Succinic dehydrogenase in the rabbit ciliary epithelium

Succinic dehydrogenase in the rabbit ciliary epithelium

Exp. Eye Res. (1963) 2, 25-27 Succinic Dehydrogenase in the Rabbit Ciliary Epithelium EVAsN CAMERON AND D . F . COLE Medical Research Council, Ophth...

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Exp. Eye Res. (1963) 2, 25-27

Succinic Dehydrogenase in the Rabbit Ciliary Epithelium EVAsN CAMERON AND D . F . COLE

Medical Research Council, Ophthalmological Research Unit, Institzae of Ophthalmology London, England (Received 18 May 1962) (1) The distribution of succinic dehydrogenase (which probably indicates sites of utilization of carbohydrates via the tricarboxylie acid cycle) has been studied histochemically in the ciliary body of albino rabbits using blue tetrazolium. (2) The enzyme is present in the epithelium but not in the stroma. (3) The enzyme is more a b u n d a n t in the anterior zone of the ciliary processes t h a n in the posterior region and the activity appears to be of an order similar to t h a t in the anterior region of the r e t i n a . . (4) Within the ciliary epithelium activity appears to be greater in the innermost epithelial layer than in the pigment-cell layer. (5) There is also a certain a m o u n t of non-specific reducing activity in the tissue, part of which is due to endogenous succinate or succinate-producing systems. 1° I n t r o d u c t i o n

T h e r e is e v i d e n c e t h a t t h e c i l i a r y e p i t h e l i u m is a r e g i o n of t h e b l o o d - a q u e o u a b a r r i e r w h e r e t h e r e occurs active t r a n s p o r t of electrolytes f r o m plasma to the posterior c h a m b e r o f t h e e y e ( L a n g h a m , 1958) a n d , b y a n a l o g y w i t h o t h e r tissues, i t s e e m e d l i k e l y t h a t t h e e n e r g y reqtfirements of active t r a n s p o r t w o u l d u l t i m a t e l y be derived from aerobic m e t a b o l i s m (eft L e a f P a g e a n d A n d e r s o n , 1959; U s s i n g , 1960). T h e p r e s e n t e x p e r i m e n t s w e r e c a r r i e d o u t ill o r d e r t o e s t a b l i s h t h e a n a t o m i c a l d i s t r i b u t i o n o f o n e o f t h e e n z y m e s c o m m o n l y i n v o l v e d in o x i d a t i v e m e t a b o l i s m - - t h e s u c c i n i e d e h y d r o g e n a s e system. The availability of succinie d e h y d r o g e n a s e (SDH), oxidizing suceinate to f u m a r a t e , w o u l d s e e m t o b e a n e c e s s a r y c o n d i t i o n for t h e u t i l i s a t i o n o f c a r b o h y d r a t e s via t h e t r i e a r b o x y l i c a c i d cycle, a n d t h e d i s t r i b u t i o n of t h i s e n z y m e s h o u l d i n d i c a t e t h e r e g i o n s w h e r e o n e of t h e m a j o r s o u r c e s of m e t a b o l i c e n e r g y is m a d e a v a i l a b l e . I n t h e s t u d y reported below t h e distribution of S D H has been e x a m i n e d using the reduct i o n of b l u e t e t r a z o l i u m [ 3 , 3 ' - d i a n i s o l e - b i s - 4 , , i ' - ( 3 , 5 - d i p h e n y l ) - t e t r a z o l i u m c h l o r i d e ] t o y i e l d a n i n s o l u b l e d i f o r m a z a n as a n i n d i c a t o r o f t h e r e g i o n s w h e r e e n z y m i c o x i d a t i o n of s u c c i n a t e t a k e s p l a c e (cf. P e a r s e , 1958, 1960). I t e x t e n d s t h e o b s e r v a t i o n s of D e B e r n a r d i n i s (1958), w h o e x a m i n e d t h e t e t r a z o l i u m s t a i n i n g of v a r i o u s r e g i o n s of t h e eye, e s p e c i a l l y t h e r e t i n a , a n d e s t i m a t e d t h e a m o u n t of d i f o r m a z a n p r o d u c e d b y tissue homogcnates. 2. M a t e r i a l and M e t h o d s

