i t i v i t y . The l y s o s o m a l s y s t e m of t h e RPE c e l l s has been r e p o r t e d t o be d e f i c i e n t i n t h e d i s e a s e c a s e r e s u l t i n g i n t h e d e s t r u c t i o n o f r o d o u t e r segments by t h e r e l e a s e o f l y t i c enzymes i n t o t h e i n t e r c e l l u l a r s p a c e . S t u d i e s by L a v a i l a s w e l l as E s s n e r and G o r r i n have d e m o n s t a t e d t h a t macrophages i n v a d e t h e l a y e r o f d e b r i s t h a t a c c u m u l a t e s i n t h e RCS i n t e r p h o t o r e c e p t o r s p a c e as t h e d e g e n e r a t i o n of t h e r e t i n a p r o g r e s s e s ( 6 , 7 ) . T h e r e a r e s e v e r a l h y p o t h e s e s as t o t h e c a u s e of t h e r o d o u t e r segment p h a g o c y t o s i s i n c a p a b i l i t y i n t h e RCS r a t ( 8 ) . F i r s t of a l l t h e p i g m e n t e p i t h e l i a l may l a c k some f a c t o r n e c e s s a r y f o r -7-phagocytosis or contain an abnormal factor that inhibits phagocytic activity. Alternately both the rod outer segments and the RPE may contain defects or complementary defects that are only expressed in connection with each other. Gery and O'Brien demonstrated that peritoneal macrophages from RCS rats exhibit phagocytic capability equal to that found in macrophages from normal strains of rat (8). Thus the genetic defect in RCS pigment epithelium is not expressed in the macrophages. Both normal and dystropic retinal pigment epithelium are capable of phagocytizing carbon particles ( 9 ) . However, when specfic photoreceptor packets are incubated with dystrophic RPE cells, no uptake occurs ( 4 ) . Muller and Lavail have shown that ROS packets from normal or dystrophic animals are equally phagocytized by normal RPE indicating that the pigment epithelium is the primary site of the phagocytic defect ( 3 ) . O'Brien proposed that RPE phagocytosis may be triggered by a change in terminal sugars such as fucose and galactose on plasma membrane glycoproteins of ROS ( 1 0 ) . It is possible that terminal sugars are not recognized by defective RPE because membrane receptors for certain sugars are lacking or masked. Williams-Seyfried and Mclaughlin's results indicate that sugar coated beads when presented to retinal pigment epithelial explants show differences in phagocytosis ( 1 1 ) . Fucose coated beads were not taken up by either normal or dystrophic explants while twice as many mannose beads are phagocytized by the normal as opposed to the dystrophic t i ssue. - 8 -PROCESS OF PHAGOCYTOSIS The p r o c e s s of p h a g o c y t o s i s o c c u r s i n t h r e e f a i r l y d i s t i n c t s t e p s ; f i r s t , r e c o g n i t i o n and a t t a c h m e n t of t h e r o d o u t e r segments t o t h e RPE c e l l ; s e c o n d , e n g u l f m e n t ; and f i n a l l y , p o s t e n g u l f m e n t phenomena w h i c h i n c l u d e s e q u e n t i a l movement and f u s i o n of l y s o s o m e s w i t h t h e phagosome, a d r o p i n p h a g o l y s o s o m e pH, and d i g e s t i o n of t h e p h a g o c y t i z e d m a t e r i a l . The r e c o g n i t i o n p r o c e s s i s most i n t e r e s t i n g t o t h i s s t u d y . I t i n v o l v e s some i l l d e f i n e d e l e c t r i c a l c h a r g e i n t e r a c t i o n s o r more s p e c i f i c i n t e r a c t i o n o f c e l l s u r f a c e m o l e c u l e s . M c l a u g h l i n e t a l . d e v e l o p e d a method f o r r e p l i c a t i n g t h e membrane s u r f a c e s of r a t r e t i n a l pigment e p i t h e l i u m e x p l a n t s d u r i n g p h a g o c y t o s i s o f l a t e x beads ( 1 2 ) . S u r f a c e r e p l i c a s o f i n i t i a l s t a g e s of p h a g o c y t o s i s show t h e a t t a c h m e n t and s p r e a d i n g of m i c r o v i l l i o v e r t h e l a t e x b e a d s . T h i s may be a m o r p h o l o g i c a l movement p r i o r t o i n g e s t i o n t h a t a l l o w s a s e q u e n t i a l and c i r c u m f e r e n t i a l i n t e r a c t i o n of r e c e p t o r s on t h e s u r f a c e of t h e RPE membranes. The beads a r e e n g u l f e d by o v e r l a p p i n g m i c r o v i l l i t h a t r e s e m b l e s "Venus f l y t r a p s " i n s u r f a c e r e p l i c a s . F o l l o w i n g s h o r t e n i n g o f t h e m i c r o v i l l i t h e beads a r e e n g u l f e d l e a v i n g d o u ghnut l i k e i m p r e s s i o n i n t h e s u r f a c e membrane f o l l o w e d by f l a t t e n e d membrane domains ( 1 2 ) . A s i m i l a r s t u d y was done w i t h human and b o v i n e RPE w i t h c o m p a r a b l e r e s u l t s ( 1 3 ) . Many s t u d i e s on p h a g o c y t o s i s have been done w i t h e m b r y o n i c RPE c e l l s i n t i s s u e c u l t u r e . C h a i t i n and H a l l u s e d a ROS a n t i s e r u m and a d o u b l e i m m u n o f l u o r e s c e n t l a b e l i n g p r o c e d u r e f o r a s s a y i n g t h e p h a g o c y t o s i s o f ROS by c u l t u r e d r a t RPE c e l l s ( 1 4 ) . -9-F o l l o w i n g t h e i n c u b a t i o n of RPE c e l l s w i t h ROS, t h e r o d o u t e r segments a t t a c h e d t o t h e s u r f a c e o f t h e c e l l s were l a b e l e d w i t h a ROS a n t i s e r u m i n c o n j u n c t i o n w i t h RH-GARG ( t e t r a m e t h y l r h o d a m i n e c o n j u g a t e d g o a t a n t i - r a b b i t I g G ) . The same c e l l s were d i s r u p t e d u nder a g r a d e d a c e t o n e s e r i e s and t h e i n t e r n a l ROS were l a b e l e d w i t h t h e ROS a n t i s e r u m f o l l o w e d by FITC-GARG ( f l u o r e s c e i n i s o t h i o c y a n a t e c o n j u g a t e d g o a t a n t i - r a b b i t I g G ) . ROS a t t a c h e d t o t h e o u t e r s u r f a c e can be d i s t i n g u i s h e d from t h e r o d s t h a t have u n d e r g o n e p h a g o c y t o s i s by u s i n g d i f f e r e n t f l u o r e s c e n t f i l t e r s . In 14 3 t h e c a s e where C mannose and H c h o l i n e r a d i o l a b e l e d o u t e r segments were u s e d t o s t u d y p h a g o c y t o s i s ( 1 5 ) , no c o n c l u s i v e c o n t r o l was us e d t o d i s t i n g u i s h a t t a c h m e n t f r o m e n d o c y t o s i s . C h a i t i n and H a l l ± s p r o c e d u r e showed t h a t t h e a t t a c h m e n t o f ROS t o d y s t r o p h i c pigment e p i t h e l i a l c e l l s o c c u r r e d a t a n o r m a l r a t e ( 1 4 ) . However, o n l y a s m a l l number of t h e s e ROS were i n g e s t e d . E x p e r i m e n t a l e v i d e n c e s u g g e s t s t h a t i n g e s t i o n o f most ROS o c c u r s w i t h i n 10 m i n u t e s t o 1 hour i n c u b a t i o n t i m e once a t t a c h m e n t has been made t o t h e pl a s m a membrane ( 1 4 , 1 5 ) . H a l l a l s o s u g g e s t s t h e p o s s i b i l i t y t h a t two c l a s s e s o f r e c e p t o r s may e x i s t f o r p h a g o c y t o s i s . One s e t o f r e c e p t o r s t h a t a l l o w s f o r r a p i d u p t a k e of ROS and a s e c o n d s l o w e r s e t of r e c e p t o r s t h a t i s a l s o c a p a b l e of p h a g o c y t o s i s of ROS. The d y s t r o p h i c c a s e would a p p e a r t o l a c k t h e f i r s t o f t h e s e two s e t s of r e c e p t o r s ( 1 4 ) . THE ROLE OF THE CYTOSKELETAL SYSTEM IN PHAGOCYTOSIS W i t h t h e c o n c l u s i o n t h a t t h e i n g e s t i o n p h a s e of p h a g o c y t o s i s i s d e f e c t i v e i n t h e d y s t r o p h i c p i g m e n t e p i t h e l i a l c e l l s , i t - 1 0 -seemed p o s s i b l e t h a t t h i s d e f e c t m i g h t i n v o l v e t h e m o b i l i z a t i o n o r f u n c t i o n i n g of t h e c o n t r a c t i l e p r o t e i n a c t i n . A c t i n i s d i r e c t l y i n v o l v e d i n t h e i n g e s t i o n mechanism i n o t h e r p h a g o c y t i c c e l l s . H a l e y d e m o n s t r a t e d w i t h 2 - d i m e n s i o n a l g e l e l e c t r o p h o r e s i s c o u p l e d t o f l u o r a g r a p h y t h a t a c t i n i s a major human RPE c e l l p r o t e i n and t h a t i t i s a c t i v e l y s y n t h e s i z e d ( 1 6 ) . O t h e r d i s t i n c t p r o t e i n s of t h e RPE may r e p r e s e n t t h e u n i q u e m a j o r c y t o s k e l e t a l p r o t e i n s . S t u d i e s w i t h a c t i n a n t i b o d i e s show t h a t a c t i n i s d i s t r i b u t e d n o r m a l l y i n d y s t r o p h i c RPE c e l l s ( 1 7 ) . However, t h e i n g e s t i o n mechanism i n v o l v i n g a c t i n becomes a c t i v a t e d a t o n l y a few s i t e s of ROS a t t a c h m e n t . The a r r a y of a c t i n f i l a m e n t s seen i n RPE p r o c e s s e s i s n o t s u i t e d f o r i n g e s t i o n o f ROS s i n c e p h a g o c y t o s i s a t t h e d i s c p a c k e t s a r e s u r r o u n d e d by l a t e r a l p r o t r u s i o n s of t h e RPE p r o c e s s e s t h a t e x t e n d i n w a r d from a l l s i d e s . The i n g e s t i o n of ROS d i s c p a c k e t s would be e x p e c t e d t o i n v o l v e a c t i n but a c t i n o t h e r t h a n t h a t i n f i l a m e n t a r r a y s ( 1 7 ) . I t may be t h e c h a n g e s i n a c t i n f i l a m e n t s t o t h e c e l l p e r i p h e r y . I n t h e c a s e o f n e u t r o p h i l i c p o l y m o r p h o n u c l e a r l e u k o c y t e s , an a b n o r m a l f u n c t i o n i n g a c t i n , c a u s e d an i m p a i r m e n t i n l o c o m o t i o n and t h e i n g e s t i o n of p a r t i c l e s ( 1 8 ) . I n d i r e c t i m m u n o f l u o r e s c e n c e and a n t i b o d i e s t o t u b u l i n were use t o s t u d y t h e d i s t r i b u t i o n o f m i c r o t u b u l e s i n r a t RPE c e l l c u l t u r e s ( 1 9 ) . They f o u n d no a p p a r e n t m i c r o t u b u l e d e f e c t i n b o t h s p r e a d i n g and f u l l y s p r e a d d y s t r o p h i c RPE c e l l s , r e n d e r i n g i t u n l i k e l y t h a t an a l t e r e d d i s t r i b u t i o n of m i c r o t u b u l e s i s r e s p o n s i b l e f o r t h e p h a g o c y t i c d e f e c t i n t h e s e c e l l s . - 1 1 -ENERGY SOURCE OF PHAGOCYTOSIS A b o v i n e RPE o r g a n c u l t u r e was us e d f o r t h e q u a n t i t a t i v e 125 a n a l y s i s of t h e p h a g o c y t o s i s of I - l a b e l e d - g l o b u l i n l a t e x p a r t i c l e s ( 2 0 ) . P h a g o c y t o s i s was s i g n i f i c a n t l y a l t e r e d by 10 M c o l c h i c i n e and 10 ug/mL c y t o c h a l a s i n B i n d i c a t i n g a p o s s i b l e c y t o s k e l e t a l mechanism i n p h a g o c y t o s i s . A l s o , a p i c a l m i c r o v i l l i a p p e a r e d t o be d e p l e t e d i n r e g i o n s where many l a t e x p a r t i c l e s were i n g e s t e d . P r e s u m a b l y t h e m i c r o v i l l i membrane was i n c o r p o r a t e d i n t o t h e phagosomal membrane d u r i n g e n d o c y t o s i s ( 2 0 ) . M a s t e r s o n and Chader i n v e s t i g a t e d t h e e f f e c t .of p h a g o c y t o s i s on t h e m e t a b o l i c m a c h i n e r y of c e l l s of t h e pi g m e n t e p i t h e l i u m ( 2 1 ) . U s i n g e m b r y o n i c c h i c k RPE c e l l s i n t i s s u e c u l t u r e t h e y were a b l e t o measure t h e p h a g o c y t o s i s of b o v i n e ROS as measured by t h e 3 u p t a k e o f H - l a b e l e d o u t e r segments. P h a g o c y t o s i s was m a r k e d l y i n h i b i t e d by t h e l a c k of g l u c o s e and by two i n h i b i t o r s of t h e t r i c a r b o x y l i c a c i d c y c l e - c y t o c h r o m e s y s t e m , d i n i t r o p h e n o l and m a l o n a t e . The o b v i o u s c o n c l u s i o n i s t h a t t r i c a r b o x y l i c a c i d and i t s a s s o c i a t e d c y t o c h r o m e s y s t e m s p l a y an i m p o r t a n t r o l e a s an e n e r g y s o u r c e f o r p h a g o c y t o s i s i n t h e s e c e l l s . LATENCY PERIOD IN PHAGOCYTOSIS S e v e r a l s t u d i e s have been p e r f o r m e d i n v i t r o and i n v i v o d e s i g n e d t o o b s e r v e t h e l a t e n c y p e r i o d i n v o l v e d i n p h a g o c y t o s i s ( 2 2 , 2 3 , 2 4 ) . When p o l y s t y r e n e s p h e r e s and n a t i v e S a r c i n a s u b f l a v a were i n j e c t e d i n t o t h e s u b r e t i n a l s p a c e of Rana p i p i e n s t a b p o l e s o n l y t h e p o l y s t y r e n e s p h e r e s were a c t i v e l y p h a g o c y t i z e d ( 2 2 ) . H o l l e y f i e l d $ s e x p e r i m e n t s c o n c l u d e d t h a t t h e n a t u r e of t h e -Im-m a t e r i a l p r e s e n t e d t o t h e pigment e p i t h e l i a l c e l l s c an d e t e r m i n e whether o r n o t t h a t m a t e r i a l w i l l be p h a g o c y t i z e d ( 2 2 ) . Q u a n t i t a t i v e s t u d i e s done on t h e p h a g o c y t o s i s of l a t e x beads showed a 12-17 h l a t e n c y p e r i o d i n e x p l a n t c u l t u r e d c a l f and r a b b i t p i g m e n t e p i t h e l i a l c e l l s w h i l e r a t RPE c e l l s showed no l a t e n t p e r i o d as i n g e s t i o n o c c u r e d i n t h e f i r s t hour ( 2 3 , 2 4 ) . The abundance of a p i c a l p r o c e s s e s d o e s n o t a p p e a r t o be d i r e c t l y r e l a t e d t o t h e r a t e of p h a g o c y t o s i s . SHEDDING OR "PINCHING OFF" OF ROS DISCS PACKETS C o n s i d e r a b l e d i s c u s s i o n has been made as t o whether ROS f r a g m e n t s a r e s h e d p r i o r t o p h a g o c y t o s i s o r whether t h e t i p s o f t h e ROS a r e p i n c h e d o f f by t h e m i c r o v i l l i of t h e RPE. R e c e n t e v i d e n c e has shown m i c r o v i l l i e v a g i n a t i n g i n t o t h e ROS, s e e m i n g l y p i n c h i n g o f f a p a c k a g e of ROS d i s c s . However, many r e s e a r c h e r s i n s i s t t h a t ROS t i p s a r e not p h a g o c y t i z e d by t h e pigment e p i t h e l i u m b e f o r e b e i n g shed i n t o t h e s u b r e t i n a l s p a ce ( 2 5 ) . S i n c e t h e p h a g o c y t i c r e s p o n s e o f t h e RPE c e l l s a p p e a r s t o be t r i g g e r e d by t h i s s h e d d i n g , some c h e m i c a l change must o c c u r t o t h e t i p o f t h e ROS t h a t a l l o w s t h e RPE t o r e c o g n i z e t h e s h e d p a c k e t of d i s c s as f o r e i g n and t o i n i t i a t e p h a g o c y t o s i s . B a s i n g e r showed t h a t f r o g s u n d e r g o a peak o f d i s c s h e d d i n g 1 h a f t e r t h e o n s e t o f l i g h t i n d i c a t i n g a l i g h t - t r i g g e r e d b u r s t ( 2 5 ) . F u r t h e r e x p e r i m e n t s i n d i c a t e t h a t a b o u t 25% of t h e p h o t o r e c e p t o r s shed t h e i r a p i c a l t i p s e a c h day ( 2 6 ) . S t u d i e s done on t h e p h a g o c y t i c p a t t e r n s i n f e t a l a n i m a l s i n d i c a t e d t h a t t h e r e was a 2-3 h p h a g o c y t i c d e l a y i n t h e f e t u s e s as o p p o s e d t o 30 m i n u t e s i n t h e - 1 3 -a d u l t , upon t h e o n s e t o f l i g h t ( 2 7 ) . T h i s r e s u l t i n d i c a t e s t h e f i r s t e v i d e n c e of a c y c l i c p a t t e r n of p h a g o c y t o s i s i n a p o p u l a t i o n o f f e t u s e s d u r i n g r e t i n a l d e v e l o p m e n t . An i n i t i a l s h o r t l a t e n t p e r i o d f o l l o w e d by a b u r s t of p h a g o c y t i c a c t i v i t y was o b s e r v e d i n c h i c k RPE c e l l s . T h i s would c o r r e s p o n d w i t h t h e i n i t i a l i n c r e a s e i n r a t e of p h a g o c y t o s i s of " p r i m e d " ROS f o l l o w e d by a s u b s e q u e n t d e c r e a s e i n t h e r a t e o f p h a g o c y t o s i s o f p o s s i b l y " u n p r i m e d " ROS ( 1 5 ) . T h i s l a t e n o n - s p e c i f i c p h a g o c y t o s i s may c o r r e s p o n d t o t h e b a s a l l e v e l s of p h a g o c y t o s i s . The p h a g o c y t i c dependence on l i g h t was s t u d i e d by e x p o s i n g r a t s t o a l i g h t - d a r k c y c l e . The RPE a b i l i t y t o p h a g o c y t i z e ROS i n c r e a s e d p r i o r t o t h e o n s e t of l i g h t i n d i c a t i n g t h a t l i g h t i s n o t a d e f i n i t e p r e r e q u i s i t e f o r p h a g o c y t o s i s ( 2 5 ) . RETINAL PIGMENT EPITHELIAL CELLS IN TISSUE CULTURE One o f t h e more r e c e n t d e v e l o p m e n t s i n t h e s t u d y o f s t r u c t u r e and f u n c t i o n of RPE c e l l s i s by t h e growth of t h e s e c e l l s i n t i s s u e c u l t u r e . RPE c e l l s grown as e x p l a n t c u l t u r e s were m a i n t a i n e d i n c u l t u r e f o r p e r i o d s up t o s i x months ( 2 8 ) . M o s a i c , t u b u l a r and s p i n d l e g r o w t h p a t t e r n s were o b s e r v e d t o e v o l v e w i t h a g e n e r a l d e c r e a s e i n p i g m e n t . M u l t i n u c l e a t e c e l l s were i n c r e a s i n g l y common a s t h e d u r a t i o n of c u l t u r e t i m e i n c r e a s e d w i t h as many as s i x o r e i g h t n u c l e i o b s e r v e d i n some c e l l s . The m o r p h o l o g i c a l p a t t e r n of t h e c o l o n i e s a p p e a r e d t o be i n f l u e n c e d t o some d e g r e e by t h e number of c e l l s p r e s e n t . The m o s a i c p a t t e r n of c e l l g r o w t h d e v e l o p e d a d j a c e n t t o t h e o r i g i n a l e x p l a n t s w h i l e s p i n d l e s h a p e d c e l l s were i n t h e p e r i p h e r y of t h e s e c o l o n i e s . I n -14-a r e a s a t some d i s t a n c e f r o m t h e o r i g i n a l e x p l a n t s , t u b u l a r a r r a n g e m e n t s o f e p i t h e l o i d c e l l s were o b s e r v e d ( 2 8 ) . The c e l l u l a r m i c r o e n v i r o n m e n t a p p e a r s t o p l a y a d e c i s i v e r o l e i n t h e f i n a l e x p r e s s i o n of c e l l d i f f e r e n t i a t i o n . W h i t t a k e r f o u n d t h a t i n c u l t u r e d c h i c k e m b r y o n i c RPE c e l l s t h e d o u b l i n g t i m e was 36 h ( 2 9 ) . T h e r e was a d i l u t i o n of m e l a n o t i c components by c e l l g r o w t h , d e c a y i n t y r o s i n a s e a c t i v i t y , and c e s s a t i o n of t y r o s i n a s e s