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The effects of sodium salicylate on the metabolism of the retina
Ea T. E!/e Zffes. (1965) 4, 364-369
T h e E f f e c t s o f S o d i u m S a l i c y l a t e o n the N I e t a b o l i s m o f the R e t i n a C~VE ~N. G ~ Y ~ o ~
AN~ :H~.'ATH~R BI~OWN
Depa.rt~nent of ~athology, Institute of 01~hthalmolocjy, University of London, J.udd Street, London., E'~ujla~ul (Received 30 A'ugust, J965) C e r t a i n a s p e c t s o f t h e ett'ecbs o f ~ a l i c y l a t ~ o n r e t i n a l m e t a b o l i s m a r c c o n s i d e r e d . I n ' v i v o a d m i n i s t r a t i o n o f s o d i u m s a l i c y l a t e i n d u c e s an i n c r e a s e in t h e a b s o l u t e level o f la~:tie a c i d in t h e r e t i n a . S u c h t r e a t m e n t is also s h o w n t o r e s u l t in a s l i g h t s t i m u l a t o r y e f f e c t o n l~ctic a c i d p r o d u c t i o u b y t h e s u b s e q u e n t l y e x c i s e d r e t i n a , bug t o h a v e n o s i g n i f i c ; r n t e f f e c t o n t h e r a t e o f g l u c o s e u p t a k e . Ineub~Ltion o f t h e n o r m a l r e t i n a , w i t h o r w i t h o u t s a l i c y l a t e , r e v e a l s t h a t u n d e r a e r o b i c eozlditio~ls b o t h g l u c o s e u p t a k e a n d lact, ic a c i d pcoduet, ion a r c s t h u u l a t ~ d ; 2,4 d i n i t r o p h e n o l i m i t a t e s t h i s e f f e c t . U n d e r a n a e r o b i c c o n d i t i o n s , b o t h t h e s e a c t i v i t i e s a r e inhibit.ed. T h e r e is n o a p p a r e n t e f f e c t o f s a l i c y h t t o o n t o ~ a l lacbie a c i d d e h y d r o g e n a s e a c t i v i t y or on ~he isoe-rlz.Vme p a t t e r n . I t is conelude, d ~b~b t h e b e n e ~ c i a l eft'cots o f s a l i e y l a t e s o n d i a b e t i c retinop~Lthy c a n n o t be e x p l a i n e d i~ t e r m s o f a l o w e r i n g o f l a c t i c a c i d c o n t e n t . r a t i o n , as h a s b e e n s u g g e s t e d . T h e s i g n i f i c a n c e o{" She i n c r e a s e d u p t a k e o f g l u c o s e is d i s c u s s e d .
I. Introduction
K e e n a n d Chlouverakis (1965) d r a w a t t e n t i o n to the possible connection between raised concentrations of lactate a n d v~scular elmnges in the retina. T h e y r e f e r to conflicting views regarding t~}e effects of lacti~te on small blood vessels a n d suggest ~ha~ there m a y be regional c{~rerences in the ~ aso-mo~or effects of lactate. As r e g a r d s bhe retina, those a u t h o r s quote the re.cent claim by I m r e (1964) to h a v e induced i n t r a v i t r e a l new vessel formation b y injection of lactic acid into the vitreous of the kitten. The reeenr finding from this l a b o r a t o r y ( G r k y ~ o r e , 1964) of a m a r k e d increase ia one of the isoenzymes of lactic acid dehydrogcnase during the vase-proliferative phase of e x p e r i m e n t a l ~etrolental fibroplasi~ is cited as corroborative evidence. K e e n a n d Chlouverakis speculate t h a t the clinical effects of salicylates on diabetic retinopa~hy migh~ be explicable in t e r m s of reducing the lactic acid c o n t e n t of the re~ina. On the basis of evidence f r o m other tissues ~hey suggest ~hat this redtmtion m i g h t be accomplished' b y lowering the blood glucose (~ilgore and 12upp, "1961), increaskng the r a t e of oxidation of p y r u v a t e by uncouplh~g oxidative phosphorylation (Smith, 1959}, and b y directly inhibiting lactic acid dehydrogenase (Baker, 1960, 1961, 1962). On the o~Imr har~d, Ashton a n d I:[enkind (personal c o m m u n i c a t i o n ) have not as y e t been able to confirm I m r e ' s observation, although only a small n u m b e r of p r e l i m i n a r y experiments h a v e s o f a r been a t t e m p t e d . Ii'urthermore, lactic acid levels h a v e been d e t e r m i n e d on rapidly frozen retinas of normal a n d aIloxan-diabetic r a t s a n d n.o Significant diffel"enees found (Gr;Lb'more and Towlsor., unpublished results). I n view of t h e circumstantial n a t u r e of much of the evidence relating to salicylates, lactic acid and vascular changes, it was t h o u g h t to be of value to investigate, directly, the effects of salicylate on certain p a r a m e t e r s of the. metabolism of the retina. 364
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2. M e t h o d s A ~lima Is T h e a n i m a l s u s e d in t h i s i~lvesbigation were a l b i n o r a t s a g e d 3 - 4 m o n t h s , of b o t h sexes a n d w e i g h i n g b e t w e e n 180 a n d 220 g. They were m a i n t a i n e d on a s t a n d a r d d i e t used in this l a b o r a t o r y ( d i e t ,t 1, Bru~cc a n d P a r k e s , 1946) a n d w e r e M l o w e d food a n d w a t e r a d lib.
1.njectious S~tlicylate w a s a d m i n i s t e r e d as t w o d o s e s , . s e p a r a t e d b y a t i m e i n t e r v a l o f I 5 hr, o f 450 m g o f s o d i u m s ~ i c y t a t e p e r kg of b o d y w e i g h t . T h e s a l i e y l a t c w a s m a d e u p in sMine at a eonecnt, rt~tion s u c h t h a t e a c h i n j e c t i o n was a p p r o x i m a t e l y 1 "0 m l ; t h e a n i m a l s w e r e killed 1 h r a f t e r t h e second i n j e c t i o n (see H u g g i n s a n d S m i t h , ]963). Preparation of. tissue Anim~tls were killed I)v c e r v i c a l d i s l o c a t i o n u n d e r l i g h t a n a e s t h e s i a a n d t h e r e t i n a s r e n m v e d wit~hitt 1-2 rain of d e a t h by c o n v e n t i o n a l t e c h n i q u e s . L i q u i d n i t r o g e n f r e e z i n g o f t h e w h o l e e y e w a s e m p l o y e d ii~ c e r t a i n of t h e d e t e r m i n a t i o n s of t o t a l lactic a c i d in o r d e r to m i n i m i z e post, m o r t c m c h a n g e s : Inc~dmt.ions All i n c u b a t i o n s were c a r r i e d o u t in f f r e b s - R i n g e r b i c a r b o n a t e buffer f o r t i f i e d w i t h 100 m g °,/o glucose. G r o u p s o f 4 r e t i n a s wcre i n c u b a t e d for 1 hr a t 37°C ia 3 . 0 ml of t h i s m e d i u m , u s i n g a c o n v e n t i o n a l W a r b u r g a p p a r a t u s . T h e gas p h a s e s w e r e tit.her 9 5 % N ~ 5o/ CO,, or o~,o~ .,o/o O2-5~/~ COo, a c c o r d i n g to choice. S a l i c y l a t e , w h e r e i n c l u d e d in t h e m e d i u m , w a s p r e s e n t in a final c o n c e n t r a t i o n of 5 × l 0 -a ~1 or 25 x 10 -a ~I as s p e c i f i e d in the t e x t a n d tables. C,lucose uptake ik p a i r of flasks was e m p l o y e d for e a c h d e t e r m i n a t i o n , o n e of w h i c h w a s s t o p p e d a f t e r e q u i l i b r i u m it1 o r d e r to p r o v i d e a n initiM r e a d i n g , t h e o t h e r o f w h i c h w a s a l l o w e d to i n c u b a t e for t.he p r e s c r i b e d 60 rain. 0-2 ml a l i q u o t s w e r e r e m o v e d f r o m t h e " i n i t i a l " a n d "fi~ml" flasks, a n d t h e i r g l u c o s e c o n t e n t d e t e r m i n e d b y t h e g l u c o s e o x i d a s e m e t h o d (see