All observatiol~s were m a d e o~ tissues from albino rabbits a n a e s t h e t i z e d with intravenous ure~hane prior to being killed. T h e eyes were r e m o v e d i m m e d i a t e l y after d e a t h a n d t h e p r e l i m i n a r y stages of t h e p r e p a r a t i o n carried o u t at r o o m t e m p e r a t u r e . The cornea was r e m o v e d a n d a disc of tissue c o n t a i n i n g the iris and c i l i a r y body o b t a i n e d by sectioning the globe coronally some 4- m m b e h i n d t h e liIubus. After r e m o v a l of t h e lens t h e disc of tissue was t h e n s t a i n e d in bulk, t h e m e t h o d being s u b s t a n t i a l l y t h e same as t h a t e m p l o y e d by K u w a b a r a a n d Cogatl (1959). I n c u b a t i o n of t h e tissue with 0-04 ~I substratc (suceinate) in 0.15 ~r p h o s p h a t e buffer a t p i t 7-2 and in t h e presence of 0.25 mM bluc 25

2{3

:EVAN

CAMEI~ON

AND

D.

F.

COLE

t e t r a z o l i u m lasted for 3 hr a t 37°C. After fixation in 7~/o formol-saline the tissue was en,bedded in 10°/(, gelatin a n d frozen sections cut a b o u t 1 5 - 2 0 / z thick. These were m o u n t e d in Kaiser's medium for e x a m i n a t i o n , a fine precipitate of the blue diformazan being present in regions of S D H a c t i v i t y . A similar piece of tissue from t h e same a n i m a l was i n c u b a t e d as described above b u t in a m e d i u m c o n t a i n i n g no added succinate, a n d served as a control. I n some e x p e r i m e n t s both t h e coutrol a n d succina-te-contailling media were m a d e up to contain 5 m moles/1 c y a n i d e or iodoacetate or 10 m moles/1 lr(Monate. Oncc the sections had been m o u n t e d a n d exazrfined a n u m b e r of t y p i c a l examples were p h o t o g r a p h e d a few d a y s later; it was found s u b s e q u e n t l y t h a t the p r e p a r a t i o n s remained stable for a t least three m o n t h s . In some cases the whole disc of tissue was p h o t o g r a p h e d immediate,~y after the i n c u b a t i o n and before sectioning in order to show t h e gross distribution of dif~rmazan in the anterior segment. 3. R e s u l t s