y n t h e s i s . S e v e r a l p r o s t a g l a n d i n s and d i b u t y r y l c y c l i c - A M P i n c r e a s e p i g m e n t a t i o n and i n d u c e d a more mature t y p e of c e l l ( 3 0 , 3 1 ) . Berman, S c h w e l l , and F e e n e y as w e l l a s S i a k o t o s have u n d e r t a k e n t h e t a s k o f p u r i f y i n g pigment e p i t h e l i a l c e l l s w i t h m o d erate s u c c e s s ( 3 2 , 3 3 ) . H e l l e r and J o n e s have s u c c e e d e d i n p u r i f y i n g r e t i n a l pigment e p i t h e l i a l c e l l s f r o m b o v i n e e y e s by d i s s o c i a t i o n i n c a l c i u m f r e e b u f f e r s , c e n t r i f u g a t i o n i n a F i c o l l d e n s i t y g r a d i e n t f o l l o w e d by r e c o v e r y i n t i s s u e c u l t u r e w i t h 90% v i a b i l i t y ( 3 4 ) . P r i m a r y g o a l s were t o d e t a c h t h e pigment e p i t h e l i a l c e l l s f r o m t h e basement membrane by as g e n t l e a p r o c e d u r e as p o s s i b l e , t o i s o l a t e t h e RPE c e l l s f r o m c o n t a m i n a t i n g r e d b l o o d c e l l s , ROS, and p i g m e n t g r a n u l e s and f i n a l l y t o o b t a i n i n t a c t and v i a b l e pigment e p i t h e l i a l c e l l s a t t h e end of t h e p r o c e d u r e . E m p h a s i s must be p l a c e d on o b t a i n i n g h i g h l y p u r i f i e d RPE c e l l s f o r s u b c e l l u l a r f r a c t i o n a t i o n . EFFECT OF AGING ON RPE CELL MORPHOLOGY AND ENZYME ACTIVITY O t h e r s t u d i e s done on RPE c e l l s have l o o k e d a t t h e m o r p h o l o g i c a l and e n z y m a t i c e f f e c t s o f a g i n g ( 3 5 ) . L i p o f u s i o n was -15-f o u n d t o a c c u m u l a t e and i n c r e a s e i n s i z e d u r i n g s e n e s c e n c e i n human and r a t RPE. A p i c a l m i c r o v i l l i a r e l o n g and t h i n up t o 11 months of age a t w h i c h p o i n t t h e y become more t u b u l a r i n f o r m . T h i s c o r r e s p o n d s w i t h t h e r e d u c t i o n i n phagosomes i n r a t RPE c e l l s f r o m 4 t o 32 month o l d r a t s . The o u t e r segment r e n e w a l r a t e may be s l o w e d i n o l d e r a n i m a l s , p e r h a p s i n r e s p o n s e t o an i m p a i r m e n t i n p h a g o c y t i c c a p a b i l i t y of t h e RPE. In t e r m s of a c i d p h o s p h a t a s e a c t i v i t y t h e r e was no d i f f e r e n c e i n t h e young and o l d r a t RPE c e l l s ( 3 5 ) . The d e g r a d a t i o n o f r h o d o p s i n by a RPE l y s o s o m a l f r a c t i o n was f o u n d t o o c c u r a f t e r t h e i n i t i a l phase of d e g r a d a t i o n of o t h e r membrane p r o t e i n s ( 3 6 ) . S u b s e q u e n t l y , r h o d o p s i n i s s l o w l y but c o m p l e t e l y d e g r a d e d as d e m o n s t r a t e d by g e l e l e c t r o p h o r e s i s . LECTIN CELL SURFACE LABELS The l a c k of ROS p h a g o c y t o s i s f o u n d i n d y s t r o p h i c RPE c e l l s may be t h e r e s u l t of d e f e c t i v e c e l l s u r f a c e r e c e p t o r s o r t o t h e l a c k o f s u c h r e c e p t o r s . C a r b o h y d r a t e s on g l y c o p r o t e i n s a r e t h o u g h t t o be i n v o l v e d i n t h e r e c o g n i t i o n of m o l e c u l e s on c e l l s u r f a c e s . R e c e n t l y , s t u d i e s u s i n g l e c t i n s have been p e r f o r m e d t o f i n d t h e d i s t r i b u t i o n and a r r a n g e m e n t of c e l l s u r f a c e c a r b o h y d r a t e s ( 3 7 ) . H e a t h and B a s i n g e r l o o k e d a t t h e h y p o t h e s i s t h a t s u g a r m o l e c u l e s m i g h t ' a c t as m a r k e r s f o r ROS d i s c p h a g o c y t o s i s i n t h e f r o g s y s t e m . L - f u c o s e , «< - m e t h y l - D -m a n n o p y r a n o s i d e , and D-mannose a l l s i g n i f i c a n t l y r e d u c e d t h e numbers of p a c k e t s o f ROS d i s c f o u n d i n t h e RPE. D - f u c o s e , L-mannose, D - f r u c t o s e , D - g a l a c t o s e , D - g l u c o s e , and s u c r o s e were - 1 6 -w i t h o u t s i g n i f i c a n t e f f e c t a t t h e same c o n c e n t r a t i o n . U l t r a s t r u c t u r a l e x a m i n a t i o n i n d i c a t e d t h a t t h e s u g a r s were e f f e c t i v e on t h e ROS d i s c s h e d d i n g p r o c e s s r a t h e r t h a n on p h a g o c y t o s i s of a l r e a d y s h e d d i s c p a c k e t s ( 3 7 ) . N i r and H a l l l o o k e d a t t h e b i n d i n g o f f e r r i t i n - l e c t i n c o m p l e x e s of R i c i n u s communis a g g l u t i n i n (RCA), wheat germ a g g l u t i n i n (WGA), and c o n c a n a v a l i n A (Con A) t o f i x e d f r o g RPE c e l l s ( 3 8 ) . They f o u n d t h a t a l l t h r e e bound t o t h e pigment e p i t h e l i a l c e l l s q u i t e c l e a r l y . Fer-WGA ( f e r r i t i n - W G A ) b i n d i n g s i t e s were somewhat more r e g u l a r l y d i s t r i b u t e d t h a n t h e Fer-RCA ( f e r r i t i n - R C A ) and F e r - C o n A ( f e r r i t i n - C o n A ) . Fer-RCA bound i n a more c l u s t e r e d p a t t e r n t o t h e m i c r o v i l l i s e p a r a t e d by a r e a s o f p l a s m a membrane wh i c h showed s p a r s e b i n d i n g of t h e l e c t i n . Rat RPE c e l l s a l s o showed an i r r e g u l a r d i s t r i b u t i o n o f t h e F e r - C o n A ( 3 9 ) . In b o t h n ormal and d y s t r o p h i c r a t s , WGA u n i f o r m l y l a b e l e d b o t h p r o x i m a l and d i s t a l membrane s u r f a c e s o f RPE m i c r o v i l l i w h e reas, RCA l a b e l e d p r i m a r i l y t h e d i s t a l r e g i o n s ( 4 0 ) . Con A l a b e l e d b o t h n ormal and d y s t r o p h i c RPE m i c r o v i l l i s p a r s e l y , and L e n s c u l i n a r i s a g g l u t i n i n ( L C A ) , s p e c i f i c f o r mannose and g l u c o s e , s t a i n e d t h e RPE m i c r o v i l l i o f n o r m a l r a t s more i n t e n s e l y t h a n t h e d i s e a s e d RCS r a t s . D i f f e r e n c e s i n t h e a c c e s s i b i l i t y o r c o m p o s i t i o n of c e r t a i n c e l l s u r f a c e s u g a r s may be r e l a t e d t o t h e d i m i n i s h e d r a t e of p h a g o c y t o s i s i n RCS r e t i n a . IMMUNOLOGICAL APPROACHES TO STUDYING MEMBRANES I m m u n o l o g i c a l s t u d i e s have been c o n c e r n e d w i t h t h e s i t e o f -17-a n t i b o d y - a n t i g e n a c t i o n . U n t i l r e c e n t l y a l l i m m u n o c y t o c h e m i s t r y p e r f o r m e d i n v o l v e d p o l y c l o n a l a n t i b o d i e s . H a l l u s e d a p o l y c l o n a l a n t i s e r a d i r e c t e d a g a i n s t r o d o u t e r segments f o r s t u d y i n g p h a g o c y t o s i s i n r e t i n a l pigment e p i t h e l i a l c e l l s ( 1 4 ) . A more s i g n i f i c a n t advancement i n i m m u n o l o g i c a l r e s e a r c h was t h e p r o d u c t i o n o f m o n o s p e c i f i c ( m o n o c l o n a l ) a n t i b o d i e s u s i n g c e l l f u s i o n t e c h n i q u e s d e v e l o p e d by R o h l e r and M i l s t e i n ( 4 1 , 4 2 ) . A n t i b o d i e s o f d e f i n e d s p e c i f i c i t y p r o d u c e d by c o n t i n u o u s c u l t u r e s o f m o n o c l o n a l c e l l l i n e s p r o v i d e e x q u i s i t e s e r o l o g i c a l and b i o c h e m i c a l p r o b e s . L a b e l e d a n t i b o d i e s f o r l i g h t and e l e c t r o n m i c r o s c o p y p r o v i d e i n s i g h t i n t o t h e l o c a t i o n and d i s t r i b u t i o n of s p e c i f i c components i n c e l l s . I m m u n o l o g i c a l s t u d i e s o v e r t h e p a s t few. y e a r s have r e s u l t e d i n s e v e r a l m o n o c l o n a l a n t i b o d i e s b e i n g r a i s e d a g a i n s t r e t i n a l t i s s u e . A n t i - r h o d o p s i n m o n o c l o n a l a n t i b o d i e s d e s i g n a t e d Rho-4A2 and Rho-1D4 r a i s e d a g a i n s t r o d o u t e r segments have been l o c a l i z e d and c h a r a c t e r i z e d f u l l y as t o t h e i r a n t i g e n i c s i t e of a c t i o n ( 4 3 ) . To d a t e no m o n o c l o n a l a n t i b o d i e s s p e c i f i c f o r t h e b o v i n e r e t i n a l pigment e p i t h e l i a l c e l l p lasma membrane have been r e p o r t e d . THESIS INVESTIGATION My t h e s i s i n v e s t i g a t i o n was d e s i g n e d t o c h a r a c t e r i z e t h e b o v i n e RPE c e l l p lasma membrane and p o s s i b l e c e l l s u r f a c e r e c e p t o r s i n v o l v e d i n t h e p h a g o c y t o s i s of r o d o u t e r segments. The f i r s t a p p r o a c h e d u s e d was t o o b t a i n a b o v i n e RPE plasma membrane p r e p a r a t i o n and t o c h a r a c t e r i z e t h e p o l y p e p t i d e s by SDS-- 1 8 -p o l y a c r y l a m i d e g e l e l e c t o p h o r e s i s . S e c o n d l y , t h e m o n o c l o n a l a n t i b o d y t e c h n i q u e was employed t o r a i s e a n t i b o d i e s t o t h e RPE e n r i c h e d p l a s m a membrane p r e p a r a t i o n and RPE c e l l s grown i n t i s s u e c u l t u r e . F i n a l l y , b o v i n e RPE c e l l s were grown i n t i s s u e c u l t u r e t o a s s i s t i n t h e i d e n t i f i c a t i o n of c e l l s u r f a c e r e c e p t o r s as w e l l as t o e s t a b l i s h a new a s s a y f o r s t u d y i n g t h e p h a g o c y t o s i s o f b o v i n e r o d o u t e r segments. -19-EXPERIMENTAL PROCEDURES 1. MATERIALS A l l g e n e r a l l a b o r a t o r y c h e m i c a l s of r e a g e n t g r a d e were o b t a i n e d f r o m Sigma C h e m i c a l , BDH C h e m i c a l s , o r F i s h e r S c i e n t i f i c . F i c o l l 400 was p u r c h a s e d from P h a r m a c i a F i n e C h e m i c a l s . D i m e t h y l s u l f o x i d e and c y a n o g e n bromide were o b t a i n e d from F i s h e r S c i e n t i f i c . BDH C h e m i c a l s s u p p l i e d t h e sodium d o d e c y l s u l p h a t e (SDS) and t h e p o l y e t h y l e n e g l y c o l ( P E G ) . B - m e r c a p t o e t h a n o l u s e d i n S D S - g e l e l e c t r o p h o r e s i s was p u r c h a s e d f r o m Eastman O r g a n i c C h e m i c a l s . Cytochrome c was s u p p l i e d by Sigma. J . T . Ba k e r C h e m i c a l s Co. s u p p l i e d t h e s i l v e r n i t r a t e . M o l e c u l a r w e i g h t s t a n d a r d s p u r c h a s e d from B i o Rad L a b o r a t o r i e s i n c l u d e d : l y s o z y m e M r=14,400, so y b e a n t r y p s i n i n h i b i t o r Mr. = 21,500, c a r b o n i c a n h y d r a s e M r=31,000, o v a l b u m i n M r = 45,000, b o v i n e serum a l b u m i n M =66,200, p h o s p h o r y l a s e b M r=92,500, B - g a l a c t o s i d a s e M =116,250, and m y o s i n M r=200,000. B o v i n e r h o d o p s i n N - t e r m i n u s p e p t i d e s were i s o l a t e d f r o m c y a n o g e n b r o m i d e d i g e s t o f r h o d o p s i n a c c o r d i n g t o t h e method o f H a r g r a v e e t a l . ( 4 4 ) . The 2-39 and 2-16 N - t e r m i n a l r h o d o p s i n p e p t i d e s w h i c h were p u r i f i e d by r e v e r s e - p h a s e h i g h p r e f o r m a n c e l i q u i d c h r o m a t o g r a p h y (HPLC) and c h a r a c t e r i z e d by amino a c i d a n a l y s i s were g e n e r o u s l y s u p p l i e d by P i o t r C z a y k o w s k i . Goat a n t i - m o u s e I g a n t i s e r a and r a b b i t a n t i - a c t i n a n t i s e r a were p u r c h a s e d from A n t i b o d i e s I n c o r p o r a t e d and M i l e s - Y e d a L t d . , r e s p e c t i v e l y . I s c o v e ' s M o d i f i e d D u l b e c c o ' s Medium (IMDM) p u r c h a s e d from - 2 0 -GIBCO L a b o r a t o r i e s was made up t o one l i t e r w i t h d i s t i l l e d water w i t h t h e a d d i t i o n of 3.7 g o f sodium b i c a r b o n a t e . The c u l t u r e medium was f i l t e r s t e r i l i z e d and s u p p l e m e n t e d w i t h p e n i c i l l i n (100 u n i t s / m L ) , s t r e p t o m y c i n (100 ug/mL), and f u n g i z o n e (1.25 ug/mL) as w e l l as 10-20% f e t a l c a l f serum ( F C S ) , a l l s u p p l i e d by GIBCO L a b o r a t o r i e s . RPMI 1640 c u l t u r e medium was p r e p a r e d i n a s i m i l a r manner. A l l c e l l c u l t u r e s were m a i n t a i n e d a t 37°C i n an a t m o s p h e r e o f 10% C 0 2 , 90% a i r i n a h u m i d i f i e d i n c u b a t o r . The t r y p s i n and c o l l a g e n a s e u s e d were p u r c h a s e d f r o m D i f c o L a b o r a t o r i e s and M i l l i p o r e C o r p o r a t i o n , r e s p e c t i v e l y . P u r i f i e d c o n c a n a v a l i n A (Con A) was p u r c h a s e d f r o m V e c t o r L a b o r a t o r i e s I n c ; R i c i n u s communis a g g l u t i n i n 1 M^ =120,000 (RCA) from M i l e s - Y e d a L t d . ; and wheat germ a g g l u t i n i n (WGA) from B o e h r i n g e r Mannheim. B u f f e r A c o n t a i n e d 0.58 g N a C l , 1.22 g T r i s , 1.02 g M g C l 2 . 6 H 2 0 , 0.11 g C a C l 2 , and 7 mg PMSF p e r l i t e r o f d i s t i l l e d w a t e r , pH 8.5. B u f f e r B c o n t a i n e d one t h i r d of t h e s e c o n c e n t r a t i o n s w i t h t h e e x c e p t i o n o f PMSF w h i c h was a t e q u a l c o n c e n t r a t i o n as B u f f e r A. P h o s p h a t e - b u f f e r s a l i n e (PBS) i n c l u d e d 8.0 g N a C l , 0.2 g K C l , 0.2 g K H 2 P 0 4 and 2.1 g N a 2 H P 0 4 p e r l i t e r of d i s t i l l e d water a t pH 7.4. RIA b u f f e r c o n s i s t e d o f 1% BSA, 0.1% NaN 3, and 2% FCS i n PBS. 2. PROTEIN ASSAYS A l l p r o t e i n c o n c e n t r a t i o n s were d e t e r m i n e d by t h e method of Lowry e t a l . ( 4 5 ) u s i n g BSA as a s t a n d a r d . 3. ISOLATION OF BOVINE RETINAL PIGMENT EPITHELIAL CELLS U s u a l l y 20 t o 50 f r e s h b o v i n e e y e s ( no more t h a n 3 h o u r s p o s t mortem) were t r a n s p o r t e d on i c e t o t h e l a b o r a t o r y where t h e y were c o o l e d t o a t e m p e r a t u r e of 10-12°C. F e e n e y - B u r n s and Berman d e m o n s t r a t e d t h a t t h e n e u r a l r e t i n a p r o p e r a d h e r e s more l o o s e l y t o t h e e p i t h e l i a l l a y e r when t h e b o v i n e e y e s a r e k e p t a t t h i s t e m p e r a t u r e ( 4 6 ) . The eye b a l l was i n i t i a l l y p u n c t u r e d w i t h t h e p o i n t o f a p a i r of c u r v e d s c i s s o r s and t h e a n t e r i o r p o r t i o n of t h e eye was c u t at. t h e l e v e l o f t h e o r a s e r r a t a . By t i l t i n g t h e eye and g e n t l y s q u e e z i n g t h e o p t i c cup, t h e l e n s and t h e aqueous and v i t r e o u s humor f e l l o ut l e a v i n g t h e s c l e r a , c h o r o i d , and r e t i n a i n t h e p o s t e r i o r p o r t i o n of t h e o p t i c c u p . The t r a n s l u c e n t r e t i n a c o m p l e t e l y c o v e r i n g t h e p o s t e r i o r p o r t i o n of t h e o p t i c c u p was c o l l e c t e d a r o u n d t h e o p t i c n e r v e and removed i n one p i e c e . C a r e was t a k e n not t o s e v e r e t h e c h o r c - i d o r s c l e r a l a y e r s . A f t e r t h e r e t i n a had been removed t h e o p t i c cup was r i n s e d once w i t h 2 mL of B u f f e r A. T h i s t r e a t m e n t removed any s t r a y p i e c e s of r e t i n a t i s s u e and most of t h e e r y t h r o c y t e c o n t a m i n a t i o n . In some c a s e s t h e o p t i c c u p was r i n s e d a g a i n w i t h an a d d i t i o n a l 2 mL of B u f f e r A c o n t a i n i n g 0.01% EDTA but w i t h o u t c a l c i u m and magnesium. T h i s s e c o n d wash h e l p e d remove some of t h e r o d o u t e r segments t h a t were l o o s e l y a t t a c h e d t o t h e RPE c e l l s . S e v e r a l methods f o r r e m o v i n g t h e RPE c e l l s f r o m t h e o p t i c c u p were t r i e d s u c h a s , a s p i r a t i n g and d i s s e c t i n g t h e c e l l s f r o m t h e t i s s u e , but t h e most e f f e c t i v e method was t o b r u s h t h e c e l l s o f f t h e o p t i c c u p . Two mL o f c o l d B u f f e r A were added t o t h e o p t i c c u p and a p p r o x i m a t e l y 1 m i n u t e of g e n t l e b r u s h i n g was u s e d t o remove t h e m e l a n o t i c and a m e l a n o t i c RPE c e l l s from t h e B r u c h ' s - 2 2 -membrane. The c e l l s u s p e n s i o n i n t h e o p t i c c u p was t r a n s f e r r e d t o a c h i l l e d 50 mL c e n t r i f u g e t u b e . A n o t h e r 1 mL of c o l d B u f f e r A was u s e d t o r i n s e t h e o p t i c cup of any r e m a i n i n g RPE c e l l s . The c e l l s u s p e n s i o n was kept on i c e f o r f u t u r e c e l l u l a r f r a c t i o n a t i o n and p l a s m a membrane i s o l a t i o n . 