M a r k s , 1959): G l u c o s e u p t a k e was c a l c u l a t e d b y difference. Lactic acid production 1-0 ml a.liquots f r o m t h e s a m e s y s t e m as u s e d for m e a s u r i n g g l u c o s e u p t a k e ware a s s a y e d for htetie acid b y t h e m e t h o d of B a r k e r ~ n d S u m m e r s o n (1941), I n t h e s e d e t e r m i n e d , ions, initial s a m p l e s w e r e n o t t a k e n , as p r e l i m i n a r y e x p e r i m e n t s s h o w e d t h a t t h e initial r e a d i n g at e q u i l i b r i u m was negligible. Total activity of lactic avid dehydrogenase A f t e r i n c u b a t i o n , t h e f o u r r e t i n a s w e r e h o m o g e n i z e d in t h e i r o w n m e d i u m a n d c e n t r i f u g e d . T h e v o l u m e of t h e s u p e r n a t a n t w a s n o t e d , a n d a n 0-1 a l i q u o t w a s r e m o v e d a n d d i l u t e d t o 10 ml w i t h d e - i o n i z e d w a t e r . 0-g rnl of t h i s d i l u e n t w a s a s s a y e d b y t h e S i g m a p r o c e d u r e - ( s e e B e t t e r a n d B r o i d a , 1960). Lactic acid dehydroyenase isoenzyme patterns T h e s e were c a r r i e d o u t on t h e s u p e r n a t a n t as g e s c r i b e d p r e v i o u s l y ( G r a y m o r e ; 1964). Total lactic acid content W h e n t h e f r e e z i n g t e c h n i q u e w a s n o t e m p l o y e d , r e t i n a s were e x c i s e d u n d e r 1 0 % t r i c M o r o a c c t i c acid (TOA). A f t e r h o m o g e n i z a t i o n in 1 0 % T C A f o l l o w e d by cengrifugatioff, t h e s u p e r n a t a n t w a s a s s a y e d for l a c t i c acid by t h e m e t h o d of B a r k e r a n d S u m m e r s o n (1941).
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3. Results
Injection of sal@flate Table I shows the r a t e s of glucose u p t a k e and lactic acid production, under aerobic T a ~L~. I
F,ffect of the 'in vivo administration of 900 ~g of sodium salicylate Ter k 9 bo~q weight, a.s a diviAed dose, on the ghtcose uptake and lactic acid outTut of the isolated reti'~a incubated in a bicarbo~mte buffer under aerobic conditions Exporimont no. i 2
3
Animal
Glucose u p t a k e (/~g/hr per retina)
L a c t i c acid p r o d u c t i o n (t~g/hr per retina)
Control Experimental
268
2(58
20,t 207
Control Experimental
272 282
25S
Con trol /~xpe~imental
2,t 7 242
207 2.t2
296
]gaoh figure represents the average of 3 tlasks.
conditions, of retinas from animals t h a t had been pro-treated with sodium salicylatc. Glucose u p t a k e is not affected, although there is a tende~my for lactic acid production to be stimulated. The validity of this latter finding is endorsed by the determinations of total lactic acid in retinas f¢om normal and salicylate treated animals. When the results are expressed on the basis of an entire retina., two experiments showed t h a t the total content was increased by 59~/o and 50~/o, respectively, following t r e a t m e n t with salieylate. When expressed in terms of per mg protein these figures became 35~/o and 33~/o. The discrepancy in values according to the method of expression undoubtedly results from the difficulties inherent in the isolation, preservation and assay of the tissue, b u t the constancy and magnitude of the i n crease would suggest t h a t this increase is r e a l Duplicate estimations were carried out in these dhterminations.
In vitro experiments Table I I shows the effects of two differex~t concentrations of salicylate on the TAnuz I!