W h e n i n c u b a t i o n w a s c a r r i e d o u t in t h e s u c c i n a t e - c o n t a i n i n g m e d i u m , d i f o r m a z a n p r e c i p i t a t i o n o c c u r r e d o n l y i n t h e e p i t h e l i u m of t h e c i l i a r y processes a n d iris, a n d w a s n o t a p p a r e n t in t h e s t r o m a . T h e d i s t r i b u t i o n w a s n o t t m i f o r m in tile p r e p a r a t i o n , t h e d i f o r m a z a n p r e c i p i t a t e b e i n g m o s t d e n s e in t h e a n t e r i o r zone ~ f t h e c i l i a r y processes. T h e m o r e l i g h t l y s t a i n i n g p o s t e r i o r zone o c c u p i e d r a t h e r m o r e t h a n one t h i r d of t h e whole c i l i a r y processes a n d t h e line of d e m a r c a t i o n w a s s u f f i c i e n t l y s h a r p to be c l e a r l y visible to t h e n a k e d e y e ( P l a t e 1). A n t e r i o r l y t h e s t a i n i n g w a s c o n t i n u e d i n t h e epit h e l i u m of t h e p o s t e r i o r s u r f a c e of t h e iris. T h i s g e n e r M d e s c r i p t i o n is in a g r e e m e n t w i t h t h e f i n d i n g of D e B e r n a r d i n i s (1958) a n d wotfld a p p l y to figures 2 a n d 4 in his paper. P o s t e r i o r l y t h e r e was v e r y f a i n t p r e c i p i t a t i o n in t h e e p i t h e l i u m b e t w e e n t h e folds of t h e c i l i a r y processes w h e r e t h e y o r i g i n a t e d in t h e p a r s p l a n a r e t i n a e a n d w h i c h w a s e v e n less a p p a r e n t in t h e e p i t h e l i u m c o v e r i n g t h e c r e s t s of t h e processes t h e m s e l v e s ( P l a t e 2). A p p r o a c h i n g t h e a n t e r i o r p a r t of t h e c i l i a r y b o d y v,...:, t h e o r a s e r r a t a r e t i n a e , t h e p r e c i p i t a t e b e c a m e d e n s e r first b e t w e e n t h e processes (t ,-o i n t h e c r y p t s ) , t h e n on t h e i r sides u n t i l , in t h e r e g i o n of m a x i ~ , ' , m s t a i n i n g , t h e e p i t h e l i u m of t h e w h o l e process, i n c l u d i n g t h e crest, w a s seen tu c o n t a i n g r a n u l e s of d i f o r m a z a n ( P l a t e 3). T h e e p i t h e l i u m c o v e r i n g ~,he c i l i a r y processes is m a d e u p of t w o l a y e r s of cells, a n i n n e r u n p i g m e n t e d l a y e r w h i c h is c o n t i n u o u s w i t h t h e p a r s o p t i c a r e t i n a e , a n d a n o u t e r l a y e r u s u a l l y c o n t a i n i n g p i g m e n t g r a n u l e s b u t wlfich are a b s e n t i n a l b i n o r a b bits. T h e c h i e f d i s a d v a n t a g e of t h i s m e t h o d w a s t h a t i t e n t a i l e d t h e use of t h i c k s e c t i o n s a n d , as a c o n s e q u e n c e , p h o t o m i c r o g r a p h y w a s difficult a n d t h e r e s u l t s w e r e n o t a= good as m i g h t be desired. S o m e d i f o r m a z a n w a s seen in t h e p i g m e n t - c e l l l a y e r , b u t m u c h m o r e d e n s e s t a i n i n g o c c u r r e d in t h e u n p i g m e n t e d e p i t h e l i a l l a y e r ( P l a t e s 4--6). I n c o n t r o l e x p e r i m e n t s , w h e r e t h e t i s s u e h a d b e e n i n c u b a t e d in p h o s p h a t e buffer and tetrazolium without added succinate, the staining with diformazan, although f a i r l y e x t e n s i v e m a c r o s c o p i c M l y ( P l a t e 1), w a s m u c h less dense, tks in t h e s u c e i n a t e series, t h e e p i t h e l i a l cells s h o w e d m o r e p r e c i p i t a t e t h a n t h e p i g m e n t cells; t h e differe n t i a t i o n of t h e c i l i a r y p r o c e s s e s into" a n t e r i o r a n d p o s t e r i o r zones w a s m o r e pron o u n c e d a n d t h e r e were m o r e processes s h o w i n g l i t t l e or no p r e c i p i t a t i o n . As in t h e s u c c i n a t e series o f e x p e r i m e n t s s l i g h t s t a i n i n g w a s f o u n d i n t h e p o s t e r i o r e p i t h e l i u m of t h e iris. C y a n i d e (5 raM) c o m p l e t e l y i n h i b i t e d t e t r a z o l i u m r e d u c t i o n in b o t h t h e c o n t r o l a n d t h e s u c c i n a t e series. M a l o n a t e (10 raM) a n d i o d o a c e t a t e (5 m~t) c a u s e d p a r t i a l

PLATE l. A n t e r i o r s e g m c p . t of r a b b i t e y e ( p o s t e r i o r view) a f t e r i n c u b a t i o n a t 3 7 ° C for 3 hr w i t h 0 . 2 5 rest b l u e t e t r a z o l i u m in 0 . 1 5 .~I p h o s p h ~ t t e at, p H 7.2: (left) e o l t t r o l , w i t h o u t a(lde(I s u c c i n a t e ; (right) p l u s 0-04 .~1 succil~atc. A ~- A~ttcrior z(me of cilittry p r o c e s s e s . P == P o s t e r i o r z o n e of c i l i a r y ])roc(.~sst~.s.