4. PREPARATION OF RETINAL PIGMENT EP I T H E L I A L CELL PLASMA MEMBRANE The b o v i n e r e t i n a l pigment e p i t h e l i a l c e l l s u s p e n s i o n was washed t w i c e i n B u f f e r A by c e n t r i f u g a t i o n a t lOOOg f o r 10 m i n u t e s a t 4°C. The p e l l e t o f RPE c e l l s f r o m 50 b o v i n e e y e s was s w e l l e d o v e r n i g h t a t 4°C i n 30 mL o f h y p o t o n i c b u f f e r c o n s i s t i n g of 0.007M T r i s , pH 7.4, and 0.01% EDTA. The f o l l o w i n g m o r n i n g t h e c e l l s were homogenized w i t h 5 s t r o k e s o f a c h i l l e d Wheaton P o t t e r - E l v e h j e m t i s s u e g r i n d e r . The n u c l e i were s u b s e q u e n t l y p e l l e t e d by c e n t i f u g a t i o n a t 480g f o r 3 m i n u t e s . An e q u a l volume of 10% F i c o l l 400 was added t o t h e homogenate and t h e t o t a l s u s p e n s i o n was l o a d e d on t o p o f 42% (w/w) s u c r o s e and spun i n a Beckman SW 27.0 r o t o r a t 25,000 rpm f o r 3 h o u r s . The c r u d e p l a s m a membrane was c o l l e c t e d a t t h e F i c o l l / s u c r o s e i n t e r f a c e w i t h a 14 gauge n e e d l e . The p l a s m a membrane s u s p e n s i o n was d i l u t e d w i t h one volume B u f f e r A and spun a t 15,000 rpm f o r 30 m i n u t e s i n a S o r v a l l SS 34 r o t o r . The p l a s m a membrane p e l l e t was r e s u s p e n d e d i n one volume B u f f e r A and one volume 20% s u c r o s e , l o a d e d o n t o a 20-50% (w/w) c o n t i n u o u s s u c r o s e g r a d i e n t and spun o v e r n i g h t a t 25,000 rpm i n a Beckman SW 27.1 r o t o r . Two bands were c o l l e c t e d between 34% and 40% (w/w) s u c r o s e w i t h a 14 gauge n e e d l e . B o t h bands were d i l u t e d s e p a r a t e l y w i t h one volume B u f f e r A and p e l l e t e d i n a S o r v a l l SS 34 r o t o r a t 15,000 rpm f o r - 2 3 -30 m i n u t e s . B o t h bands were a s s a y e d f o r pla s m a membrane e n r i c h m e n t b a s e d on 5$ n u c l e o t i d a s e and N a + R + A T P a s e s p e c i f i c a c t i v i t y . C o n t a m i n a t i o n o f t h e p r e p a r a t i o n by m i t o c h o n d r i a and e n d o p l a s m i c r e t i c u l u m were q u a n t i t a t e d by s u c c i n a t e c y t o c h r o m e c r e d u c t a s e and NADH c y t o c h r o m e c r e d u c t a s e , r e s p e c t i v e l y as d e s c r i b e d below. In o r d e r t o remove t h e c o n t a m i n a t i n g r h o d o p s i n from t h e membrane p r e p a r a t i o n , d e t e r g e n t s o l u b i l i z e d membrane was c h r o m a t o g r a p h e d on an a n t i - r h o d o p s i n a f f i n i t y c o l u m n . Rho-1D4 m o n o c l o n a l a n t i b o d y J s p e c i f i c f o r r h o d o p s i n as d e s c r i b e d by Molday and M a c K e n z i e ( 4 3 ) [ was c o v a l e n t l y c o u p l e d t o S e p h a r o s e 2BC1 by t h e CNBr- a c t i v a t i o n method as d e s c r i b e d by C u a t r e c a s a s ( 4 7 ) . G e n e r a l l y 1 mL of membrane s u s p e n s i o n (1 mg/mL) was s o l u b i l i z e d i n 1% CHAPS and i n c u b a t e d w i t h 1 mL of Rho-1D4 a n t i b o d y - S e p h a r o s e 2BC1 beads f o r 30 m i n u t e s a t room t e m p e r a t u r e i n a B i o a n a l y t i c a l Systems I n c . m i c r o f i l t r a t i o n u n i t . The c o n t a m i n a t i n g r h o d o p s i n bound t o t h e S e p h a r o s e beads w h i l e t h e r e m a i n i n g membrane components were e l u t e d by c e n t r i f u g a t i o n i n a c l i n i c a l b e n c h t o p c e n t r i f u g e a t 2,500 rpm f o r 10 m i n u t e s . The e l u t e d m a t e r i a l was s t o r e d a t -20°C u n t i l r e q u i r e d . 5. ENZYME ASSAYS  A. 5'NUCLEOTIDASE 5' N u c l e o t i d a s e was a s s a y e d , w i t h m o d i f i c a t i o n s , a c c o r d i n g t o S o l y am e t a l . ( 4 8 ) . Membrane p r o t e i n (40-60 ug) was i n c u b a t e d i n 0.5 M T r i s b u f f e r , 10 mM M g C l 2 and 10 mM a d e n o s i n e 5' monophosphate, pH 7.5, i n a t o t a l volume of 1 mL. A f t e r 20 m i n u t e s a t 37°C t h e r e a c t i o n was s t o p p e d by a d d i n g 1 mL of c o l d - 2 4 -10% TCA and t h e p r e c i p i t a t e was p e l l e t e d by c e n t r i f u g a t i o n a t 3000 rpm f o r 5 m i n u t e s . A h e a t d e n a t u r e d membrane c o n t r o l was r u n under t h e same c o n d i t i o n s . The i n o r g a n i c p h o s p h a t e g e n e r a t e d was d e t e r m i n e d by t h e F i s k e and Subbarow method ( 4 9 ) . To a 0.5 mL volume o f t h e a s s a y sample 0.5 mL o f t h e a s s a y r e a g e n t , (0.1g ammonium m o l y b d a t e d i s s o l v e d i n 10.0 mL of 0.5 M H^SO^with 0.4 g of F e S 0 4 . 7 H 2 0 ) was added and v o r t e x e d . The a b s o r b a n c e a t 700 nm was measured f o r a l l samples between 15-30 m i n u t e s . P h o s p h a t e c o n c e n t r a t i o n s were c a l c u l a t e d f r o m a s t a n d a r d c u r v e run s i m u l t a n e o u s l y u s i n g i n o r g a n i c p h o s p h a t e . B. N a + K + ATPASE N a + K + ATPase was a s s a y e d by a m o d i f i c a t i o n of t h e method o f C o s t a n t i n o - C e c c a r i n e t a l . ( 5 0 ) . T h i r t y uL samples (30-60 ug p r o t e i n ) were i n c u b a t e d i n a t o t a l volume o f 0.5 mL c o n s i s t i n g o f 50 mM T r i s - C l , pH 7.4, 100 mM N a C l , and 3 mM M g C l 2 w i t h 3 mM T r i s - A T P f o r 15 m i n u t e s a t 37°C. The r e a c t i o n was s t a r t e d by t h e - 4 a d d i t i o n o f 20 uL of 0.5 M KC1 i n t h e p r e s e n c e o r a b s e n c e o f 10 M o u a b a i n . The r e a c t i o n was run f o r 30 m i n u t e s p r i o r t o b e i n g t e r m i n a t e d by t h e a d d i t i o n o f 0.5 mL o f i c e c o l d 10% TCA. The sampl e s were c h i l l e d on i c e f o r f i v e m i n u t e s and t h e n c e n t r i f u g e d a t 3000 rpm f o r 5 m i n u t e s t o p e l l e t t h e p r e c i p i t a t e . S u p e r n a t a n t a l i q u o t s o f 0.5 mL from e a c h sample were a s s a y e d f o r i n o r g a n i c p h o s p h a t e by t h e method of F i s k e and Subbarrow (49) as p r e v i o u s l y d e s c r i b e d . C. NADPH CYTOCHROME C REDUCTASE NADPH c y t o c h r o m e c r e d u c t a s e a c t i v i t y was a s s a y e d by t h e method o f R a g n o t t i e t a l . ( 5 1 ) . A 50 uL sample ( 50-100 ug -25-p r o t e i n ) was added t o 1.42 mL o f a s o l u t i o n c o n s i s t i n g of 66 umoles of p o t a s s i u m p h o s p h a t e , 0.05 umoles KCN, 100 umoles KC1, and 0.075 umoles c y t o c h r o m e c, a t pH 7.6. The r e a c t i o n was s t a r t e d by t h e a d d i t i o n o f 30 uL of 3 mM NADPH. The i n c r e a s e i n a b s o r b a n c e a t 550 nm was r e c o r d e d f o r 4 m i n u t e s . The s p e c i f i c a c t i v i t y was c a l c u l a t e d from t h e m o l a r e x t i n c t i o n c o e f f i c i e n t of 3 -1 - 1 18.7 X 10 M cm f o r c y t o c h r o m e c ( r e d u c e d minus o x i d i z e d ) a t 550 nm. D. SUCCINATE CYTOCHROME C REDUCTASE S u c c i n a t e c y t o c h r o m e c r e d u c t a s e a c t i v i t y was measured by t h e method o f K i n g ( 5 2 ) . A 50 uL sample ( 50-100 ug p r o t e i n ) was adde d t o 0.92 mL of a s o l u t i o n c o n s i s t i n g of 100 umoles p o t a s s i u m p h o s p h a t e , 2.5 umoles KCN, 0.3 umoles EDTA, and 0.1 umoles c y t o c h r o m e c, a t pH 7.4. The r e a c t i o n was s t a r t e d by t h e a d d i t i o n o f 30 uL o f 0.6 M s u c c i n a t e . The i n c r e a s e i n a b s o r b a n c e a t 550 nm was r e c o r d e d f o r 3 m i n u t e s . The s p e c i f i c a c t i v i t y was c a l c u l a t e d 3 - 1 - 1 f r o m t h e m o l a r e x t i n c t i o n c o e f f i c i e n t of 18.7 X 10 M cm f o r c y t o c h r o m e c ( r e d u c e d minus o x i d i z e d ) a t 550 nm. 6. SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS AND GEL TRANSFER RPE c e l l plasma membrane o r r o d o u t e r segment p r e p a r a t i o n s (1.0-5.0 mg/mL) were s o l u b i l i z e d i n an e q u a l volume o f d e n a t u r i n g s o l u t i o n c o n s i s t i n g o f 5% SDS, 40% s u c r o s e , 0.01 M T r i s , pH 6.8, 10% B - m e r c a p t o e t h a n o l and 4% bromophenol b l u e . Samples (15-25 uL) were a p p l i e d t o w e l l s o f a 6.5-15% p o l y a c r y l a m i d e g r a d i e n t s l a b g e l o r a 10% c o n t i n u o u s p o l y a c r y l a m i d e s l a b g e l (0.75 mm X 5.0 cm or 0.75 mm X 12.0 cm) and e l e c t r o p h o r e s i s was c a r r i e d o u t a c c o r d i n g t o t h e p r o c e d u r e of Laemmli ( 5 3 ) . G e l s l i c e s were e i t h e r s t a i n e d w i t h c o o m a s s i e b l u e a c c o r d i n g t o F a i r b a n k s e t  a l . ( 5 4 ) o r s t a i n e d w i t h s i l v e r a c c o r d i n g t o Wray e t a l . ( 5 5 ) . In some c a s e s g e l s were s u b j e c t e d t o e l e c t r o p h o r e t i c t r a n s f e r as a d a p t e d from b l o t t i n g t r a n s f e r p r o c e d u r e of Towbin e t a l . ( 5 6 ) . B r i e f l y , S D S - g e l s were washed t w i c e w i t h 100 mL c h a n g e s o f 20 mM T r i s - A c e t a t e , 2 mM NaEDTA, and 0.01% SDS ( t r a n s f e r b u f f e r ) . The washed g e l s were s a n d w i c h e d a g a i n s t a wet s h e e t of p u r e n i t r o c e l l u l o s e membrane and p l a c e d i n a B i o Rad t r a n s b l o t a p p a r a t u s . E l e c t r o p h o r e t i c t r a n s f e r was c a r r i e d out a t 19 V and 0.5 A a t 4°C i n t r a n s f e r b u f f e r f o r 12-16 h. F o l l o w i n g t r a n s f e r t h e n i t r o c e l l u l o s e p a p e r was q u e n c h e d of any r e m a i n i n g b i n d i n g s i t e s f o r 2-16 h a t room t e m p e r a t u r e w i t h immunoblot b u f f e r c o n s i s t i n g of 0.15 M N a C l , 10 mM s o d i um p h o s p h a t e , 1 mM EDTA, 1 mM NaN 3, and 2% T r i t o n - X 100 w i t h 4% BSA a t pH 7.5. The n i t r o c e l l u l o s e p a p e r was r i n s e d i n d i s t i l l e d w a t e r and i n c u b a t e d i m m e d i a t e l y w i t h a m o n o c l o n a l a n t i b o d y . In o t h e r c a s e s , g e l s l i c e s underwent e l e c t r o p h o r e t i c t r a n s f e r t o C N B r - a c t i v a t e d p a p e r p r e p a r e d by t h e method o f C l a r k e t  a l . ( 5 7 ) . P r i o r t o t h e t r a n s f e r t h e g e l was washed i n t h r e e 100 mL c h a n g e s of 0.1 M s o d i um p h o s p h a t e , pH 7.4, c o n t a i n i n g 0.1% SDS f o r 5 m i n u t e s i n e a c h , f o l l o w e d by two 100 mL c h a n g e s i n 0.02 M s odium p h o s p h a t e , pH 7.4, f o r 5 m i n u t e s e a c h . The t r a n s f e r t o o k p l a c e i n a B i o Rad t r a n s b l o t a p p a r a t u s a t 35 V and 1.5 A a t 4°C i n 0.02 M sodium p h o s p h a t e , pH 7.4, f o r 2-4 h. A f t e r t h e t r a n s f e r t h e r e m a i n i n g CNBr- r e a c t i v e g r o u p s were q u e n c h e d i n T r i s b u f f e r , pH 9.0, w i t h 0.06 M g l y c i n e and 1% BSA o v e r n i g h t a t room t e m p e r a t u r e . The f o l l o w i n g m o r n i n g , t h e f i l t e r p a p e r s were r i n s e d - 2 7 -i n d i s t i l l e d w a ter and i n c u b a t e d w i t h t h e a p p r o p r i a t e m o n o c l o n a l a n t i b o d y . 7. PREPARATION OF GOAT ANTI-MOUSE Ig AND GOAT ANTI-RABBIT I g  ANTIBODY REAGENTS Goat a n t i - m o u s e I g and goat a n t i - r a b b i t I g were p u r i f i e d by a f f i n i t y c h r o m a t o g r a p h y on a mouse I g o r r a b b i t I g S e p h a r o s e 4B col u m n , r e s p e c t i v e l y ( 5 8 ) . B o t h g o a t a n t i - m o u s e I g and g o a t a n t i -1 25 r a b b i t I g a n t i b o d i e s were l a b e l e d w i t h I by t h e c h l o r a m i n e T method (59) t o g i v e s p e c i f i c a c t i v i t i e s of 1-2 x 10^ dpm/ug. One mg o f t h e a n t i b o d y was d i s s o l v e d i n 0.1 M PBS f o l l o w e d by t h e . . . 125 a d d i t i o n of 500 u C i of Na I . The r e a c t i o n was i n i t i a t e d by t h e a d d i t i o n o f 5 uL o f c h l o r a m i n e T (4 mg/mL) a t room t e m p e r a t u r e . 1 25 A f t e r 10 m i n u t e s t h e f r e e Na I was removed by c e n t r i f u g i n g t h e r e a c t i o n m i x t u r e a t 1500-2000 rpm f o r 10 m i n u t e s t h r o u g h a C e n t r e x M i c r o f i l t e r u n i t l o a d e d w i t h 0.5 g of AG 1—X10 a n i o n e xchange r e s i n . 8. POLYPEPTIDE DETECTION BY ANTIBODY T r a n s f e r p a p e r s were r i n s e d i n RIA b u f f e r and i n c u b a t e d w i t h 10 mL of h y b r i d o m a c u l t u r e f l u i d or r a b b i t a n t i - a c t i n . a n t i s e r a (100 f o l d d i l u t e d ) f o r 1-2 h a t 23°C. The p a p e r s were t h e n washed f o r 1-2 h w i t h s e v e r a l c h a n g e s of immunoblot b u f f e r w i t h o u t BSA f o l l o w e d by two c h a n g e s i n wash b u f f e r c o n s i s t i n g of 2 M u r e a , 0.1 M g l y c i n e , and 1% T r i t o n X-100. The p a p e r s were s u b s e q u e n t l y 125 125 i n c u b a t e d w i t h I - l a b e l e d g o a t a n t i - m o u s e I g or I - l a b e l e d g o a t a n t i - r a b b i t I g (1-2 x 1 0 6 dpm/mL) f o r 1-2 h a t 23°C f o l l o w e d by e x t e n s i v e w a s h i n g as b e f o r e . The n i t r o c e l l u l o s e p a p e r s were a i r d r i e d and s u b j e c t e d t o a u t o r a d i o g r a p h y on p r e f l a s h e d Kodak -28-R o y a l X-Omat f i l m w i t h an X - r a y i n t e n s i f y i n g s c r e e n f o r 12-48 h a t 23°C. In t h e c a s e of CNBr- a c t i v a t e d p a p e r s , a l l washing t o o k p l a c e i n s e v e r a l c h a n g e s of PBS w i t h 0.4% N - l a u r o y l s a r c o s i n e . 9. SILVER STAINING TECHNIQUE SDS g e l s were washed f o r 2 h i n 50% m e t h a n o l / 1 0 % a c e t i c a c i d a nd s i l v e r s t a i n e d a c c o r d i n g t o t h e p r o t o c o l of Wray e t a l . ( 5 5 ) . The f r e s h s t a i n i n g s o l u t i o n c o n t a i n e d 0.8 g s i l v e r n i t r a t e 21 mL of 0.36% sodium h y d r o x i d e i n a t o t a l volume o f 100 mL. The g e l was s t a i n e d f o r 15 m i n u t e s w i t h c o n s t a n t g e n t l e a g i t a t i o n f o l l o w e d by a 5 mi n u t e wash i n d e i o n i z e d d i s t i l l e d w a t e r . The s t a i n was d e v e l o p e d i n 2.5 mL of 1% c i t r i c a c i d a nd 0.25 ml o f 38% f o r m a l d e h y d e i n a t o t a l volume of 500 mL f o r l e s s t h a n 10 m i n u t e s . The d e v e l o p m e n t was s t o p p e d by p l a c i n g t h e g e l i n 45% m e t h a n o l / 1 0 % a c e t i c a c i d . The g e l was s w e l l e d t o normal s i z e i n d i s t i l l e d water and p h o t o g r a p h e d w i t h Kodak h i g h c o n t r a s t f i l m . 10. MONOCLONAL ANTIBODY TECHNIQUES A. IMMUNIZATION OF BALB/C MICE T e c h n i q u e f o r t h e p r o d u c t i o n of m o n o c l o n a l a n t i b o d i e s were d e v e l o p e d by M i l s t e i n ( 4 1 ) . M a l e o r f e m a l e BALB/C mice were immunized w i t h a 100 ug o f RPE p l a s m a membrane p r e p a r a t i o n or RPE c e l l s grown i n t i s s u e c u l t u r e , e m u l s i f i e d w i t h 0.1 mL of F r e u n d ' s c o m p l e t e a d j u v a n t . T h r e e i n t r a p e r i t o n e a l i n j e c t i o n s were c a r r i e d out t h r e e weeks a p a r t w i t h t h e s p l e e n c e l l - myeloma c e l l f u s i o n t a k i n g p l a c e f o u r d a y s a f t e r t h e l a s t i m m u n i z a t i o n . B. PREPARATION OF TISSUE CULTURE MYELOMA CELLS One v i a l of l i q u i d n i t r o g e n f r o z e n NS-1 (myeloma) c e l l s c o n t a i n i n g 1 x 10^ c e l l s was thawed i n a 37°C w a t e r b a t h . The -29-c e l l s were washed once i n 10 mL o f IMDM medium s u p p l e m e n t e d w i t h 10% f e t a l c a l f serum (FCS) and p e l l e t e d a t 1500 rpm f o r 5 m i n u t e s . The c e l l s were r e s u s p e n d e d i n 5 mL o f s u p p l e m e n t e d medium. A sample of t h e c e l l s was d i l u t e d one t o one w i t h 0.4% t r y p a n b l u e and t h e v i a b l e c e l l s were c o u n t e d on a 5 h a e m o c y t o m e t e r . The c e l l s were p l a t e d o ut a t 1 x 10 v i a b l e c e l l s p e r mL i n 10 mL p e t r i d i s h e s . The c e l l s were m a i n t a i n e d between 5 4 6 x 10 - 5 x 10 c e l l s p e r mL f o r 7-10 d a y s b e f o r e s u f f i c i e n t numbers were a v a i l a b l e f o r a c e l l f u s i o n . C PRODUCTION OF HYBRIDOMA CELLS The myeloma c e l l s grown i n t i s s u e c u l t u r e were a s p i r a t e d o f f t h e 10 mL p e t r i d i s h e s and c o l l e c t e d i n p o l y s t y r e n e s t e r i l e t u b e s . The c e l l s were spun down a t 1500 rpm f o r 5 m i n u t e s and c o l l e c t i v e l y r e s u s p e n d e d i n 10 mL of serum f r e e medium. 7 A p p r o x i m a t e l y 2 x 1 0 v i a b l e c e l l s were u s e d f o r a c e l l f u s i o n . A t h y m o c y t e c e l l s u s p e n s i o n was p r e p a r e d from one weaning r a t w h i c h a c t e d as a f e e d e r l a y e r f o r c e l l g r o w t h . The thymus was e x c i s e d from above t h e r a t h e a r t and s u b s e q u e n t l y b r o k e n up i n 10 mL o f serum f r e e IMDM medium by s q u e e z i n g t h e thymus t h r o u g h a s t e r i l e p i e c e o f c h e e s e c l o t h . The c e l l s u s p e n s i o n was d i s p e r s e d t h r o u g h a 20.5 gauge n e e d l e i n t o a 10 mL p o l y s t y r e n e t u b e and c e n t r i f u g e d a t 1500 rpm f o r 10 m i n u t e s . The thy m o c y t e p e l l e t was r e s u s p e n d e d i n 10 mL o f IMDM medium s u p p l e m e n t e d w i t h 100 uM h y p o x a n t h i n e , 0.4 uM a m i n o p t e r i n , 16 uM t h y m i d i n e (1 x HAT) and 20% FCS and s t o r e d a t 37°C . A v i a b l e c e l l c o u n t was a l s o p e r f o r m e d on t h e haemocytometer as d e s c r i b e d a b o v e . The s p l e e n c e l l s u s p e n s i o n was p r e p a r e d from t h e immunized -30-BALB/C mouse. The s p l e e n was removed from t h e mouse under s t e r i l e c o n d i t i o n s and p l a c e d i n a 10 mL p e t r i d i s h c o n t a i n i n g serum f r e e IMDM medium. The s p l e e n was f i n e l y c h o p p e d i n o r d e r t o d i s p e r s e t h e c e l l s . The c e l l s u s p e n s i o n was f o r c e d t h r o u g h a 20.5 gauge n e e d l e i n t o a 10 mL p o l y s t y r e n e t u b e and spun a t 1500 rpm f o r 5 m i n u t e s . The p e l l e t was r e s u s p e n d e d i n 5 mL o f serum f r e e IMDM medium and i n c u b a t e d a t 37°C. A sample of serum from t h e mouse was s a v e d f o r a s o l i d - p h a s e radioimmune a s s a y o f t h e t i t r e . The c e l l f u s i o n was c a r r i e d o u t a c c o r d i n g t o G a l f r e -et a l . ( 6 0 ) . Immune s p l e e n c e l l s (2 x 10 ) and myeloma c e l l s (2 x 10 ) were mixed i n a c e n t r i f u g e t u b e and p e l l e t e d a t 1500 rpm f o r 5 m i n u t e s . The c e l l s were washed i n serum f r e e IMDM and r e p e l l e t e d . The p e l l e t o f c e l l s was p l a c e d i n a 37°C water b a t h and 1 mL of 50% p o l y e t h y l e n e g l y c o l (PEG) 1500 was added o v e r a p e r i o d of 1 m i n u t e . The s u s p e n s i o n was s t i r r e d f o r 1 m i n u t e f o l l o w e d by t h e a d d i t i o n o f 2 mL o f serum f r e e IMDM o v e r t h e f o l l o w i n g 2 m i n u t e s . An a d d i t i o n a l 7 mL o f serum f r e e medium was added t o t h e c e l l s u s p e n s i o n o v e r t h e n e x t 2-3 m i n u t e s . The c e l l s u s p e n s i o n was spun a t 1500 rpm f o r 5 m i n u t e s and t h e p e l l e t was r e s u s p e n d e d i n t h e same IMDM medium and r e p e l l e t e d . F i n a l l y , t h e p e l l e t was r e s u s p e n d e d i n 100 mL o f IMDM 1 x HAT medium s u p p l e m e n t e d w i t h 20% FCS and 2 x 10^ t h y m o c y t e s p e r mL. The c e l l s were p l a t e d b u t i n 1 mL volumes i n m u l t i w e l l d i s h e s . A f t e r i n c u b a t i o n a t 37°C f o r one week, t h e o l d medium was a s p i r a t e d o f f th e w e l l s and f r e s h IMDM 1 x HAT medium s u p p l e m e n t e d w i t h 20% FCS was a d d e d . The w e l l s were a s s a y e d f o r p o s i t i v e a n t i b o d y p r o d u c i n g c e l l s by t h e s o l i d - p h a s e radioimmune a s s a y method, 10-14 d a y s -31-a f t e r t h e f u s i o n . D. STANDARD RIA A s t a n d a r d s o l i d - p h a s e radioimmune a s s a y was u s e d t o d e t e c t a n t i b o d y s e c r e t i n g h y b r i d o m a s a c c o r d i n g t o t h e p r o c e d u r e d e v e l o p e d by M a c K e n z i e and Mol d a y ( 5 8 ) . The b o v i n e RPE plasma membrane p r e p a r a t i o n o r a r o d o u t e r segment p r e p a r a t i o n (1-2 mg/mL p r o t e i n ) was s o l u b i l i z e d w i t h 1% T r i t o n X-100 and d i l u t e d t o 0.25 mg/mL w i t h d i s t i l l e d w a t e r . A l i q u o t s o f 25 uL were d r i e d down on f l e x v i n y l m i c r o t i t r e w e l l s a t 60°C f o r 2 h. The p l a t e was washed i n d i s t i l l e d w a t e r f o l l o w e d by i n c u b a t i o n w i t h RIA b u f f e r f o r 1 h t o q u e n c h t h e n o n - s p e c i f i c b i n d i n g s i t e s . The v i n y l p l a t e was r i n s e d b r i e f l y i n PBS b u f f e r p r i o r t o i n c u b a t i o n w i t h 25 uL o f c u l t u r e f l u i d f o r 1 h a t room t e m p e r a t u r e . The p l a t e was washed e x t e n s i v e l y w i t h PBS t o remove unbound a n t i b o d y 1 25 and s u b s e q u e n t l y i n c u b a t e d w i t h 25 uL of I - l a b e l e d g oat a n t i -mouse I g ( 15-40 ug/mL; 1-2 x 1 0 6 dpm/ug) f o r 1 h a t room t e m p e r a t u r e . The p l a t e was washed e x t e n s i v e l y a s b e f o r e and c o u n t e d i n a Beckman 8000 Gamma C o u n t e r . In some c a s e s r h o d o p s i n , a c t i n o r o t h e r p r o t e i n s were u s e d a s t h e s o l i d phase a n t i g e n . F i x e d b o v i n e RPE c e l l s , p r e v i o u s l y grown i n v i n y l f l a t - b o t t o m m i c r o t i t r e w e l l s , were a l s o used as an a n t i g e n i n radioimmune a s s a y s . E . HYBRIDOMA CLONING Hybridoma c e l l s f r o m p o s i t i v e m i c r o t i t r e w e l l s were a s p i r a t e d o f f t h e f l a t b ottomed w e l l s and t r a n s f e r r e d t o a c e n t r i f u g e t u b e and spun a t 1500 rpm f o r 5 m i n u t e s . The p e l l e t was r e s u s p e n d e d i n 1 mL o f IMDM 1 x HT medium s u p p l e m e n t e d w i t h 20% FCS and c o u n t e d - 3 2 -on a haemocytometer. A p p r o x i m a t e l y 750-1000 c e l l s were p l a t e d o u t i n 0.1 mL m u l t i w e l l p l a t e s r e s u l t i n g i n 5 c e l l s / w e l l or 1 c e l l / w e l l w i t h 2 x 1 0 ^ t h y m o c y t e s /mL. The r e m a i n i n g u n c l o n e d c e l l s were p i p e t e d i n t o a 5 mL p e t r i d i s h w i t h 2 x 105" thymocytes/mL. One week l a t e r t h e w e l l s were marked f o r s i n g l e c o l o n y g r o w t h . The c l o n e d c e l l s were a s s a y e d 10-14 d a y s l a t e r by RIA and t h e p o s i t i v e w e l l s were r e c l o n e d as b e f o r e u n t i l v i r t u a l l y a l l w e l l s were p o s i t i v e . The c e l l s were t h e n expanded f o r m o n o c l o n a l a n t i b o d y p r o d u c t i o n e i t h e r i n c u l t u r e f l u i d o r i n a s c i t e s f l u i d . F. STORAGE OF VIABLE HYBRIDOMA CELLS Hybridoma c e l l s g r o w i n g i n l o g phase were a s p i r a t e d o f f t h e p e t r i d i s h e s , t r a n s f e r r e d t o a c e n t r i f u g e t u b e and p e l l e t e d a s b e f o r e . The p e l l e t was r e s u s p e n d e d i n 1.0 mL of 10% FCS s u p p l e m e n t e d growth medium p l u s 10% DMSO. The c e l l s were f r o z e n s l o w l y t o a v o i d i c e c r y s t a l f o r m a t i o n by p l a c i n g v i a l s i n a foam i n s u l a t e d c e l l box l o c a t e d i n a - 7 0 ° C f r e e z e r . A f t e r 24 h o u r s t h e v i a l s were s t o r e d i n d e f i n i t e l y i n l i q u i d n i t r o g e n . G. SOLID-PHASE RADIOIMMUNE COMPETITION ASSAYS C o m p e t i t i o n a s s a y s were p e r f o r m e d i n o r d e r t o more s p e c i f i c a l l y d e f i n e t h e a n t i b o d y s i t e o f a c t i o n . B r i e f l y , 50 uL o f v a r y i n g c o n c e n t r a t i o n s of c o m p e t i n g a n t i g e n was i n c u b a t e d a t 23° C w i t h 50 uL o f h y b r i d o m a c u l t u r e f l u i d . The c u l t u r e f l u i d u s e d was p r e v i o u s l y d i l u t e d t o a c o n c e n t r a t i o n w h i c h gave 80-90% s a t u r a t i o n o f b i n d i n g by t h e s t a n d a r d - s o l i d phase RIA. A f t e r 1 h 50 uL o f t h e m i x t u r e was removed and s c r e e n e d f o r r e m a i n i n g a n t i b o d y a c t i v i t y by t h e s t a n d a r d s o l i d - p h a s e radioimmune a s s a y - 3 3 -d e s c r i b e d a b o v e . H. LOWICRYL THIN SECTION LABELING R e t i n a t i s s u e was f i x e d i n 1.25% g l u t a r a l d e h y d e i n 0.1 M c a c o d y l a t e b u f f e r , pH 7.2, p l u s 0.25 M s u c r o s e f o r 30-60 m i n u t e s a t 4°C. A f t e r w ashing i n t h e same b u f f e r f o r 1 h, t h e t i s s u e was embedded i n L o w i c r y l r e s i n a c c o r d i n g t o t h e method o f R o t h e t a l . ( 6 1 ) . B r i e f l y , t h e t i s s u e was d e h y d r a t e d i n a g r a d e d s e r i e s of a queous d i m e t h y l f o r m a m i d e (DMF) a t p r o g r e s s i v e l y l o w e r t e m p e r a t u r e s : 50% a t 4°C, 70% a t - 2 0 ° C , 90% a t - 3 5 ° C , and 2 c h a n g e s of 100% a t -35°C a l l f o r 30 m i n u t e s i n e a c h . The t i s s u e was t r a n s f e r r e d t o a m i x t u r e of 2 volumes o f 100% DMF and 1 volume L o w i c r y l K4M r e s i n f o r 1 h a t -35°C f o l l o w e d by 1 volume 100% DMF and 1 volume L o w i c r y l K4M f o r 1 h a t - 3 5 ° C . A f t e r i n c u b a t i n g t h e t i s s u e i n 100% L o w i c r y l K4M r e s i n o v e r n i g h t a t 35°C, t h e L o w i c r y l K4M r e s i n was p o l y m e r i z e d by u l t r a v i o l e t i r r a d i a t i o n o v e r n i g h t a t - 3 5 ° C . F o l l o w i n g p o l y m e r i z a t i o n of t h e L o w i c r y l r e s i n embedded t i s s u e , t h e b l o c k s were trimmed and u l t a t h i n s e c t i o n s (60-70 nm) were c u t and c o l l e c t e d on c l e a n c o p p e r g r i d s . The g r i d s were p r e -i n c u b a t e d i n 50 uL of PBS w i t h 0.1% BSA f o r 10 m i n u t e s t o b l o c k n o n - s p e c i f i c b i n d i n g s i t e s . The g r i d s were t h e n i n c u b a t e d i n 50 uL o f 10 f o l d d i l u t e d Rho-5A3 c u l t u r e f l u i d f o r 30 m i n u t e s a t room t e m p e r a t u r e f o l l o w e d by e x t e n s i v e w a s h i n g i n PBS and a 10 m i n u t e i n c u b a t i o n i n PBS w i t h 0.1% BSA. F i n a l l y , t h e g r i d s were i n c u b a t e d i n 50 uL of 5 f o l d d i l u t e d g o a t a n t i - m o u s e I g d e x t r a n -g o l d f o r 30 m i n u t e s a t room t e m p e r a t u r e ( 6 2 ) . A f t e r w ashing t h e g r i d s e x t e n s i v e l y i n PBS, t h e y were s t a i n e d w i t h s a t u r a t e d u r a n y l - 3 4 -a c e t a t e f o r 5-7 m i n u t e s and s a t u r a t e d l e a d c i t r a t e f o r 1.5-2 m i n u t e s a t room t e m p e r a t u r e . The s e c t i o n s were v i e w e d on a P h i l i p s 200 e l e c t r o n m i c r o s c o p e . 11. BOVINE RETINAL PIGMENT E P I T H E L I A L CELLS IN TISSUE CULTURE A. CULTURING OF BOVINE RPE CELLS B o v i n e RPE c e l l s were c u l t u r e d i n v i t r o by m o d i f i c a t i o n s t o t h e method o f Basu e t a l . ( 6 3 ) . F r e s h b o v i n e e y e s were d i s s e c t e d as p r e v i o u s l y d e s c r i b e d under s t e r i l e c o n d i t i o n s . The o p t i c c u p was r i n s e d once i n s t e r i l e B u f f e r A. The o p t i c cup was s u b s e q u e n t l y i n c u b a t e d w i t h 2 mL of s t e r i l e PBS w i t h 0.25% t r y p s i n and 70 u n i t s / m L c o l l a g e n a s e a t 37°C w i t h c o n s t a n t g e n t l e a g i t a t i o n f o r 60-80 m i n u t e s . The RPE c e l l s were s u b s e q u e n t l y a s p i r a t e d o f f t h e o p t i c c u p and t r a n s f e r r e d t o a c e n t r i f u g e t u b e . The c e l l s u s p e n s i o n was d i l u t e d w i t h 3 v o l u m e s o f c u l t u r e medium c o n s i s t i n g o f RPMI-1640 medium s u p p l e m e n t e d w i t h 10 mM Hepes, pH 7.2, b u f f e r , a n t i b i o t i c s ( p e n i c i l l i n 100 u n i t s / m L , s t r e p t o m y c i n 100 ug/mL, and f u n g i z o n e 50 ug/mL) and 10% FCS. The c e l l s u s p e n s i o n was spun a t 1500 rpm f o r 5 m i n u t e s . The p e l l e t was washed i n c u l t u r e medium and r e p e l l e t e d as b e f o r e . The RPE c e l l s were p l a t e d o u t ( a p p r o x i m a t e l y 5 x 1 0 c e l l s / m L ) i n 10 mL Kimax g l a s s p e t r i d i s h e s o r 10 mL p l a s t i c p e t r i d i s h e s c o n t a i n i n g a l c o h o l s t e r i l i z e d g l a s s c o v e r s l i p s . The c e l l s were grown a t 37°C f o r 7-14 d a y s or u n t i l c o n f l u e n t g r o w t h was r e a c h e d a t w h i c h p o i n t t h e c e l l s were f i x e d i n 0.2% g l u t a r a l d e h y d e , s u b - c u l t u r e d , o r h a r v e s t e d and s t o r e d f o r f u t u r e u s e . B. FLUORESCENT DETECTION OF ACTIN IN RPE CELLS B o v i n e RPE c e l l s were grown on g l a s s c o v e r s l i p s i n t i s s u e -35-c u l t u r e f o r 2 weeks or u n t i l c o n f l u e n t g r o w t h was a c h i e v e d . The c o v e r s l i p s were r i n s e d i n PBS b u f f e r w i t h 0.81 mM MgSO^ and 1.27 mM C a C l 2 p r i o r t o f i x a t i o n i n 1% p a r a f o r m a l d e h y d e i n t h e same b u f f e r f o r 30 m i n u t e s a t 4°C. The c o v e r s l i p s were washed i n PBS w i t h M g + + and C a + + f o r 30 m i n u t e s . The c e l l s were t r e a t e d i n 50% a c e t o n e f o r 3 m i n u t e s f o l l o w e d by 5 m i n u t e s i n a b s o l u t e a c e t o n e and r e p l a c e d i n 50% a c e t o n e f o r 3 m i n u t e s , a l l a t 4°C. A g a i n t h e c e l l s were washed i n PBS w i t h M g + + and C a + + f o r 30 m i n u t e s . The RPE c e l l s were i n c u b a t e d i n 100 t i m e s d i l u t e d , r a b b i t a n t i - a c t i n a n t i s e r a f o r 40 m i n u t e s a t 4°C. The c o v e r s l i p s were washed e x t e n s i v e l y i n t h r e e c h a n g e s of PBS w i t h M g + + and C a + + . The c e l l s were i n c u b a t e d f o r 40 m i n u t e s a t 4°C w i t h 100 uL of F I T C - l a b e l e d g o a t a n t i - r a b b i t I g a n t i b o d y . The c e l l s were washed as b e f o r e and mounted w i t h 50% g l y c e r o l on a g l a s s s l i d e , p r i o r t o b e i n g v i e w e d and p h o t o g r a p h e d w i t h a L e i t z f l u o r e s c e n t m i c r o s c o p e . Two c o n t r o l s were r u n w i t h t h e t e s t e x p e r i m e n t . In one c o n t r o l t h e p r i m a r y a n t i b o d y was o m i t t e d i n t h e f i r s t l a b e l i n g s t e p ; i n t h e s e c o n d c o n t r o l , a s o l u t i o n of t h e p r i m a r y a n t i b o d y i n c u b a t e d w i t h 100 ug o f p u r i f i e d a c t i n was u s e d i n t h e f i r s t s t e p . C. FLUORESCENT LECTIN LABELED BOVINE RPE CELLS IN VITRO F l u o r e s c e n t l e c t i n s a r e p r e p a r e d a c c o r d i n g t o Maher and Molday (64,65) by r e a c t i n g t h e l e c t i n w i t h F I T C (0.05 mg/mg l e c t i n ) i n 0.02 M sodium c a r b o n a t e , pH 9.0, f o r 2 h a t 25°C . The f l u o r e s c e n t l e c t i n s were s e p a r a t e d from f r e e d y es on t h e a p p r o p r i a t e a f f i n i t y column: o v o m u c o i d - S e p h a r o s e , f o r WGA: S e p h a r o s e 4B, f o r RCA: and Sephadex G-200, f o r Con A. F I T C - l e c t i n p r e p a r a t i o n s t y p i c a l l y had 495 nm/280 nm a b s o r b a n c e r a t i o s of -36-0.5-1.0. C o v e r s l i p s of 1-2 week o l d b o v i n e RPE c e l l s were f i x e d w i t h 1% p a r a f o r m a l d e h y d e i n 0.1 M c a c o d y l a t e b u f f e r , pH 7.2, w i t h 0.18 M s u c r o s e f o r 20 m i n u t e s a t 4°C. The f i x e d or u n f i x e d RPE c e l l s were r i n s e d i n PBS w i t h M g + + and C a + + f o l l o w e d by i n c u b a t i o n a t 37°C f o r 5 m i n u t e s w i t h 100 ug/mL o f t h e a p p r o p r i a t e f l u o r e s c e n t l e c t i n . The e x c e s s or unbound l e c t i n was washed o f f by s u c c e s s i v e washes i n t h e same b u f f e r . C o n t r o l s were run by c o m p e t i n g o f f t h e bound l e c t i n f o r 15 m i n u t e s , a t 37°C, w i t h 0.2 M m e t h y l m a n n o s i d e , 0.02 M N - N - d i a c e t y l c h i b i t o s e , o r 0.2 M D - g a l a c t o s e f o r Con A, WGA, o r RCA, r e s p e c t i v e l y . F l u o r e s c e n t l e c t i n t r e a t e d RPE c e l l s were o b s e r v e d and p h o t o g r a p h e d under a L e i t z f l u o r e s c e n t m i c r o s c o p e . D. DISCONTINUOUS FLUORESCENT LECTIN LABELING OF BOVINE RPE CELLS U n f i x e d b o v i n e RPE c e l l s grown i n t i s s u e c u l t u r e f o r 2 weeks were r i n s e d i n PBS w i t h M g + + and C a + + and i n c u b a t e d w i t h 100 ug/mL of t h e a p p r o p r i a t e f l u o r e s c e n t l e c t i n f o r 5 m i n u t e s , a t 37°C. The c o v e r s l i p s were r i n s e d i n PBS w i t h M g + + and C a + + f o l l o w e d by a 60 mi n u t e i n c u b a t i o n p e r i o d i n t h e same b u f f e r a t 37°C. S u b s e q u e n t l y t h e c e l l s were e i t h e r r i n s e d i n t h e same b u f f e r o r i n c u b a t e d f o r an a d d i t i o n a l 15 m i n u t e s , a t 37°C, w i t h t h e a p p r o p r i a t e l e c t i n i n h i b i t o r and r i n s e d p r i o r t o o b s e r v a t i o n . E. CONTINUOUS FI T C - L E C T I N LABELING OF BOVINE RPE CELLS U n f i x e d b o v i n e RPE c e l l s grown i n t i s s u e c u l t u r e f o r two weeks were l a b e l e d w i t h f l u o r e s c e n t l e c t i n s as b e f o r e w i t h t h e e x c e p t i o n t h a t t h e c e l l s were c o n t i n u o u s l y l a b e l e d f o r 60 m i n u t e s a t 3 7 ° C . The c o v e r s l i p s were washed e x t e n s i v e l y i n PBS w i t h M g + + - 3 7 -and Ca p r i o r t o o b s e r v a t i o n under t h e f l u o r e s c e n t m i c r o s c o p e . 11. PHAGOCYTOSIS OF BOVINE ROS BY BOVINE RPE CELLS IN VITRO A. PREPARATION OF SEALED DARK ADAPTED BOVINE ROS T w e n t y - f i v e f r e s h b o v i n e e y e s c o l l e c t e d f r o m t h e l o c a l s l a u g h t e r house were d a r k a d a p t e d a t room t e m p e r a t u r e f o r 30 m i n u t e s . The r e t i n a s were c a r e f u l l y removed and p l a c e d i n 10-15 mL of h o m o g e n i z i n g s o l u t i o n c o n s i s t i n g of 20% s u c r o s e , 20 mM T r i s , pH 7.2, 0.25 mM M g C l 2 , 5 mM t a u r i n e , and 10 mM g l u c o s e . The ROS were b r o k e n l o o s e f r o m t h e r e t i n a by g e n t l y i n v e r t i n g t h e s o l u t i o n a p p r o x i m a t e l y t h i r t y t i m e s , f o l l o w e d by f i l t r a t i o n t h r o u g h c h e e s e c l o t h . The p r o c e d u r e was r e p e a t e d w i t h an a d d i t i o n a l 1-2 mL of h o m o g e n i z i n g s o l u t i o n . The s o l u t i o n was c e n t r i f u g e d f o r 2 m i n u t e s a t 1000 rpm and t h e p e l l e t d i s c a r d e d . The s u p e r n a t a n t was f i l t e r e d t h r o u g h c h e e s e c l o t h once more, l a y e r e d on a 27-60% (w/w) c o n t i n u o u s s u c r o s e g r a d i e n t and spun i n a Beckman SW 27.0 r o t o r a t 25,000 rpm f o r 1.5 h. The band a t t h e t o p of t h e g r a d i e n t ( a p p r o x i m a t e l y 30%) was removed and 1 volume was d i l u t e d w i t h 2 volumes 0.01 M T r i s b u f f e r , pH 7.4. The s o l u t i o n was c e n t r i f u g e d a t 5000 rpm a t 4°C f o r 5 m i n u t e s . The p e l l e t was washed t w i c e i n Hank's B a l a n c e S a l t S o l u t i o n (HBSS) and c e n t r i f u g a t i o n was r e p e a t e d . The ROS were r e s u s p e n d e d i n 1 mL o f RPMI 1640 medium s u p p l e m e n t e d w i t h 10% FCS. B. PHAGOCYTOSIS OF ROS BY ADULT BOVINE RPE CELLS IN VITRO Two week o l d b o v i n e RPE c e l l s grown on g l a s s c o v e r s l i p s were ++ ++ i n i t i a l l y washed i n PBS w i t h Mg and Ca p r i o r t o i n c u b a t i o n w i t h d a r k a d a p t e d ROS. The c e l l s were i n c u b a t e d i n t h e d a r k f o r 5 h a t 37°C f o l l o w e d by e x t e n s i v e w a s h i n g i n t h e same b u f f e r . In -38-o r d e r t o enhance p h a g o c y t o s i s 10 mM g l u c o s e was a l s o added t o t h e ROS s u s p e n s i o n i n some c a s e s . The c e l l s were f i x e d i n 0.2%-1% g l u t a r a l d e h y d e i n 0.1 M c a c o d y l a t e b u f f e r , pH 7.2, and p r e p a r e d f o r t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y (TEM). C. PREPARATION OF SAMPLES FOR TRANSMISSION ELECTRON MICROSCOPY T i s s u e samples were f i x e d i n 2.5% g l u t a r a l d e h y d e i n 0.1 M c a c o d y l a t e b u f f e r , pH 7.2, c o n t a i n i n g 0.25 M s u c r o s e f o r 30-60 m i n u t e s a t 4°C. The sa m p l e s were washed i n t h e same b u f f e r p r i o r t o p o s t f i x i n g i n 1% osmium t e t r o x i d e f o r 30-60 m i n u t e s a t 4°C. A f t e r t h e samples were washed i n t h e same b u f f e r f o r 1 h, t