PLA'rt.: 2. i a n c , ent, lal sect.ion of , ~ t . e r i o r s e g m e n t t h r o u g h t h e p o s t e r i o r z o n e of (:ili,~ry p r o c e s s e s . I n tile r e g i o n S t h e s u c c i t d e d e h y ( l r o g e J l a s e r e a c t i o n is m o r e evi(lenl~ i n tile f o l d s b e t w e e , t t h e p r o c e s s e s t h a n i n t h e e p i t h e l i u m of t h e c r e s t s . rl

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SDH

IN RABBIT CILIARY EPITHELIUM

27

inhibition of diformazan staining both with and without added succinate, the effect of malonate being greater than that of iodoacetate. The distinction between the anterior and posterior zones of the ciliary processes described above also occurred when malonate or icdoacetate were present. Under the conditions described it is reasonable to regard malonate as a selective inhibitor of SDH (Krebs, 1943) and hence as a means of identifying the enzyme. 4. Discussion

From the foregoing resuEs it appears t h a t SDH is present in the epithelium of the ciliary process and the posterior surface of the iris. The precipitation of diformazan that occurred without the addition of succinate to the medium indicates that the tissues contain a certain amount of endogenous succinate or some system capable of producing succinate (by oxidation of isocitrate for example). This would account for the finding t h a t dfformazan coloration in the control series, without added succinate, was decreased in the presence of malonate acting as a specific SDtt h~hibitor. Cyanide caused a more extensive inhibition than malonate, indicating t h a t oxidations other thA~ those directly involving SDH were taking place in the tissue. In addition it is also possible that there are present non-specific tetrazolium-reducing agents (nothing dehydrogenases) described by Racker (1955) and by Zimmerman and Pearse (1959). Despite the relatively large amount of non-specific tetrazolium reduction the ciliary epithelium displays a marked ability to oxidize added succinate, and comparison with the anterior region of the retina in those specimens in which both tissues had been stained simultaneously would seem to indicate that the SDH activity of the two are of the same order of magnitude (Plate 7). This is supported by the work of De Bernardinis (1958) who extracted the formazan and estimated it colorimetrically. The observation t h a t S D t t activity is less in the pigment cell layer than in the epithelial cell layer is in keeping with the distribution in the retina and pigment epithelium noted by Cogan and Kuwabara (1960). This difference of SDH activity between the epithelial and pigmented cells is suggestive of a metabolic difference, either of rate or orientation, between the adjacent cell layers of the ciliary processes. REFERENCES Cogan, 'D. G. and Kuwabara, T. (1960). J. Histochem. Cytochem. 8, 380. De Bernardinis, E. (1958). Ann. Ottalm. 84, 312. I4rebs, H. A. (1943). Advanc. Enzymol. 3, 191. Kuwabara, T. and Cogan, I). G. (1959). J. Histochem. Cytochem. 7, 329. Langham, M. E. (1958). Physiol. Rev. 38, 215. Leaf, A., Page, L. B. and Anderson, ft. (1959). J. biol. Chem. 234, 1625. Pearse, A. G. E. (1958). J. din. Path. 11, 520. Pearse, A. G. E. (1960). Histochemistry, 2nd Ed. Churchill, London. .Racker, E. (1955). Physiol. Rev. 35, 1. Ussing, H. BE. (1960). In Handbuch der experimenteUen Pharmakologie, ed. by O. Eich[er and A. Farah, Vol. 13, p. 1. Springer, Berlin. Zimmerman, H. and Pearse, A. G. E. (1959). J. Hi~tochem. Cytochem. 7. 271.