h e y were d e h y d r a t e d i n an e t h a n o l s e r i e s (50%,70%,90%, and two c h a n g e s of a b s o l u t e e t h a n o l ) f o r 15 m i n u t e s i n e a c h a t room t e m p e r a t u r e . D e h y d r a t i o n was c o n t i n u e d i n a 50:50 m i x t u r e o f a b s o l u t e e t h a n o l and p r o p y l e n e o x i d e f o r 15 m i n u t e s a t room t e m p e r a t u r e f o l l o w e d by a 15 m i n u t e i n c u b a t i o n i n p r o p y l e n e o x i d e . A r a l d i t e / E p o n r e s i n p e n e t r a t i o n was f a c i l i t a t e d by i n c u b a t i n g samples i n a 50:50 mix of p r o p y l e n e o x i d e and r e s i n f o r 1-2 h f o l l o w e d by 24-48 h i n 100% r e s i n . The embedded samples were p l a c e d i n a 60°C oven and a l l o w e d t o h a r d e n f o r 24 h. S e v e n t y nanometer t h i c k s e c t i o n s were c u t on a S o r v a l l 5000 U l t r a M i c r o t o m e and s u p p o r t e d on f o r m v a r c o a t e d S200 c o p p e r g r i d s o r u n c o a t e d S200 c o p p e r g r i d s . P r i o r t o v i e w i n g , t h e g r i d s were s t a i n e d w i t h s a t u r a t e d u r a n y l a c e t a t e f o r 5-7 m i n u t e s a t room t e m p e r a t u r e . The g r i d s were a l s o s t a i n e d w i t h s a t u r a t e d l e a d c i t r a t e f o r 1.5-2 m i n u t e s b e f o r e b e i n g o b s e r v e d on a P h i l i p s 200 e l e c t r o n m i c r o s c o p e . D. PREPARATION OF SAMPLES FOR SCANNING ELECTRON MICROSCOPY - 3 9 -U n l a b e l e d o r l a b e l e d c e l l s were washed i n PBS and p r e p a r e d f o r s c a n n i n g e l e c t r o n m i c r o s c o p y as f o l l o w s ( 6 6 ) . The RPE c e l l s were f i r s t f i x e d i n 1% g l u t a r a l d e h y d e i n 0.1 M c a c o d y l a t e b u f f e r f o r 1 h a t 4°C f o l l o w e d by w a s h i n g i n t h e same b u f f e r f o r 1 h. The RPE c e l l s were s t a i n e d w i t h 1% osmium t e t r o x i d e (0) and 1% t h i o c a r b o h y d r a z i d e (T) i n t h e s e q u e n c e 0:T:0:T:0 f o r 30 m i n u t e s i n e a c h w i t h e x t e n s i v e w a s h i n g i n c a c o d y l a t e b u f f e r between e a c h s t e p . The c e l l s were d e h y d r a t e d i n a g r a d e d e t h a n o l s e r i e s (50%,70%,90% , and 2 c h a n g e s of a b s o l u t e e t h a n o l ) f o r 15 m i n u t e s e a c h a t room t e m p e r a t u r e . The c e l l s were d r i e d f r o m CO^ i n a P o l a r o n c r i t i c a l p o i n t d r y i n g a p p a r a t u s (67) and c o a t e d w i t h 200 nm o f g o l d - p a l l a d i u m i n a T e c h n i c s Hummer V S p u t t e r - C o a t e r . Samples were v i e w e d and p h o t o g r a p h e d on a Cambridge 250 S c a n n i n g E l e c t r o n M i c r o s c o p e (SEM). - 4 0 -SECTION 1 ANALYSIS OF BOVINE RETINAL PIGMENT EPITHELIAL  PLASMA MEMBRANE PREPARATIONS  RESULTS 1. PREPARATION OF BOVINE RPE PLASMA MEMBRANE The f r a c t i o n a t i o n of b o v i n e RPE c e l l membranes was m o n i t o r e d by f o l l o w i n g t h e d i s t r i b u t i o n o f m a r k e r s f o r d i f f e r e n t s u b c e l l u l a r components; N a + K + ATPase (50,68,69) and 5$ n u c l e o t i d a s e ( 4 8 , 7 0 ) , f o r t h e pla s m a membrane; NADPH c y t o c h r o m e c r e d u c t a s e (51,69) f o r e n d o p l a s m i c r e t i c u l u m ; and s u c c i n a t e c y t o c h r o m e c r e d u c t a s e (52,69) f o r m i t o c h r o n d i a . T a b l e 1 f o l l o w s t h e t o t a l a c t i v i t y and s p e c i f i c a c t i v i t y f o r e a c h of t h e s e enzymes as w e l l a s t h e i r e n r i c h m e n t and r e c o v e r y w i t h r e s p e c t t o t h e c e l l homogenate. A f t e r t h e i n i t i a l h y p o t o n i c d i s r u p t i o n of t h e RPE c e l l s , t h e n u c l e i were p e l l e t e d y i e l d i n g t h e 480g s u p e r n a t a n t . C e n t r i f u g a t i o n of t h e s u p e r n a t a n t on a F i c o l l / 42% s u c r o s e d i s c o n t i n u o u s g r a d i e n t y i e l d e d a 3.5 t o 5.0 f o l d e n r i c h e d p l a s m a membrane p r e p a r a t i o n . S u b s e q u e n t c e n t r i f u g a t i o n on a 20-50% c o n t i n u o u s s u c r o s e g r a d i e n t r e s u l t e d i n two p l a s m a membrane e n r i c h e d bands, t h e l o w e r of w h i c h had a more t u r b i d a p p e a r a n c e . The p l a s m a membrane m a r k e r s were e n r i c h e d by 7-9 f o l d w i t h r e s p e c t t o t h e c e l l homogenate w i t h a r e c o v e r y o f about 18%. NADPH c y t o c h r o m e c r e d u c t a s e s p e c i f i c a c t i v i t y d e c r e a s e d a p p r o x i m a t e l y 2-5 f o l d b a s e d on t h e homogenate. S u c c i n a t e c y t o c h r o m e c r e d u c t a s e s p e c i f i c a c t i v i t y was h i g h e r a t t h e F i c o l l / s u c r o s e i n t e r f a c e but l o w e r i n t h e bands o b t a i n e d from -41-TABLE 1 DISTRIBUTION OF MARKERS IN VARIOUS FRACTIONS RECOVERED DURING MEMBRANE ISOLATION F r a c t i o n TA 5 ' N u c l e o t i d a s e %R SA PF TA + + Na K ATPase %R SA PF Homogenate 936 100 15.6 1 852 1 00 14.2 1 480 g s u p e r n a t a n t 915 97.0 18.3 1.2 841 98.0 16.8 1 .2 I n t e r f a c e 360 38.5 60.0 3.8 451 53.0 76.2 5.3 25,000 rpm Top Band 100 10.7 125 8.0 101 11.8 1 27 8.9 25,000 rpm 72.1 7.8 90.0 5.7 71.4- 8.3 88.8 6.3 Bottom Band F r a c t i o n NADPH C y t o C R e d u c t a s e TA %R SA PF S u c c i n a t e C y t o C R e d u c t a s e TA %R SA PF Homogenate 1632 100 34.3 1 912 100 19.0 1 480g 1497 91.7 39.0 1.13 S u p e r n a t a n t 916 100 23.5 1.2 I n t e r f a c e 197 12.0 47.0 1.14 182 20.0 43.3 2.27 25,000 rpm 27.2 1.6 16.0 0.46 Top Band 15.7 1.7 9.1 0.47 25,000 rpm 17.6 1. 1 8.0 0.23 Bottom Band 54.3 5.9 24.7 1.30 -42-T a b l e 1. D i s t r i b u t i o n of m a r k e r s i n v a r i o u s f r a c t i o n s r e c o v e r e d d u r i n g membrane i s o l a t i o n . The homogenate r e p r e s e n t s t h e c e l l u l a r s u s p e n s i o n a f t e r h y p o t o n i c d i s r u p t i o n , w h i l e t h e 480g s u p e r n a t a n t was t h e c e l l s u s p e n s i o n a f t e r p e l l e t i n g t h e n u c l e i . The i n t e r f a c e i s t h e membrane band c o l l e c t e d f r o m t h e F i c o l l / 4 2 % s u c r o s e i n t e r f a c e . The 25,000 rpm t o p and b o t t o m bands a r e t h e two bands c o l l e c t e d f r o m t h e 20-50% (w/w) c o n t i n u o u s s u c r o s e g r a d i e n t . TA = t o t a l a c t i v i t y ( n m o l e s / m i n ) . %R = r e p r e s e n t s t h e e n z y m a t i c r e c o v e r y b a s e d on t h e c e l l homogenate. SA = s p e c i f i c a c t i v i t y (nmoles/min/mg). and PF = p u r i f i c a t i o n f o l d b a s e d on t h e c e l l homogenate. -43-t h e c o n t i n u o u s s u c r o s e g r a d i e n t . However, t h e lower band had a h i g h e r s u c c i n a t e c y t o c h r o m e c r e d u c t a s e s p e c i f i c a c t i v i t y t h a n t h e homogenate i n d i c a t i n g a m i t o c h o n d r i a membrane c o n t a m i n a t i o n of t h i s band. A n a l y s i s of t h e membrane p r e p a r a t i o n s by c o o m a s s i e b l u e s t a i n e d p o l y a c r y l a m i d e g e l s i n d i c a t e d t h e wide d i v e r s i t y of p o l y p e p t i d e bands f o u n d i n t h e RPE p r e p a r a t i o n s r a n g i n g from an a p p a r e n t M r=20,000 t o 200,000 ( F i g . 3 l a n e s b and c ) . In F i g . 3 l a n e s b and c r e p r e s e n t t h e t o p and bottom bands c o l l e c t e d from t h e c o n t i n u o u s s u c r o s e g r a d i e n t w h i c h d i f f e r c o n s i d e r a b l y i n s t a i n i n g p a t t e r n f r o m a r o d o u t e r segment p r e p a r a t i o n ( F i g . 3 l a n e d ) . However, t h e RPE membrane p r e p a r a t i o n s had a l a r g e r h o d o p s i n c o n t a m i n a t i o n as seen by a major band a t a p p a r e n t M r=34,000. A f r a c t i o n a t i o n s t u d y o f a RPE c e l l homogenate spun on a 30-50% (w/w) c o n t i n u o u s s u c r o s e g r a d i e n t r e s u l t e d i n a h i g h Rho-1D4 ( a n t i - r h o d o p s i n m o n o c l o n a l a n t i b o d y ) b i n d i n g l e v e l a t t h e f r a c t i o n where 5 ' n u c l e o t i d a s e a c t i v i t y was a t a peak ( F i g . 4 ) . T h i s i n d i c a t e d t h a t b o t h t h e RPE and ROS membranes c o m i g r a t e i n t h e s u c r o s e g r a d i e n t . In an a t t e m p t t o remove some of t h e r h o d o p s i n c o n t a m i n a t i o n , t h e membrane p r e p a r a t i o n was s o l u b i l i z e d i n 1% CHAPS and p a s s e d t h r o u g h an i m m u n o a f f i n i t y column c o n s i s t i n g of Rho-1D4 a n t i b o d y c o v a l e n t l y l i n k e d t o S e p h a r o s e 4B b e a d s . The i m m u n o a f f i n i t y column removed most o f t h e r h o d o p s i n as shown by a c o o m a s s i e b l u e s t a i n e d S D S - p o l y a c r y l a m i d e g e l ( F i g . 3 , l a n e a ) . Once t h e membrane p r e p a r a t i o n p a s s e d t h r o u g h t h e a f f i n i t y column t h e p o l y p e p t i d e s above an a p p a r e n t 1^=100,000 and below M =45,000 were n o t seen by c o o m a s s i e b l u e s t a i n i n g due t o s —dye a b c d e C B S I L V E R S T A I N F i g u r e 3. C o o m a s s i e b l u e and s i l v e r s t a i n e d S D S - p o l y a c r y l a m i d e g e l s of v a r i o u s membrane f r a c t i o n s . Membrane samples were s o l u b i l i z e d i n SDS i n t h e p r e s e n c e o f 2 - m e r c a p t o e t h a n o l and 25 uL were a p p l i e d t o e a c h w e l l . E l e c t r o p h o r e s i s was c a r r i e d out on a 5-16% g r a d i e n t S D S - p o l y a c r y l a m i d e g e l . G e l s were s t a i n e d w i t h e i t h e r c o o m a s s i e b l u e (CB) o r s i l v e r . a) 1% CHAPS s o l u b i l i z e d b o v i n e r e t i n a l pigment e p i t h e l i a l p l a s m a membrane p r e p a r a t i o n p a s s e d t h r o u g h an a n t i - r h o d o p s i n (Rho-1D4) S e p h a r o s e i m m u n o a f f i n i t y c o l u m n . The s u s p e n s i o n was c e n t r i f u g e d and 25 uL of t h e e l u d e n t were l o a d e d on t h e g e l b) 25 uL o f t h e t o p band c o l l e c t e d from a 20-50% (w/w) c o n t i n u o u s s u c r o s e g r a d i e n t c e n t r i f u g e d i n an Beckman SW 27.1 r o t o r a t 25,000 rpm f o r 14-16 h. c) 25 uL of t h e l o w e r band c o l l e c t e d from t h e same c o n t i n u o u s g r a d i e n t , d) 25 uL of a r o d o u t e r segment p r e p a r a t i o n . e) m o l e c u l a r w e i g h t s t a n d a r d s . L a n e s b-e were o v e r d e v e l o p e d when s t a i n e d w i t h s i l v e r t o i d e n t i f y t h e p o l y p e p t i d e bands i n l a n e a. -45-1 2 3 4 5 6 7 8 9 10 11 12 Fraction Number F i g u r e 4. A RPE c e l l homogenate f r a c t i o n a t i o n p r o f i l e of a 30-50% (w/w) c o n t i n u o u s s u c r o s e g r a d i e n t c e n t r i f u g e d f o r 14 h a t 25,000 rpm. F r a c t i o n s were c o l l e c t e d w i t h a f r a c t i o n c o l l e c t o r i n 2 ml v o l u m e s . 5 1 n u c l e o t i d a s e s p e c i f i c a c t i v i t y (• • ) was i n nmoles/min/mg. Mouse a n t i - r h o d o p s i n Rho-1D4 h y b r i d o m a a s c i t e s 125 f l u i d i n c o n j u n c t i o n w i t h I - l a b e l e d g o a t a n t i - m o u s e Ig was u s e d t o d e t e r m i n e t h e amount of b i n d i n g t o e a c h f r a c t i o n by t h e s t a n d a r d RIA method ( • • ) as d e s c r i b e d i n E x p e r i m e n t a l P r o c e d u r e s . S u c r o s e d e n s i t i e s were d e t e r m i n e d by m e a s u r i n g t h e r e f r a c t i v e i n d e x o f e a c h f r a c t i o n . - 4 6 -p o l y p e p t i d e d i l u t i o n . U s i n g a much more s e n s i t i v e s i l v e r s t a i n method most of t h e g e l bands had been c o n s e r v e d a f t e r p a s s i n g t h r o u g h t h e i m m u n o a f f i n i t y column ( F i g . 3 l a n e a ) . The g e l t h a t was s i l v e r s t a i n e d had t o be o v e r d e v e l o p e d i n o r d e r t o i d e n t i f y t h e p o l y p e p t i d e bands i n l a n e a. 2. DETECTION OF ACTIN IN THE RPE PLASMA MEMBRANE PREPARATION W i t h t h e knowledge t h a t a c t i n i s an i m p o r t a n t c y t o s k e l e t a l p r o t e i n , i t was e x p e c t e d t h a t a c t i n would be a major RPE c e l l component due t o t h e r o l e of RPE c e l l s i n t h e p h a g o c y t o s i s of r o d o u t e r segments. The RPE pla s m a membrane p r e p a r a t i o n was r u n on a S D S - p o l y a c r y l a m i d e g e l and s u b s e q u e n t l y t r a n s f e r r e d t o n i t r o c e l l u l o s e where a c t i n was d e t e c t e d by a r a b b i t a n t i - a c t i n a n t i s e r a a t an a p p a r e n t M r=46,000 ( F i g . 5 ) . I t was a l s o n o t e d t h a t l i t t l e a c t i n was d e t e c t e d i n t h e ROS p r e p a r a t i o n a s oppo s e d t o t h e b o v i n e RPE pl a s m a membrane p r e p a r a t i o n . The p o s s i b l e s i g n i f i c a n c e of a c t i n i n t h e s e c e l l s w i l l be d i s c u s s e d l a t e r . 3. TEM OBSERVATION OF SUBCELLULAR FRACTIONATION R h o d o p s i n was d e t e c t e d i n t h e p l a s m a membrane p r e p a r a t i o n by S D S - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s w h i c h l e d t o t h e p o s s i b i l i t y t h a t t h e membrane p r e p a r a t i o n was c o n t a m i n a t e d w i t h r o d o u t e r segment d i s c m a t e r i a l . At t h e l e v e l of TEM, ROS d i s c -l i k e m a t e r i a l was f o u n d i n t h e p l a s m a membrane e n r i c h e d bands c o l l e c t e d from t h e s u c r o s e g r a d i e n t ( F i g . 6 ) . The p l a s m a membrane f r a g m e n t s a p p e a r e d as v e s i c l e s o f v a r y i n g s i z e . - 4 7 -F i q u r e 5. D e t e c t i o n o f a c t i n i n ROS and b o v i n e RPE pla s m a membrane p r e p a r a t i o n by S D S - p o l y a c r y l a m i d e g e l e l e t r o p h o r e s i s i n c o n j u n c t i o n w i t h immunoblot. Rod o u t e r segments (a) and b o v i n e RPE c e l l p lasma membrane p r e p a r a t i o n s (b) were s o l u b i l i z e d i n SDS i n t h e p r e s e n c e of 2 - m e r c a p t o e t h a n o l and 25 uL of e a c h was s u b j e c t e d t o e l e c t r o p h o r e s i s on a 5-16% g r a d i e n t SDS-p o l y a c r y l a m i d e g e l . The g e l s were s t a i n e d w i t h c o o m a s s i e b l u e (CB) or e l e c t r o p h o r e t i c a l l y t r a n s f e r r e d t o n i t r o c e l l u l o s e p a p e r . The n i t r o c e l l u l o s e p a p e r was i n c u b a t e d f o r two h o u r s w i t h r a b b i t a n t i - a c t i n a n t i s e r a , washed i n immunoblot b u f f e r a n d i n c u b a t e d 1 2 5 f o r 1 hour i n I - l a b e l e d g o a t a n t i - r a b b i t I g a n t i b o d y (1-2 X 10^ dpm/mL). The p a p e r was washed as b e f o r e and s u b j e c t e d t o a u t o r a d i o g r a p h y . -48-F i g u r e 6. T r a n s m i s s i o n e l e c t r o n m i c r o g r a p h s of b o v i n e r e t i n a l p igment e p i t h e l i a l c e l l p l a s m a membrane p r e p a r a t i o n s c o l l e c t e d from t h e t o p band of a 20-50% (w/w) c o n t i n u o u s s u c r o s e g r a d i e n t . A r rows d e n o t e a r e a s of p o s s i b l e c o n t a m i n a t i n g ROS d i s c m a t e r i a l i s o l a t e d w i t h t h e b o v i n e r e t i n a l p i gment e p i t h e l i a l c e l l p l a s m a membrane. -49-DISCUSSION The RPE pla s m a membrane p r e p a r a t i o n was 7-9 f o l d e n r i c h e d b a s e d on t h e c e l l u l a r homogenate. B o t h N a + K + ATPase and 5 ' n u c l e o t i d a s e plasma membrane m a r k e r s showed s i m i l a r f o l d p u r i f i c a t i o n . S i m i l a r r e c o v e r i e s and i n c r e a s e s i n s p e c i f i c a c t i v i t i e s o f plasma membrane m a r k e r s have been f o u n d w i t h s e v e r a l o t h e r c e l l l i n e s w i t h s i m i l a r methods of p u r i f i c a t i o n ( 6 8 , 7 1 ) . A m i n o r c o n t a m i n a t i o n o f t h e membrane p r e p a r a t i o n was m i t o c h o n d r i a l membrane i n t h e low e r s u c r o s e band. However t h e p r i m a r y s o u r c e o f i m p u r i t i e s i n t h e RPE membrane p r e p a r a t i o n was r o d o u t e r segment membrane from r o d p h o t o r e c e p t o r c e l l s . In t h e a d u l t b o v i n e eye i t a p p e a r s t h a t t h e i n d i g i t a t e d j u n c t i o n between t h e RPE c e l l s and t h e p h o t o r e c e p t o r c e l l s i s e x t r e m e l y t i g h t . The l o n g and numerous m i c r o v i l l i o f t h e RPE p r e v e n t t h e c l e a n r e m o v a l of a l l ROS w i t h t h e r e t i n a . S u b sequent w a s h i n g w i t h B u f f e r A c o n t a i n i n g EDTA f a i l e d t o r e l e a s e t h e t i g h t l y bound ROS from t h e RPE. A n a l y s i s o f t h e f r a c t i o n s from t h e c o n t i n u o u s s u c r o s e g r a d i e n t d e s i g n e d t o s e p a r a t e s u b c e l l u l a r o r g a n e l l e s d e m o n s t r a t e d t h e c o m i g r a t i o n of ROS membrane and RPE plas m a membrane. D i s r u p t e d RPE c e l l s i n 10% F i c o l l were l a y e r e d on t o p of 42% s u c r o s e and c e n t r i f u g e d i n an a t t e m p t t o f l o a t t h e " s a c k l i k e " s e a l e d ROS d i s c s on t o p of t h e F i c o l l w h i l e c o l l e c t i n g a c r u d e membrane band a t t h e i n t e r f a c e . The m a t e r i a l p e l l e t e d t h r o u g h t h i s c e n t r i f u g a t i o n was unbroken m i t o c h o n d r i a , r e s i d u a l n u c l e i , and p i g m e n t . T h i s method p r o v e d t o be s u c c e s s f u l i n r e m o v i n g a p p r o x i m a t e l y h a l f t h e d i s c m a t e r i a l c o n s i s t i n g -50-p r i m a r i l y o f r h o d o p s i n b a s e d on c o o m a s s i e b l u e s t a i n e d g e l s -C o n s i d e r a b l e plasma membrane marker a c t i v i t y (50-60%) was l o s t when t h e membrane p r e p a r a t i o n was spun on t o p of t h e 42% s u c r o s e as much of t h e plasma membrane g o t t r a p p e d w i t h t h e pigment and p e l l e t e d t h r o u g h t h e s u c r o s e . Plasma membrane marker a c t i v i t y was f o u n d i n b o t h of t h e p r i m a r y bands c o l l e c t e d from t h e c o n t i n u o u s s u c r o s e g r a d i e n t . The p l a s m a membrane i n t h e low e r band p r o b a b l y m i g r a t e d f u r t h e r i n t h e s u c r o s e due i t s a g g r e g a t i o n w i t h m i t o c h o n d r i a l membrane f r a g m e n t s . A n a l y z i n g t h e c r u d e p l a s m a membrane and t h e upper and lo w e r bands c o l l e c t e d o f f of t h e c o n t i n u o u s s u c r o s e g r a d i e n t by SDS g e l e l e c t r o p h o r e s i s r e v e a l e d t h a t many of t h e p o l y p e p t i d e bands were c o n s e r v e d t h r o u g h out t h e pla s m a membrane p u r i f i c a t i o n p r o c e d u r e . Most o f t h e p o l y p e p t i d e s seen i n t h i s SDS p o l y a c r y l a m i d e g e l e l e c t r o p h o r e t i c s y s t e m have a p p a r e n t 'M r a n g i n g from 20,000 t o 200,000. One p r i m a r y band seen on c o o m a s s i e b l u e s t a i n e d SDS g e l s and i d e n t i f i e d by t h e i m m u n o b l o t t i n g t e c h n i q u e was a c t i n w i t h a p p a r e n t M r= 46,000. A c t i n has been r e p o r t e d t o be an i m p o r t a n t c y t o s k e l e t a l p r o t e i n i n human RPE c e l l s ( 1 6 , 1 7 ) . U l t r a s t r u c t u r a l e x a m i n a t i o n of t h e RPE p l a s m a membrane p r e p a r a t i o n s i n d i c a t e d p o s s i b l e ROS d i s c m a t e r i a l l o c a t e d amongst t h e e n c l o s e d membrane v e s i c l e s . In an a t t e m p t t o remove t h e r h o d o p s i n c o n t a m i n a t i o n i n t h e p l a s m a membrane p r e p a r a t i o n , t h e membrane was s o l u b i l i z e d and p a s s e d t h r o u g h an a n t i - r h o d o p s i n i m m u n o a f f i n i t y c olumn. The s o l u b i l i z e d membrane was d i l u t e d 2-3 f o l d w i t h PBS w h i c h r e s u l t e d i n a r e q u i r e m e n t f o r a more s e n s i t i v e s i l v e r s t a i n f o r p o l y p e p t i d e band i d e n t i f i c a t i o n on SDS p o l y a c r y l a m i d e g e l s . T h i s p r o c e d u r e r e t a i n e d t h e m a j o r i t y of p o l y p e p t i d e bands w i t h a c o n s i d e r a b l e r e d u c t i o n i n t h e r h o d o p s i n c o n t a m i n a t i o n . The p r i m a r y g o a l f o r p e r f o r m i n g a RPE c e l l s u b c e l l u l a r f r a c t i o n a t i o n s t u d y was t o o b t a i n a r e a s o n a b l y e n r i c h e d plasma membrane p r e p a r a t i o n f o r use as an immunogen i n m o n o c l o n a l a n t i b o d y p r o d u c t i o n . C o n s e q u e n t l y , t h e t o p band from t h e s u c r o s e g r a d i e n t and t h e a n t i - r h o d o p s i n i m m u n o a f f i n i t y column e l u d e n t were u s e d t o immunize BALB/C mice f o r t h e p r o d u c t i o n of m o n o c l o n a l a n t i b o d i e s a g a i n s t RPE pl a s m a membrane components. - 5 2 -SECTION 2 THE PRODUCTION AND CHARACTERIZATION OF A MONOCLONAL ANTIBODY RESULTS 1. PRODUCTION OF MONOCLONAL ANTIBODIES S e v e r a l a t t e m p t s were made t o r a i s e m o n o c l o n a l a n t i b o d i e s a g a i n s t b o v i n e RPE pl a s m a membrane p r e p a r a t i o n s . P a r t i a l l y p u r i f i e d p l a s m a membrane was i n j e c t e d i n t o BALB/C m i c e and s c r e e n e d f o r a n t i b o d y p r o d u c t i o n by RIA. The mouse b l o o d t i t r e was f o u n d t o have a h a l f maximum a n t i b o d y b i n d i n g t o t h e RPE plas m a membrane p r e p a r a t i o n when d i l u t e d 350 t i m e s i n b u f f e r i n d i c a t i n g t h a t membrane p r e p a r a t i o n was a n t i g e n i c t o t h e mouse immune s y s t e m ( F i g . 7 ) . When mouse myeloma c e l l s were f u s e d w i t h s p l e e n l y m p h o c y t e s from an immunized mouse, h y b r i d o m a c e l l l i n e s were o b t a i n e d w h i c h s e c r e t e d a n t i b o d i e s r e a c t i v e t o w a r d s T r i t o n X-100 s o l u b i l i z e d and SDS s o l u b i l i z e d RPE pl a s m a membrane p r e p a r a t i o n s . F u r t h e r t e s t i n g i n d i c a t e d t h a t t h e s e m o n o c l o n a l a n t i b o d i e s c r o s s - r e a c t e d w i t h p r e p a r a t i o n s o f T r i t o n X-100 and SDS s o l u b i l i z e d ROS. 2. CHARACTERIZATION OF RHQ-5A3 One p a r t i c u l a r m o n o c l o n a l a n t i b o d y d e s i g n a t e d a s Rho-5A3 was f o u n d t o be a g a i n s t r h o d o p s i n . Rho-5A3 h y b r i d o m a c u l t u r e f l u i d had a h a l f maximum b i n d i n g t o s o l u b i l i z e d r o d o u t e r segments a t a 60 f o l d d i l u t i o n ( F i g . 8 ) . T a b l e 2 shows t y p i c a l r e s u l t s f o r a RIA i n w h i c h d e t e r g e n t s o l u b i l i z e d ROS membranes i m m o b i l i z e d i n m i c r o t i t e r w e l l s were s e q u e n t i a l l y t r e a t e d w i t h h y b r i d o m a c e l l 125 s u p e r n a t a n t and I - l a b e l e d g o a t a n t i - m o u s e I g a n t i b o d y . Rho-5A3 a n t i b o d y showed a 26% r e d u c t i o n i n a c t i v i t y t o ROS s o l u b i l i z e d i n - 5 3 -F i q u r e 7. A n t i b o d y t i t r a t i o n c u r v e f o r b l o o d from a BALB/C mouse immunized w i t h b o v i n e RPE pla s m a membranes. B o v i n e r e t i n a l p i g ment e p i t h e l i a l c e l l p l a s m a membrane p r e p a r a t i o n s o l u b i l i z e d i n T r i t o n X - 1 0 0 was i m m o b i l i z e d on m i c r o t i t e r w e l l s and i n c u b a t e d w i t h s e r i a l d i l u t i o n s of t h e b l o o d serum i n i t i a l l y d i l u t e d 5 f o l d i n RIA b u f f e r . A f t e r w a s h i n g i n PBS t h e w e l l s were i n c u b a t e d w i t h 1 2 5 I - l a b e l e d g o a t a n t i - m o u s e I g a n t i b o d y . - 5 4 -0 0 I c o C Q CL O 16 64 256 1024 4096 Reciprocal Dilution F i g u r e 8. T i t r a t i o n c u r v e o f Rho-5A3 m o n o c l o n a l a n t i b o d y c u l t u r e f l u i d a g a i n s t i m m o b i l i z e d T r i t o n X-100 s o l u b i l i z e d r o d o u t e r s e g m e n t s . S o l u b i l i z e d r o d o u t e r s e g m e n t s were d r i e d down on m i c r o t i t e r w e l l s and i n c u b a t e d w i t h s e r i a l d i l u t i o n s o f Rho-5A3 m o n o c l o n a l a n t i b o d y c u l t u r e f l u i d . A f t e r w a s h i n g w i t h PBS, t h e 125 m i c r o t i t e r w e l l s were i n c u b a t e d w i t h I - l a b e l e d g o a t a n t i - m o u s e I g a n t i b o d y . -55-TABLE 2 SOLID-PHASE RADIOIMMUNE ASSAY FOR A ROS S P E C I F I C ANTIBODY 1 25 I-LABELED GOAT ANTI-MOUSE Ig BOUND (DPM) HYBRIDOMA CELL TRITON X-100 SDS SUPERNATANT SOLUBILIZED ROS SOLUBILIZED ROS RHO-5A3 38,246 28,244 CONTROL 2,940 1 , 450 C o n t r o l = n o n - s p e c i f i c a n t i b o d y T a b l e 2. A c o m p a r i s o n of R h o - 5 A 3 m o n o c l o n a l a n t i b o d y b i n d i n g t o T r i t o n X-100 s o l u b i l i z e d and SDS s o l u b i l i z e d r o d o u t e r segments. Rod o u t e r segments ( 2 . 5 mg/mL) were s o l u b i l i z e d i n e i t h e r 1% T r i t o n X-100 or 1% SDS, d i l u t e d 10 f o l d i n d i s t i l l e d w a t e r and 25 uL were d r i e d down i n m i c r o t i t e r w e l l s . A f t e r b l o c k i n g t h e non-s p e c i f i c s i t e s i n RIA b u f f e r , t h e w e l l s were i n c u b a t e d i n R h o ~ 5 A 3 h y b r i d o m a c u l t u r e f l u i d , f o l l o w e d by wa s h i n g and i n c u b a t i o n w i t h 1 2 5 I - l a b e l e d g o a t a n t i - m o u s e I g a n t i b o d y . The c o n t r o l was t r e a t e d i n a s i m i l a r manner u s i n g a n o n - s p e c i f i c a n t i b o d y . -56-SDS as opposed to ROS s o l u b i l i z e d in Triton X-100. The control used was a primary non-specific antibody to ROS. 3. SUBCLASSIFICATION OF RHQ-5A3 MONOCLONAL ANTIBODY Solid-phase radioimmune assays were performed, as previously described, using Triton X-100 s o l u b i l i z e d ROS immobilized on microtiter wells. The wells were incubated with Rho-5A3 culture f l u i d with an additional subclass Ig antibody incubation followed 1 25 by detection with I-labeled goat anti-rabbit Ig antibody. Results in Table 3 indicated that the Rho-5A3 monoclonal is an IgG^ kappa l i g h t chain antibody. 4. IDENTIFICATION OF RHQ-5A3 ANTIGEN OF BOVINE ROS MEMBRANES In order to identi f y the antigenic s i t e for Rho-5A3, ROS were subjected to trypsin and S.Aureus V-8 protease treatment with separation of the p r o t e o l y t i c fragments by SDS gel electrophoresis followed by transfer of the polypeptides to CNBr-activated paper. The paper s t r i p s were then d i r e c t l y treated with Rho-5A3 or Rho-1D4 antibodies and detection was made by using 1 25 I-labeled goat anti-mouse Ig as a second antibody. Results are shown in Fig.9, along with the coomassie blue staining pattern. Both antibodies Rho-1D4 and Rho-5A3 bind to rhodopsin, the major coomassie blue staining glycoprotein of apparent M^= 34,000. When ROS were treated with S.Aureus protease three major rhodopsin bands were located at apparent Mf= 33,000 ,25,000 and 12,000 (72,73). Rho-5A3 bound to the Mr=33,000 and 25,000 cleaved rhodopsin bands while Rho-1D4 only bound residual undigested rhodopsin (74). When ROS were subjected to proteolysis by trypsin, .one - 5 7 -TABLE 3 IDENTIFICATION OF I g SUBTYPE OF RHQ-5A3  MONOCLONAL ANTIBODY R a b b i t a n t i - m o u s e Ig s u b t y p e s p e c i f i c a n t i b o d y b i n d i n g t o Rho-5A3 Ant i - ( « . ) -(y,) -<**> - (*> -(X ) ) c o n t r o l DPM bound X 1 0 ~ 3 2.2 2.7 3.0 2.5 6.0 2.2 9.6 2.6 2.9 T a b l e 3. Immunoassay f o r t h e s c r e e n i n g o f Rho-5A3 m o n o c l o n a l Ig a n t i b o d y s u b t y p e s . Rho-5A3 h y b r i d o m a c u l t u r e f l u i d was i n c u b a t e d w i t h T r i t o n X-100 s o l u b i l i z e d r o d o u t e r segments d r i e d down on m i c r o t i t e r w e l l s . A f t e r w a s h i n g i n PBS b u f f e r t h e w e l l s were i n c u b a t e d w i t h s u b c l a s s s p e c i f i c r a b b i t a n t i - m o u s e i m m u n o g l o b u l i n 1 25 f o l l o w e d by w a s h i n g as b e f o r e and i n c u b a t i o n w i t h I - l a b e l e d g o a t a n t i - r a b b i t I g a n t i b o d y . -58-Figure 9. A n a l y s i s of polypeptides from untreated and protease digested rod outer segments which bind Rho-1D4 and Rho-5A3 a n t i -rhodopsin monoclonal a n t i b o d i e s . Rod outer segments were digested with S.aureus protease (lane b) and t r y p s i n (lane c ) , washed by c e n t r i f u g a t i o n , s o l u b i l i z e d and e l e c t r o p h o r e s i z e d on a 10% SDS-polyacrylamide g e l . The polypeptides were e i t h e r s t a i n e d by coomassie blue (CB) or t r a n s f e r r e d to CNBr- a c t i v a t e d paper. The l a t t e r was t r e a t e d with Rho-1D4 or Rho-5A3 c u l t u r e f l u i d . 1 25 I d e n t i f i c a t i o n was made using I - l a b e l e d goat anti-mouse Ig antibody. Lane a was untreated rod outer segments. -59-fragment migrated just ahead of the leading edge of undigested rhodopsin. The Rho-5A3 monoclonal antibody binds strongly to t h i s large (apparent Mr=32,000) fragment while Rho-1D4 binds only residual undigested rhodopsin. It has been shown that t h i s fragment corresponds to rhodopsin with a 9 amino acid segment removed from the carboxyl terminus (74). 5. COMPETITIVE INHIBITION OF RHQ-5A3 MONOCLONAL ANTIBODY A competition assay was performed with Rho-1D4-Sepharose immunoaffinity p u r i f i e d rhodopsin (43) to determine the approximate concentration of rhodopsin required to give half maximum Rho~5A3 antibody binding. The i n h i b i t i o n p r o f i l e (Fig. 10) indicated that half maximum binding was achieved at a concentration of 5 ug/mL. The effectiveness of sealed ROS discs, frozen-thawed ROS discs, and Triton X-100 s o l u b i l i z e d ROS discs to i n h i b i t antibody binding to Triton X-100 treated, immobilized ROS discs was studied in order to determine the a c c e s s i b i l i t y of the antigenic s i t e (Fig. 11). Sealed discs and frozen-thawed discs showed no competition for the antibody even at high concentrations. Triton X-100 s o l u b i l i z e d discs were found to i n h i b i t Rho-5A3 antibody binding to immobilized and Triton X-100 s o l u b i l i z e d discs at concentrations above 0.1 mg/mL. 6. ANALYSIS OF RHQ-5A3 ANTIBODY BINDING PEPTIDE Rhodopsin peptides were also used in an attempt to i n h i b i t Rho-5A3 binding to Triton X-100 s o l u b i l i z e d immobilized rod outer segments. When Rho-5A3 was competed with a 2-39 N-terminal polypeptide from rhodopsin, i n h i b i t i o n of Rho-5A3 binding to - 6 0 -10 «-5 r4 I O ' 3 10 ~ 10 Rhodopsin (mg/ml) 10 r2 10 -1 F i g u r e 10. I n h i b i t i o n of Rho-5A3 monoclonal a n t i b o d y b i n d i n g t o T r i t o n X-100 s o l u b i l i z e d r o d o u t e r segments by i n c u b a t i o n w i t h r h o d o p s i n . S e r i a l d i l u t i o n s of Rho-1D4 i m m u n o a f f i n i t y p u r i f i e d r h o d o p s i n were i n c u b a t e d w i t h Rho-5A3 c u l t u r e f l u i d i n m i c r o t i t e r w e l l s . T w e n t y - f i v e uL from each w e l l was i n c u b a t e d w i t h d r i e d down s o l u b i l i z e d rod o u t e r segments. F o l l o w i n g washing i n PBS, 125 a n t i b o d y d e t e c t i o n was made u s i n g I - l a b e l e d goat anti-mouse I g a n t i b o d y . - 6 1 -F i q u r e 11. I n h i b i t i o n of Rho~5A3 m o n o c l o n a l a n t i b o d y b i n d i n g t o T r i t o n X - 1 0 0 s o l u b i l i z e d r o d o u t e r segment d i s c p r o t e i n by f r o z e n / t h a w e d d i s c s ( A A ) , s e a l e d d i s c s ( • • ) , and T r i t o n X - 1 0 0 s o l u b i l i z e d d i s c s ( • • ). S e r i a l d i l u t i o n s of i n h i b i t o r were i n c u b a t e d w i t h Rho-5A3 h y b r i d o m a c u l t u r e f l u i d i n m i c r o t i t e r w e l l s . T w e n t y - f i v e uL were removed from e a c h w e l l and i n c u b a t e d w i t h T r i t o n X - 1 0 0 s o l u b i l i z e d d i s c p r o t e i n s d r i e d down i n m i c r o t i t e r w e l l s p r e v i o u s l y i n c u b a t e d i n RIA b u f f e r . A f t e r 1 25 w a s h i n g i n PBS t h e w e l l s were i n c u b a t e d w i t h I - l a b e l e d g o a t a n t i - m o u s e I g a n t i b o d y . - 6 2 -i m m o b i l i z e d T r i t o n X-100 s o l u b i l i z e d r o d o u t e r segments was o b s e r v e d a t c o n c e n t r a t i o n s above 0.5 uM, but no i n h i b i t i o n was o b s e r v e d w i t h t h e 2-16 N - t e r m i n a l r h o d o p s i n p o l y p e p t i d e ( F i g . 1 2 ) . 7. LOWICRYL THIN SECTION LABELING T h i n s e c t i o n s of b o v i n e p h o t o r e c e p t o r c e l l s , embedded i n L o w i c r y l r e s i n , were l a b e l e d w i t h Rho-5A3 m o n o c l o n a l a n t i b o d y f o l l o w e d by g o a t a n t i - m o u s e I g g o l d - d e x t r a n . U l t r a s t r u c t u r a l e x a m i n a t o n by TEM, r e v e a l e d t h a t t h e r o d o u t e r segments were h e a v i l y and randomly l a b e l e d w h i l e t h e r o d i n n e r segments h a d v e r y l i t t l e l a b e l ( F i g . 1 3 ) . -63-F i g u r e 12. I n h i b i t i o n o f Rho-5A3 m o n o c l o n a l a n t i b o d y b i n d i n g t o T r i t o n X-100 s o l u b i l i z e d r o d o u t e r segments by r h o d o p s i n 2-16 N-t e r m i n u s p o l y p e p t i d e ( • • ) and r h o d o p s i n 2-39 N - t e r m i n u s p o l y p e p t i d e ( # # ). S e r i a l d i l u t i o n s o f p o l y p e p t i d e s were i n c u b a t e d w i t h Rho-5A3 h y b r i d o m a c u l t u r e f l u i d i n m i c r o t i t e r w e l l s . T w e n t y - f i v e uL from e a c h w e l l was i n c u b a t e d w i t h T r i t o n X-100 s o l u b i l i z e d r o d o u t e r segments d r i e d down i n m i c r o t i t e r w e l l s p r e v i o u s l y i n c u b a t e d i n RIA b u f f e r . A f t e r w a s h i n g i n PBS t h e 125 w e l l s were i n c u b a t e d i n I - l a b e l e d g o a t a n t i - m o u s e Ig a n t i b o d y . -64-F i g u r e 13, T r a n s m i s s i o n e l e c t r o n m i c r o g r a p h of a g l u t a r a l d e h y d e f i x e d b o v i n e r o d p h o t o r e c e p t o r c e l l embedded i n L o w i c r y l and l a b e l e d w i t h Rho-5A3 m o n o c l o n a l a n t i b o d y h y b r i d o m a c u l t u r e f l u i d f o l l o w e d by g o a t a n t i - m o u s e I g - A u 2 n d e x t r a n . N o t i c e t h e heavy l a b e l i n g on t h e r o d o u t e r segment w i t h l i t t l e l a b e l i n g on t h e r o d i n n e r segment. -65-DISCUSSION S e v e r a l c e l l f u s i o n s were p e r f o r m e d w i t h s p l e e n c e l l s f r o m BALB/C mice p r e v i o u s l y immunized w i t h RPE p l a s m a membrane p r e p a r a t i o n s f o r t h e p r o d u c t i o n of m o n o c l o n a l a n t i b o d y s e c r e t i n g h y b r i d o m a c e l l l i n e s . When the i m m u n o a f f i n i t y p u r i f i e d RPE p l a s m a membrane p r e p a r a t i o n was u s e d t o immunize BALB/C m i c e , no h y b r i d o m a c e l l l i n e s were p r o d u c e d t h a t s e c r e t e d m o n o c l o n a l a n t i b o d i e s s p e c i f i c f o r RPE plasma membrane p r e p a r a t i o n s . T h i s r e s u l t p r o b a b l y r e f l e c t s t h e l a c k of s u f f i c i e n t p r o t e i n i n i t i a l l y i n j e c t e d i n t o t h e mouse. A l t e r n a t i v e l y t h e s o l u b i l i z e d p r o t e i n s i n j e c t e d were not a n t i g e n i c t o t h e immune s y s t e m of t h e mouse. However, t h e f u s i o n o f s p l e e n c e l l s f r o m a BALB/C mouse immunized w i t h t h e RPE plasma membrane p r e p a r a t i o n t o p band c o l l e c t e d f r o m t h e c o n t i n u o u s s u c r o s e g r a d i e n t and NS-1 (myeloma) c e l l s r e s u l t e d i n a h y b r i d o m a c e l l l i n e w hich s e c r e t e d a m o n o c l o n a l a n t i b o d y s p e c i f i c f o r t h e membrane p r e p a r a t i o n . The m o n o c l o n a l a n t i b o d y , d e s i g n a t e d Rho-5A3, however was f o u n d t o be s p e c i f i c f o r r h o d o p s i n as d e t e r m i n e d by radioimmune l a b e l i n g o f ROS p r o t e i n s e l e c t r o p h o r e t i c a l l y t r a n s f e r r e d from SDS g e l s t o CNBr- a c t i v a t e d p a p e r . Radioimmune c o m p e t i t i o n s t u d i e s l o c a l i z e d t h e a n t i g e n i c s i t e t o t h e N - t e r m i n u s i n t r a d i s k a l r e g i o n of t h e r h o d o p s i n m o l e c u l e ( F i g . 14). A n a l y s i s of i m m u n o g l o b u l i n s u b t y p e s i n d i c a t e d t h a t t h e Rho-5A3 m o n o c l o n a l a n t i b o d y was an IgG^ kappa l i g h t c h a i n a n t i b o d y . IgG a n t i b o d i e s a r e t h e most u s e f u l t y p e of a n t i b o d y f o r s e r o l o g i c a l l a b e l i n g and i m m u n o l o g i c a l s t u d i e s as t h e i r non-s p e c i f i c b i n d i n g t e n d s t o be q u i t e low. -66-F i g u r e 14. A d i a g r a m m a t i c model o f r h o d o p s i n , m o d i f i e d f r o m H a r g r a v e (75), and t h e l o c a t i o n o f t h e a n t i g e n i c s i t e s f o r a v a r i e t y o f m o n o c l o n a l a n t i b o d i e s . N ote t h e a p p r o x i m a t e l o c a t i o n of t h e Rho-5A3 m o n o c l o n a l a n t i b o d y on t h e N - t e r m i n u s of t h e r h o d o p s i n m o l e c u l e . The Rho-1D4 m o n o c l o n a l a n t i b o d y has been shown t o be l o c a t e d on t h e c a r b o x y l t e r m i n u s (43). - 6 7 -The a n t i g e n i c s i t e f o r t h e m o n o c l o n a l a n t i b o d y , Rho-1D4, i s a l o n g t h e f i r s t s e v e n amino a c i d s on t h e c a r b o x y l t e r m i n u s o f r h o d o p s i n . ( 4 3 ) W i t h Rho-1D4 as a c o n t r o l , l i m i t e d p r o t e o l y t i c d i g e s t i o n s t u d i e s were p e r f o r m e d t o l o c a l i z e t h e s i t e of Rho-5A3 a c t i o n . D i g e s t i o n o f ROS m a t e r i a l w i t h S . a u r e u s V-8 p r o t e a s e o r t r y p s i n w h i c h removed a sev e n o r n i n e amino a c i d p e p t i d e from t h e C - t e r m i n u s of r h o d o p s i n r e s u l t e d i n t h e l o s s o f Rho-1D4 b i n d i n g w h i l e t h e a n t i g e n i c s i t e f o r Rho-5A3 b i n d i n g was m a i n t a i n e d . ( F i g . 9 ) . In t h e c a s e of S . a u r e u s V-8 d i g e s t i o n , t h e Rho~5A3 m o n o c l o n a l a n t i b o d y a l s o bound t o t h e r h o d o p s i n p r o t e o l y t i c f r a g ment (M r=25,000) d e s i g n a t e d t h e F fr a g m e n t w h i c h i s known t o c o n t a i n t h e N - t e r m i n u s ( 7 3 ) . T h e s e r e s u l t s i n d i c a t e t h a t t h e Rho-5A3 a n t i b o d y b i n d s t o an a n t i g e n i c s i t e a l o n g t h e amino-t e r m i n a l t w o - t h i r d s o f r h o d o p s i n ( F i g . 1 4 ) . W i t h t h e s e r e s u l t s , i t was i n t e r e s t i n g t o e x p l o r e t h e a c c e s s i b i l i t y of t h e Rho-5A3 b i n d i n g s i t e . The Rho~5A3 a n t i b o d y d i d n o t b i n d t o s e a l e d or f r o z e n - t h a w e d ROS d i s c r h o d o p s i n m a t e r i a l even a t h i g h c o n c e n t r a t i o n s . However, Rho~5A3 bound v e r y w e l l t o r h o d o p s i n when s o l u b i l i z e d i n T r i t o n X-100 w h i c h i s known t o c a u s e i r r e v e r s i b l e s t r u c t u r a l c h a n g e s . B a s e d on t h e s e r e s u l t s i t a p p e a r s t h a t t h e Rho-5A3 a n t i g e n i c d e t e r m i n a n t i s i n a c c e s s i b l e t o t h e a n t i b o d y when r h o d o p s i n i s i n t h e d i s c membrane. The n a t i v e r h o d o p s i n p r o t e i n c o n f o r m a t i o n o r t h e p h o s p h o l i p i d b i l a y e r may c a u s e t h e i n a c c e s s i b i l i t y of t h e Rho-5A3 d e t e r m i n a n t . In an a t t e m p t t o f u r t h e r i d e n t i f y t h e a n t i g e n i c s i t e of Rho-5A3 a c t i o n , c o m p e t i t i o n a s s a y s were p e r f o r m e d w i t h s p e c i f i c a l l y p r e p a r e d r h o d o p s i n p o l y p e p t i d e s . P r e v i o u s l y , Rho-5A3 a n t i b o d y had -68-been shown t o compete w i t h r h o d o p s i n w i t h h a l f maximum a n t i b o d y b i n d i n g when t h e r h o d o p s i n c o n c e n t r a t i o n was 5 ug/mL, q u i t e s i m i l a r t o Rho-4A2 ( a n o t h e r N - t e r m i n a l r h o d o p s i n b i n d i n g m o n o c l o n a l a n t i b o d y ) ( 4 3 ) . S o l i d - p h a s e c o m p e t i t i o n a s s a y s d e m o n s t r a t e d Rho-5A3 a n t i b o d y ' s a b i l i t y t o compete w i t h t h e 2-39 r h o d o p s i n N - t e r m i n a l p e p t i d e a t a c o n c e n t r a t i o n above 0.5 uM. However, no c o m p e t i t i o n was o b s e r v e d w i t h t h e 2-16 r h o d o p s i n N-t e r m i n a l p o l y p e p t i d e up t o a c o n c e n t r a t i o n of 10 uM. T h e s e p r e l i m i n a r y s t u d i e s i n d i c a t e t h a t t h e Rho-5A3 a n t i g e n i c s i t e i s l o c a t e d i n t h e 17-38 r h o d o p s i n N - t e r m i n a l p e p t i d e r e g i o n . B o v i n e ROS, p r e v i o u s l y f i x e d and embedded i n L o w i c r y l , were l a b e l e d e x t e n s i v e l y and randomly by g o a t a n t i - m o u s e I g g o l d d e x t r a n a f t e r t r e a t m e n t o f t h e s e c t i o n s w i t h t h e Rho-5A3 a n t i b o d y ( F i g . 1 3 ) . In t h e s e s e c t i o n s t h e a n t i g e n i c s i t e s of Rho-5A3 a c t i o n a r e a c c e s s i b l e t o l a b e l i n g t e c h n i q u e s . R h o d o p s i n was f o u n d a b u n d a n t l y i n t h e r o d o u t e r , segments and not f o u n d a t a l l i n t h e r o d i n n e r segments. The q u e s t i o n r e m a i n e d a s t o t h e d i f f i c u l t y i n o b t a i n i n g a n t i b o d i e s s p e c i f i c f o r t h e b o v i n e RPE p l a s m a membrane. A n o t h e r a p p r o a c h u s e d t o p r o d u c e m o n o c l o n a l a n t i b o d i e s s p e c i f i c f o r b o v i n e RPE c e l l s was t o i n t r o d u c e t h e c e l l s i n t o t i s s u e c u l t u r e p r i o r t o i m m u n i z a t i o n . B o v i n e RPE c e l l s were grown i n t i s s u e c u l t u r e and i n j e c t e d i n t o BALB/C m i c e . Subsequent s p l e e n c e l l and myeloma c e l l f u s i o n s r e s u l t e d i n two more h y b r i d o m a c e l l l i n e s w h i c h s e c r e t e d a n t i b o d i e s a g a i n s t ROS p r e p a r a t i o n s but n e i t h e r had r h o d o p s i n as t h e i r a n t i g e n i c d e t e r m i n a n t . I t a p p e a r s t h a t ROS and r e s i d u a l ROS p r o t e i n s a s s o c i a t e d w i t h t h e RPE c e l l s -69-a r e h i g h l y a n t i g e n i c w h i l e RPE p l a s m a membranes a r e n o t , p o s s i b l y due t o a b l o o d - b r a i n b a r r i e r . -70-SECTION 3 ANALYSIS OF BOVINE RPE CELLS IN VITRO RESULTS 1. ISOLATION OF BOVINE RPE CELLS S e v e r a l p r o c e d u r e s f o r i s o l a t i n g RPE c e l l s f r o m t h e b o v i n e eye o p t i c cup were e x p l o r e d . T e c h n i q u e s s u c h as s c r a p i n g t h e RPE c e l l s f r o m t h e o p t i c c u p or d i s s e c t i n g out t h e RPE c e l l s f r o m t h e B r u c h ' s membrane were p e r f o r m e d w i t h m i n i m a l r e s u l t s due t o p o o r y i e l d s and c e l l b r e a k a g e . The method w h i c h p r o d u c e d t h e b e s t y i e l d was e n z y m a t i c d i g e s t i o n w i t h t r y p s i n and c o l l a g e n a s e . The RPE c e l l s were p l a t e d o u t on a g l a s s s u b s t r a t e w h i c h p r o v i d e d an e x c e l l e n t s u p p o r t f o r c e l l g r o w t h . 2. THE MORPHOLOGY OF BOVINE RPE CELLS IN VITRO The b o v i n e RPE c e l l s a f t e r e n z y m a t i c t r e a t m e n t were c u b o i d a l i n shape and c o n s i d e r a b l y r e d u c e d i n s i z e . A f t e r 4 d a y s i n t i s s u e c u l t u r e t h e c e l l s s t i l l r e t a i n e d a c u b o i d a l shape ( F i g . 1 5 a ) . The b o v i n e RPE c e l l s became e s t a b l i s h e d i n t i s s u e c u l t u r e a f t e r a p p r o x i m a t e l y 1 week ( F i g . 15b) and b o t h t h e m e l a n o t i c and a m e l a n o t i c c e l l s became more d i s p e r s e d i n a p p e a r a n c e ( F i g . 1 5 c ) . As t h e c e l l u l a r g r o w t h became c o n f l u e n t w i t h a d o u b l i n g t i m e o f 52 h o u r s t h e p i g m e n t a t i o n of t h e c e l l s became l e s s d e n s e . A t t h e l e v e l of SEM, RPE c e l l s a p p e a r e d f l a t t e n e d a f t e r 10 d a y s i n t i s s u e c u l t u r e w i t h s e v e r a l m i c r o v i l l i a p p a r e n t on a p p r o x i m a t e l y one h a l f of t h e c e l l s u r f a c e ( F i g . 16). The RPE c e l l s a l s o had s c a t t e r e d 1-2 urn wide v e s i c l e s a t t a c h e d t o t h e c e l l s u r f a c e ( F i g . 1 7 ) . U n d e r l y i n g melanosomes b u l g e d out a g a i n s t - 7 1 -F i g u r e 15. B o v i n e r e t i n a l p i gment e p i t h e l i a l c e l l s grown i n RPMI-1640 medium on g l a s s c o v e r s l i p s as s e e n by phase c o n t r a s t m i c r o s c o p y . a) 4 days ( 1 0 0 0 X ) . b) 7 d a y s ( 1 4 6 0 X ) . c) 18 d a y s ( 1 6 0 0 X ) . B o v i n e RPE c e l l s were i s o l a t e d f r o m t h e o p t i c c u p by e n z y m a t i c t r e a t m e n t w i t h 0.25% t r y p s i n and 70 u n i t s / m L c o l l a g e n a s e d i s s o l v e d i n B u f f e r A. The c e l l s were i n c u b a t e d a t o 5 37"C ( a p p r o x i m a t e l y 5 x 10 c e l l s / m L ) i n 10 mL p e t r i d i s h e s c o n t a i n i n g g l a s s c o v e r s l i p s . - 7 2 -F i g u r e 16. S c a n n i n g e l e c t r o n m i c r o g r a p h o f a b o v i n e r e t i n a l p igment e p i t h e l i a l c e l l grown on g l a s s f o r 10 days i n RPMI-1640 t i s s u e c u l t u r e medium. C l e a r arrow i n d i c a t e s a r e a w i t h few m i c r o v i l l i w h i l e dark a r r o w i n d i c a t e s a r e a w i t h dense m i c r o v i l l i . - 7 3 -F i g u r e 17. S c a n n i n g e l e c t r o n m i c r o g r a p h of a b o v i n e r e t i n a l p i gment e p i t h e l i a l c e l l grown on g l a s s i n RPMI-1640 c u l t u r e medium f o r 10 d a y s . A r r o w s d e n o t e v e s i c l e s , p o s s i b l y r o d o u t e r segments a t t a c h e d t o t h e c e l l s u r f a c e . - 7 4 -t h e plasma membrane due t o c e l l s h r i n k a g e d u r i n g c r i t i c a l p o i n t d r y i n g of t h e samples f o r SEM ( F i g . 1 8 ) . The g e n e r a l m o r p h o l o g i c a l f e a t u r e s o f t h e c e l l , however, were m a i n t a i n e d a f t e r d r y i n g . Extreme c a u t i o n had t o be u s e d i n c r i t i c a l p o i n t d r y i n g of t h e RPE c e l l s i n t h e p r e p a r a t i o n f o r SEM as t h e plasma membrane t e n d s t o be q u i t e f r a g i l e and e a s i l y d i s r u p t e d . 3. FLUORESENT DETECTION OF ACTIN IN BOVINE CULTURED RPE CELLS P a r a f o r m a l d e h y d e f i x e d b o v i n e RPE c e l l s were t r e a t e d w i t h a c e t o n e and t h e c e l l s were i n c u b a t e d w i t h r a b b i t a n t i - a c t i n a n t i s e r a f o l l o w e d by F I T C - l a b e l e d g o a t a n t i - r a b b i t Ig a n t i b o d y . R e s u l t s c o n f i r m e d b i o c h e m i c a l s t u d i e s t h a t a c t i n was a major component o f t h e b o v i n e RPE c e l l . F l u o r e s c e n c e was c o n c e n t r a t e d t h r o u g h o u t t h e c e l l w i t h heavy f l u o r e s c e n c e a s s o c i a t e d w i t h t h e p l a s m a membrane. When r a b b i t a n t i - a c t i n a n t i s e r a p r e i n c u b a t e d w i t h p u r i f i e d a c t i n was u s e d as t h e p r i m a r y a n t i b o d y , o n l y m i n i m a l f l u o r e s c e n c e was d e t e c t e d i n t h i s c o n t r o l sample ( F i g . 1 9 ) . 4. DISCONTINUOUS FLUORESCENT LECTIN LABELING OF BOVINE RPE CELLS V a r i o u s f l u o r e s c e n t l e c t i n s were use t o s t u d y t h e a r r a n g e m e n t , d i s t r i b u t i o n , and r e d i s t r i b u t i o n of s u g a r r e s i d u e s on t h e s u r f a c e of b o v i n e of RPE c e l l s . Such l e c t i n s i n c l u d e F I T C -Con A, s p e c i f i c f o r *s-D-mannose and °<-D-glucose; FITC-WGA, s p e c i f i c f o r N- a c e t y l g l u c o s a m i n e o l i g o m e r s ; and FITC-RCA, s p e c i f i c f o r D- g a l a c t o s e . S t u d i e s w i t h 2 week o l d RPE c e l l s i n c u b a t e d f o r 5 m i n u t e s a t 37°C, w i t h FITC-Con A showed e x t e n s i v e l a b e l i n g on t h e c e l l s u r f a c e ( F i g . 2 0 a ) . S i m i l a r r e s u l t s were f o u n d w i t h c e l l s f i x e d - 7 5 -F i g u r e 18. S c a n n i n g e l e c t r o n m i c r o g r a p h of a b o v i n e r e t i n a l e p i t h e l i a l c e l l grown on g l a s s f o r 10 d a y s . Note t h e r o u g h t e x t u r e of t h e c e l l s u r f a c e due t o t h e abundance of melanosomes. - 7 7 -F i g u r e 19. F l u o r e s c e n t m i c r o g r a p h s o f p a r a f o r m a l d e h y d e f i x e d 2 week o l d b o v i n e r e t i n a l pigment e p i t h e l i a l c e l l s i n c u b a t e d w i t h r a b b i t a n t i - a c t i n a n t i s e r a . a) RPE c e l l s grown on g l a s s c o v e r s l i p s were f i x e d i n 1% p a r a f o r m a l d e h y d e f o l l o w e d by t r e a t m e n t i n a c e t o n e . A f t e r w a s h i n g i n PBS b u f f e r c o n t a i n i n g M g + + and C a + + t h e c o v e r s l i p s were i n c u b a t e d w i t h r a b b i t a n t i - a c t i n a n t i s e r a f o l l o w e d by w a s h i n g as b e f o r e and i n c u b a t i o n w i t h F I T C -l a b e l e d g o at a n t i - r a b b i t I g a n t i b o d y . (2000x) b) As w i t h a ) , w i t h t h e e x c e p t i o n t h a t t h e p r i m a r y a n t i b o d y was p r e v i o u s l y i n c u b a t e d w i t h 100 ug of p u r i f i e d a c t i n . ( 2 0 0 0 x ) . -78-w i t h 1% p a r a f o r m a l d e h y d e . As a c o n t r o l , t h e FITC-Con A l e c t i n was competed o f f t h e c e l l s u r f a c e w i t h 0.25 M <* m e t h y l mannoside ( F i g . 2 0 b ) . L a b e l i n g w i t h FITC-Con A l e c t i n f o r 5 m i n u t e s f o l l o w e d by a 60 m i n u t e i n c u b a t i o n p e r i o d i n b u f f e r r e s u l t e d i n p a t c h y f l u o r e s c e n c e i n d i c a t i n g a p o s s i b l e i n t e r n a l i z a t i o n of t h e Con A r e c e p t o r s ( F i g . 2 0 c ) . V e r i f i c a t i o n of r e c e p t o r r e d i s t r i b u t i o n and i n t e r n a l i z a t i o n a t t h e l e v e l o f f l u o r e s c e n c e , was a c h i e v e d by an a t t e m p t t o compete o f f t h e FITC-Con A l e c t i n w i t h m e t h y l mannoside w h i c h p r o v e d t o be u n s u c c e s s f u l ( F i g . 20d) . A s i m i l a r s t u d y was p e r f o r m e d u s i n g FITC-WGA and FITC-RCA. L a b e l i n g of u n f i x e d RPE c e l l s f o r 5 m i n u t e s gave a v e r y u n i f o r m and i n t e n s e f l u o r e s c e n c e f o r FITC-WGA ( F i g . 2 1 a ) , but t h e l a b e l i n g w i t h FITC-RCA was c o n s i d e r a b l y l e s s i n t e n s e ( F i g . 2 2 a ) . In t h e c o n t r o l e x p e r i m e n t s l a b e l i n g w i t h FITC-WGA was competed o f f t h e c e l l s u r f a c e by t r e a t i n g t h e c e l l s w i t h t h e s a c c h a r i d e i n h i b i t o r 0.02 M N-N d i a c e t y l c h i t o b i o s e a t 37°C, f o r 15 m i n u t e s ( F i g . 21 b ) . FITC-RCA was competed o f f i n a s i m i l a r manner by 0.2 M D-g a l a c t o s e ( F i g . 2 2 b ) . L a b e l i n g RPE c e l l s f o r 5 m i n u t e s w i t h F I T C -WGA f o l l o w e d by 60 m i n u t e s i n b u f f e r r e s u l t e d i n p a r t i a l r e c e p t o r r e d i s t r i b u t i o n w i t h some a p p a r e n t i n t e r n a l i z a t i o n due t o t h e l a c k of c o m p e t i t i o n w i t h t h e a p p r o p r i a t e s a c c h a r i d e i n h i b i t o r ( F i g . 2 1 c - d ) . S i m i l a r l y , FITC-RCA when t r e a t e d i n t h e same way y i e l d e d p a t c h y f l u o r e s c e n c e due t o r e c e p t o r r e d i s t r i b u t i o n and i n t e r n a l i z a t i o n ( F i g . 2 2 c ) . 5. CONTINUOUS FLUORESCENT LECTIN LABELING OF RPE CELLS IN VITRO B o v i n e RPE c e l l s were l a b e l e d c o n t i n u o u s l y f o r 60 m i n u t e s F i g u r e 21 . Figure 2 2 . a. b. c. - 8 3 -F i q u r e 20. F l u o r e s c e n t m i c r o g r a p h s o f u n f i x e d RPE c e l l s l a b e l e d w i t h F ITC-Con A f o r : a) 5 min.; b) 5 min. f o l l o w e d by a 15 min. i n c u b a t i o n i n 0.25 M *<- m e t h y l mannoside; c) 5- min. f o l l o w e d by wash i n b u f f e r and 60 min. i n c u b a t i o n i n b u f f e r a t 37°C; d) as i n c) f o l l o w e d by a 15 min. i n c u b a t i o n w i t h 0.25 M ^ - m e t h y l m a n n o s i d e . F i g u r e 21. F l u o r e s c e n t m i c r o g r a p h s of u n f i x e d RPE c e l l s l a b e l e d w i t h FITC-WGA f o r : a) 5 min.; b) 5 min. f o l l o w e d by a 15 min. i n c u b a t i o n w i t h 0.020 M N - N - d i a c e t y l c h i t o b i o s e ; c) 5 min. f o l l o w e d by wash and 60 min. i n c u b a t i o n i n b u f f e r a t 37°C; d) as i n c ) f o l l o w e d by i n c u b a t i o n i n 0.020 M N - N - d i a c e t y l c h i t o b i o s e f o r 15 min. F i g u r e 22. F l u o r e s c e n t m i c r o g r a p h s of u n f i x e d RPE c e l l s l a b e l e d w i t h FITC-RCA f o r : a) 5 min.; b) 5 min. f o l l o w e d by a 15 min. i n c u b a t i o n i n 0.2 M D - g a l a c t o s e ; c ) 5 min. f o l l o w e d by wash and 60 min. i n c u b a t i o n i n b u f f e r a t 37°C, p l u s 15 min. i n 0.2 M D - g a l a c t o s e . F i g u r e 23. F l u o r e s c e n t m i c r o g r a p h s of RPE c e l l s l a b e l e d f o r 60 m i n u t e s a t 37°C w i t h ; a) F ITC-Con A b) FITC-WGA c) FITC-RCA. - 8 4 -w i t h F I T C - l e c t i n s , a t 37°C. In t h e c a s e o f Con A, t h e r e was e x t e n s i v e and i n t e n s e f l u o r e s c e n c e f o u n d t h r o u g h out t h e c e l l w i t h some p r o m i n e n t l a r g e f l u o r e s c e n t p a t c h e s ( F i g . 2 3 a ) . T h e s e r e s u l t s i n d i c a t e d t h a t Con A r e c e p t o r s were i n d u c e d t o i n t e r n a l i z e but a t t h e same t i m e new r e c e p t o r s became a v a i l a b l e r e s u l t i n g i n a heavy c o a t of l a b e l on . t h e c e l l s u r f a c e . C o n t i n u o u s l a b e l i n g w i t h FITC-WGA and FITC-RCA r e s u l t e d i n heavy f l u o r e s c e n c e on t h e membrane w i t h some a p p a r e n t f l u o r e s c e n t p a t c h e s ( F i g s . 23b-c) 6. PROBING BOVINE RPE CULTURED CELLS WITH RHQ-1D4 Due t o t h e p r e s e n c e of a h i g h c o n c e n t r a t i o n of r h o d o p s i n i n th e RPE pla s m a membrane p r e p a r a t i o n from f r e s h b o v i n e e y e s , i t was i n t e r e s t i n g t o e x p l o r e whether r h o d o p s i n c o u l d be d e t e c t e d i n RPE c e l l s grown i n t i s s u e c u l t u r e . U s i n g Rho-1D4 m o n o c l o n a l a n t i b o d y , s p e c i f i c f o r t h e C t e r m i n u s of r h o d o p s i n , b o v i n e RPE c e l l s grown i n m i c r o t i t r e w e l l s were s o l u b i l i z e d i n 0.1% T r i t o n X-100 and a s s a y e d by t h e s t a n d a r d RIA method. R e s u l t s i n d i c a t e d t h a t Rho-1D4 bound t o a r e s i d u a l r h o d o p s i n component i n t h e RPE c e l l s a t a h i g h l e v e l f o r o v e r 10 d a y s i n v i t r o ( F i g . 2 4 ) . P r e s u m a b l y , e i t h e r r o d o u t e r segments a r e s t i l l a s s o c i a t e d w i t h t h e RPE c e l l s or r h o d o p s i n has n o t been c o m p l e t e l y d e g r a d e d by t h e c e l l s i n t i s s u e c u l t u r e . 7. PHAGOCYTOSIS OF ROS BY BOVINE RPE CELLS IN VITRO When b o v i n e ROS were i n c u b a t e d f o r 5 h w i t h b o v i n e RPE c e l l s , SEM o b s e r v a t i o n r e v e a l e d t h a t t h e ROS were a t t a c h e d t o t h e c e l l s u r f a c e ( F i g . 2 5 ) . P r e l i m i n a r y r e s u l t s i n d i c a t e d t h a t t h e b o v i n e RPE c e l l s were r e c o g n i z i n g and a t t a c h i n g t o t h e r o d o u t e r -85-F i g u r e 24. Rho-1D4 m o n o c l o n a l a n t i b o d y b i n d i n g t o T r i t o n X-100 s o l u b i l i z e d b o v i n e RPE c e l l s grown i n m i c r o t i t e r w e l l s . B o v i n e RPE c e l l s grown i n f l a t b o t t o m m i c r o t i t e r w e l l s were s o l u b i l i z e d i n 0.1% T r i t o n X-100 and i n c u b a t e d w i t h Rho-1D4 h y b r i d o m a c u l t u r e f l u i d . F o l l o w i n g w a s h i n g i n b u f f e r , t h e w e l l s were i n c u b a t e d w i t h 1 2 5 I - l a b e l e d g oat a n t i - m o u s e I g a n t i b o d y . - 8 6 -F i q u r e 25. S c a n n i n g e l e c t r o n m i c r o g r a p h of a 2 week o l d b o v i n e RPE c e l l i n c u b a t e d f o r 5 h w i t h s e a l e d b o v i n e r o d o u t e r segments. A r r o w s i n d i c a t e some of t h e numerous bound r o d o u t e r segments amongst t h e mat of m i c r o v i l l i . - 8 7 -segments. An assay condit ion was establ i shed for the phagocytosis of dark adapted ROS by 2 week old bovine RPE c e l l s . Sealed ROS were c o l l e c t e d from a sucrose gradient and incubated for 5 h , at 3 7 ° C , with RPE c e l l s grown on glass c o v e r s l i p s . Qual i tat ive observations made by TEM indicated that some, but not a l l , adult bovine RPE c e l l s in v i t r o were capable of phagocytizing ROS (Figs.26,27) These resul t s establ ished a new system for studying the phagocytosis of rod outer segments. -90-F i g u r e 26. T r a n s m i s s i o n e l e c t r o n m i c r o g r a p h s of 2 week o l d b o v i n e RPE c e l l s i n c u b a t e d w i t h d a r k a d a p t e d s e a l e d r o d o u t e r segments f o r 5 h. Arrow d e n o t e s t h e p o s i t i o n of t h e pla s m a membrane. Note t h a t t h e phagosome (PH) of ROS d i s c membrane i s c o m p l e t e l y i n s i d e t h e c e l l . The n u c l e u s (N) i s q u i t e e v i d e n t a l o n g w i t h s e v e r a l melanosomes (M). F i g u r e 27. T r a n s m i s s i o n e l e c t r o n m i c r o g r a p h s of a n o t h e r 2 week o l d b o v i n e RPE c e l l i n c u b a t e d w i t h d a r k a d a p t e d s e a l e d r o d o u t e r segments w h i c h f u r t h e r i l l u s t r a t e s ROS p h a g o c y t o s i s . N o t i c e t h e a r r a n g e m e n t of s t a c k s o f ROS d i s c membrane f o r m i n g t h e l a r g e phagosomes (PH). The n u c l e u s (N) and melanosomes (M) a r e a l s o e v i d e n t . - 9 1 -DISCUSSION The i n t r o d u c i n g o f a d u l t b o v i n e RPE c e l l s i n t o t i s s u e c u l t u r e opened up a whole new a p p r o a c h i n s t u d y i n g RPE c e l l s u r f a c e s and RPE-ROS i n t e r a c t i o n s . Once t h e RPE c e l l s r e c o v e r e d from t h e t r a u m a t i c e n z y m a t i c t r e a t m e n t t h e i r c e l l m o r p h o l o g y became l e s s c u b o i d a l as t h e y e s t a b l i s h e d r a p i d growth i n v i t r o as seen by o t h e r r e s e a r c h e r s ( 2 8 , 3 0 , 3 1 ) . The no r m a l and m u l t i n u c l e a t e d c e l l s , w i t h p r i m a r i l y m o s a i c and s p i n d l e g r o w t h p a t t e r n s d e v e l o p e d a h a r d y g r o w t h r a t e ( d o u b l i n g t i m e 52 h ) u n t i l c o n f l u e n t c u l t u r e s were o b t a i n e d . At t h e l e v e l of l i g h t m i c r o s c o p y t h e c e l l s d e m o n s t r a t e d a f l a t h i g h l y p i g m e n t e d n a t u r e as o b s e r v e d i n i n t a c t t i s s u e . M i c r o v i l l i were f o u n d o v e r a l a r g e s e c t i o n of t h e b o v i n e RPE c e l l s u r f a c e w i t h i n t e r s p e r s e d a t t a c h e d v e s i c l e s . T h e s e v e s i c l e s on t h e c e l l s u r f a c e have t h e a p p e a r a n c e of ROS w h i c h s t i l l may be t i g h t l y a s s o c i a t e d w i t h RPE c e l l s even a f t e r 10 days i n t i s s u e c u l t u r e . From t h e f r a c t i o n a t i o n and SDS g e l e l e c t r o p h o r e s i s of RPE c e l l s grown i n t i s s u e c u l t u r e t h e r e was s t i l l c o n s i d e r a b l e r h o d o p s i n a s s o c i a t e d w i t h t h e RPE c e l l s . P r o b i n g w i t h t h e Rho-1D4 a n t i b o d y ( s p e c i f i c f o r c a r b o x y l t e r m i n a l o f r h o d o p s i n ) , r h o d o p s i n was f o u n d t o be a s s o c i a t e d w i t h t h e b o v i n e RPE c e l l s i n t i s s u e c u l t u r e f o r as l o n g a s 2 weeks. I t a p p e a r s t h a t t h e membrane p r o t e i n , r h o d o p s i n , o r a t l e a s t t h e Rho-1D4 a n t i g e n i c s i t e , i s s l o w l y d e g r a d e d i n t i s s u e c u l t u r e w i t h a p o s s i b l e i n i t i a l l a t e n t p e r i o d . T h i s would s u p p o r t t h e p r o p o s e d i d e a t h a t t h e d e g r a d a t i o n of r h o d o p s i n by RPE l y s o s o m a l f r a c t i o n s o c c u r s a f t e r t h e i n i t i a l p hase of d e g r a d a t i o n of o t h e r membrane p r o t e i n s ( 3 6 ) . - 9 2 -P r e l i m i n a r y s t u d i e s l o o k e d a t t h e a r r a n g e m e n t of c e r t a i n c e l l s u r f a c e g l y c o p r o t e i n s t h a t may be i m p o r t a n t i n ROS-RPE i n t e r a c t i o n s . F l u o r e s c e n t l e c t i n s were u s e d t o l o o k a t t h e d i s t r i b u t i o n of s u g a r r e s i d u e s on b o v i n e RPE c e l l s . L a b e l i n g b o t h f i x e d and u n f i x e d m e l a n o t i c and a m e l a n o t i c b o v i n e RPE c e l l s i n v i t r o w i t h F I T O C o n A, WGA, and RCA a t 37°C r e s u l t e d i n a d ense u n i f o r m l a b e l i n g a t t h e f l u o r e s c e n t l e v e l o f o b s e r v a t i o n . L a b e l i n g RPE c e l l s w i t h f l u o r e s c e n t l e c t i n s f o l l o w e d by 60 m i n u t e s i n b u f f e r r e s u l t e d i n t h e e n e r g y d e p e n d e n t r e d i s t r i b u t i o n of t h e l a b e l , as most of t h e f l u o r e s c e n c e a p p e a r e d as i n t e n s e s p o t s c e n t r a l i z e d i n t h e e x t r a n u c l e a r r e g i o n of t h e c e l l . The s t a i n i n g p a t t e r n s r e f l e c t an i n t e r n a l i z a t i o n of t h e l a b e l s i n c e a h i g h c o n c e n t r a t i o n o f i n h i b i t o r f a i l e d t o remove t h e s p e c i f i c l a b e l . The c e l l s t r e a t e d w i t h WGA s t i l l a p p e a r e d t o have l a b e l e d r e c e p t o r s on t h e membrane even a f t e r 60 m i n u t e s i n b u f f e r i n d i c a t i n g t h a t not a l l t h e l a b e l e d WGA r e c e p t o r s were i n d u c e d t o i n t e r n a l i z e or r e d i s t r i b u t e as seems t o be t h e c a s e w i t h Con A and RCA. RCA p e r o x i d a s e l a b e l e d s i t e s on c u l t u r e d e m b r y o n a l n e u r o n s were shown by TEM t o i n t e r n a l i z e ( 7 6 ) . C e l l s u r f a c e b i n d i n g s i t e s c o n t i n u o u s l y l a b e l e d w i t h l e c t i n s a t 37°C i n d u c e d r e a r r a n g e m e n t f o r a l l t h r e e l e c t i n s u s e d . At t h e l e v e l o f f l u o r e s c e n c e , a l l t h r e e l e c t i n r e c e p t o r s a p p e a r t o r e d i s t r i b u t e i n d e p e n d e n t l y of t h e i r u n l a b e l e d c o u n t e r p a r t s when s u b s a t u r a t i n g c o n c e n t r a t i o n s o f l e c t i n s were u s e d . R e d i s t r i b u t i o n and i n t e r n a l i z a t i o n i s c o n s t a n t l y o c c u r i n g so t h a t p r e v i o u s l y u n l a b e l e d r e c e p t o r s become a c c e s s i b l e f o r l a b e l i n g r e s u l t i n g i n a dense p a t t e r n o f l a b e l on t h e c e l l s u r f a c e b e i n g m a i n t a i n e d . -93-F u r t h e r e x p e r i m e n t s u s i n g l e c t i n s a r e r e q u i r e d t o c o n c l u s i v e l y p r e d i c t t h e mode o f l e c t i n r e c e p t o r i n t e r n a l i z a t i o n . D i f f e r e n c e s i n t u r n o v e r r a t e s of l a b e l e d r e c e p t o r s may p l a y a r o l e i n t h e r e d i s t r i b u t i o n p a t t e r n s . D o u b l e l a b e l i n g e x p e r i m e n t s would f u r t h e r d e f i n e t h e a c t i o n of m u l t i p l e r e c e p t o r r e d i s t r i b u t i o n and i n t e r n a l i z a t i o n a s Con A i t s e l f b i n d s t o many plas m a membrane p r o t e i n s ( 7 7 ) . L e c t i n s t u d i e s a r e i n t e r e s t i n g i n t h i s p a p e r as t o t h e i r p o s s i b l e r o l e i n r e c e p t o r - r e c e p t o r i n t e r a c t i o n between r o d o u t e r segments and RPE c e l l s u r f a c e s . I t i s p o s s i b l e t h a t s u g a r r e c e p t o r a r r a n g e m e n t may be i n v o l v e d i n t h e r e c o g n i t i o n of ROS d i s c p a c k e t s r e l e a s e d from t h e r o d o u t e r segments. C a r b o h y d r a t e s as f o u n d on g l y c o p r o t e i n s s u c h as L - f u c o s e and D-mannose s i g n i f i c a n t l y r e d u c e d t h e ROS d i s c p a c k e t s p h a g o c y t i z e d i n t h e f r o g s y s t e m ( 3 7 ) . In t h e f u t u r e , f u r t h e r s t u d i e s a t t h e l e v e l of TEM a r e r e q u i r e d t o q u a n t i t a t i v e l y l o c a l i z e v a r i o u s l e c t i n s on t h e c e l l s u r f a c e s of n o r m a l and d y s t r o p h i c r a t RPE. From t h e RPE p l a s m a membrane p r e p a r a t i o n i t was n o t e d t h a t a c t i n was d e t e c t e d by immunoblot t e c h n i q u e s . A c e t o n e t r e a t e d RPE c e l l s a l s o d e m o n s t r a t e d s p e c i f i c a n t i - a c t i n f l u o r e s c e n c e c o n c e n t r a t e d p r i m a r i l y on t h e p l a s m a membrane o f t h e c e l l s b u t a l s o f o u n d t h r o u g h o u t t h e c e l l . The h i g h d e g r e e o f p i g m e n t a t i o n t e n d s t o o b s c u r e much of t h e f l u o r e s c e n t p r e v e n t i n g a good o b s e r v a t i o n of t h e i n t e r n a l c y t o s k e l e t a l s y s t e m . A n t i - a c t i n f l u o r e s c e n c e was n o n - u n i f o r m b e i n g more c o n c e n t r a t e d near t h e n u c l e a r r e g i o n i n t e r s p e r s e d w i t h b r i g h t e r f l u o r e s c e n t p a t c h e s w h i c h may r e p r e s e n t s u r f a c e m i c r o v i l l i . - 9 4 -I t a p p e a r s from l i t e r a t u r e t h a t t h e i n g e s t i o n phase of p h a g o c y t o s i s i s d e f e c t i v e i n t h e d y s t r o p h i c pigment e p i t h e l i a l c e l l s ( 3 , 9 , 1 4 ) . C o n s e q u e n t l y , t h i s d e f e c t may i n v o l v e t h e m o b i l i z a t i o n o r f u n c t i o n i n g of a c t i n . E v i d e n c e from t h e a r r a n g e m e n t of a c t i n f i l a m e n t s i n RPE c e l l s p o i n t s t o needed a c t i n a c t i v a t i o n and r e a r r a n g e m e n t t o t h e c e l l p e r i p h e r y f o r t h e m e c h a n i s t i c p h a g o c y t o s i s of ROS ( 1 7 ) . P o s s i b l y , i n t h e d y s t r o p h i c c a s e , a c t i n i s o n l y a c t i v a t e d i n a few s i t e s when ROS become a t t a c h e d t o t h e c e l l s u r f a c e . In o r d e r t o f u r t h e r s t u d y t h e ROS-RPE r e c e p t o r i n t e r a c t i o n a s u i t a b l e a s s a y s y s t e m had t o be d e v e l o p e d t h a t c o u l d be u s e d r e p e a t e d l y . A s s a y s y s t e m s u s i n g c h i c k and r a t embryonic RPE e x p l a n t s have been u s e d i n t h e p a s t t o s t u d y t h e p h a g o c y t o s i s of n o n - s p e c i f i c p a r t i c l e s as w e l l as ROS ( 4 , 5 , 1 4 , 1 5 ) , but b o v i n e RPE c e l l s i n v i t r o have not been u s e d p r e v i o u s l y i n t h e s t u d y of p h a g o c y t o s i s of ROS. By u s i n g s p e c i a l l y p r e p a r e d d a r k a d a p t e d , s e a l e d ROS, two week o l d RPE c e l l s have been shown, not o n l y t o a t t a c h ROS, but a l s o t o p h a g o c y t i z e t h e s e same ROS. Rod o u t e r segments have been seen a t t a c h e d t o RPE c e l l s by SEM ( F i g . 2 5 ) . At t h e l e v e l of TEM, l a r g e ROS p a c k e t s were o b s e r v e d a t t a c h e d and a l s o c o m p l e t e l y e n d o c y t o s e d by t h e b o v i n e RPE c e l l s ( F i g s . 2 6 , 2 7 ) . O b s e r v a t i o n s by TEM f a i l e d t o p r o v i d e any q u a n t i t a t i v e i n f o r m a t i o n i n terms of t h e number of ROS p h a g o c y t i z e d . Some c e l l s i n v i t r o f a i l e d t o r e c o g n i z e or p h a g o c y t i z e ROS due p o s s i b l y t o a l o s s of r e c e p t o r s i t e s t h r o u g h c e l l d i v i s i o n s or a l o s s of needed m i c r o v i l l i . T h i s new p h a g o c y t o s i s a s s a y s y s t e m opens up a whole new s e r i e s o f f u t u r e e x p e r i m e n t s f o r s t u d y i n g -95-ROS r e c e p t o r - R P E r e c e p t o r i n t e r a c t i o n s . CONCLUSIONS A 7-9 f o l d e n r i c h e d RPE plasma membrane p r e p a r a t i o n was p r e p a r e d and c h a r a c t e r i z e d by e n z y m a t i c a n a l y s i s . The membrane p r e p a r a t i o n was f o u n d t o have a l a r g e r h o d o p s i n c o n t a m i n a t i o n w h i c h was p a r t i a l l y removed by i m m u n o a f f i n i t y c h r o m a t o g r a p h y . A m o n o c l o n a l a n t i b o d y , d e s i g n a t e d Rho-5A3, was r a i s e d a g a i n s t t h e RPE membrane p r e p a r a t i o n , but i t was f o u n d t o be s p e c i f i c f o r r h o d o p s i n . The Rho-5A3 a n t i b o d y bound t o s o l u b i l i z e d r o d o u t e r segments and was l o c a l i z e d t o t h e N - t e r m i n u s o f t h e r h o d o p s i n m o l e c u l e . In o r d e r t o f u r t h e r s t u d y b o v i n e RPE c e l l s , t h e c e l l s were i n t r o d u c e d i n t o t i s s u e c u l t u r e where t h e y m a i n t a i n e d many .of t h e i r i n v i v o c h a r a c t e r i s t i c s . F l u o r e s c e n t l e c t i n l a b e l e d Con A, WGA, and RCA s i t e s on t h e c e l l s u r f a c e of b o v i n e RPE were i n d u c e d t o c o n s t a n t l y r e d i s t r i b u t e and i n t e r n a l i z e m a i n t a i n i n g a d e n s e p a t t e r n o f l a b e l , on t h e c e l l s u r f a c e . A c t i n was i d e n t i f i e d a s a major RPE pla s m a membrane and c y t o s k e l e t a l p r o t e i n . F i n a l l y a new a s s a y s y s t e m was e s t a b l i s h e d f o r s t u d y i n g t h e p h a g o c y t o s i s of ROS by b o v i n e RPE c e l l s i n v i t r o . F u t u r e i n v e s t i g a t i o n c an t a k e s e v e r a l a p p r o a c h e s . F i r s t o f a l l , a newly o b t a i n e d RPE plasma membrane s p e c i f i c m o n o c l o n a l a n t i b o d y d e s i g n a t e d RPE-3A6 c o u l d be c o m p l e t e l y c h a r a c t e r i z e d a l o n g w i t h i t s b o v i n e RPE d e t e r m i n a n t . S e c o n d l y , an a n t i - N -t e r m i n u s r h o d o p s i n m o n o c l o n a l antibody', d e s i g n a t e d Rho-4D2 w h i c h has t h e a b i l i t y t o b i n d t o s e a l e d ROS, c o u l d be u s e d t o -96-q u a n t i t a t i v e l y s t u d y p h a g o c y t o s i s o f ROS i n c o n j u n c t i o n w i t h r a d i o a c t i v e l a b e l s . T h i r d l y , t h e r o l e o f c e l l s u r f a c e g l y c o p r o t e i n s can be f u r t h e r s t u d i e d by l e c t i n s a t t h e f l u o r e s c e n t , SEM, and TEM l e v e l s . F i n a l l y , t h e p r o b l e m s of p r o d u c i n g h y b r i d o m a c e l l l i n e s t h a t s e c r e t e m o n o c l o n a l a n t i b o d i e s a g a i n s t RPE plasma membrane components s h o u l d be s o l v e d and new m o n o c l o n a l a n t i b o d i e s r a i s e d . 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