Effects of salicyIat~, in vitro, on the glucose uptake and lactic acid production of the retina i~zcubated ez'ther anaerobically or aerobically Salioylate 5 × I0 -a.~i Anaerobic Aerobic Giucov~ upt.ako /zg/hr p~r r~tina
Control ExImriment
L a c t i c acid
production t~g/hr p e r retina
Control E~x p e r m ~ c n t
S~licylato 25 × 10 -a~I Anaerobic Aerobic
410 _+ 44.6 (6) 374 + 40-4 (6)
(15) 287.4 _ 17.0 (15)
475.5 +- 9-2 (S) 272-4 +- 25.3 (9)
263-5 +- 24.1 (6) 293 _-4-33-6 (6)
401-1 ± 17-2 (6) 380-5 +- 25.6 (6)
190-2 ± 8-4 (12) 221-9 ± 13-S (12)
3 9 7 - 4 + 28-3 (8) 218.8 ± 28"3 (8)
213-0 ± 5-5 ({i) 285.0 ± 21-3 (6)
2 2 5 . 7 4- 15-0
Tho n u m b e r o f d e t e r m i n a t i o n s ar~-sbown in parenthesis.
SOl)lUll
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A~NI) H E T I N A L
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glucose u p t a k e and lactic acid production of retinas incubated either aerobically or amterobicully. Under.aerobic conditions both these parameters are increased, bhe most striking stimulation occurring in lactic acid p r o d u c t i o n in the presence of the higher concc~atration of salicylate (3'/~/o). This situt~tion is reversed under anaerobic conditions, b o t k ghtcosc.'4pt,ake and lactic acid p r o d u c t i o n being inhibited by salicylate. At~ the higher concentration, this amount, s to an inhibition of 43°/0 and ,15°/oi respectively. Table l l I shows She results of two series of experiments in which retintts 'I'ABL~ . I l l
Stimulatory effect o f 0.5 × 10 -J ~t 2,4 dinitro2)henol and/or 5 × 10 -~ M salicylate on the aerobic metabolism of the retina (?;?ucose Salicvlat¢~ 2,,1 1 ) i n i t . r o p h c n o l Salieylate -" 2,-1 1) i n i t r o p h c n o l
ul)take ( % )
l . a c t i e aci(i production (%)
63.4 125-3
,51 "2 ! 10.2
72-ti
74 "8
Rcsl~lts a r e e x p r e s s e d a s percentage s t i m u l a t i o n o f t h e n o r m a l a c t i v i t y a n d r e p r e s e n t t i m means Of two
runs.
were i~mubated aerobically in the presence of salieylate a n d / o r 2,4 d i n i t r o p h e n o l (DNP), and t.he metabolic activities or" these tissues compared wi~h t h a t of the norm~d retina incubated alone. The results of the determinatio~s are expressed as the pcrcent flge s t i m u l a t i o a of the normal control level. 2,4 D N P stimulates the a c t i v i t y of the retina to an even more marked degq'ec t h a n salicylate. The addition of ] ) N t ) to a system a l r e a d y containing salicylate induces a slight increase in the stimulator), effect of the kttter. Table IV shows t h e in vitro effect of salicylatc on the T,~ ~LS I V
E ~ c t of saIi.cylate in vitro on total lactic atilt dehydrogen.ase activity in rat retina '.Pre~, t,m e n t
}';× p c r i m c n t a l
Normal
25 )." l 0 - a 5.~ Sodium salieylate Anaerobic
:2110) 2 3 3 0 ~ 2283 2-t07)
2113~ 2218~ 231,1
5 ~< 10 - z .-,t Sodium salicylate Aerobic
22,q3"~ 235S~ 2333)
OO2l)
21}12)
2750)
R e s u l t s e x p r e s s e d a s :B.B. u n i t s ] r e t i n a .
total lactic acid dehydrogenase of the retina. The results are expressed as B.J3. units, each uni~ being equivalen~ to t h a t amounb of enzyme t h a t would cause a decreaze in optical d e n s i t y a t 340 m~t of 0-00I per rain i~x a reaction mixture of 3.0 ml (see Berger aIld Broida, 1960). There is no evidence of ally change. The isoenzyme p a t t e r n s did n o t a p p e a r to differ significantly from n o r m a l
36B
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N. G I l A Y , ' I I O I I F - " A N D
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I~I~.OWN
4. Discussion
The lower concentration of salicylate employed iu the in vitro investigations is recommended by Manchester, I{andle and Smith (1958) as being c o m m e n s u r a t e with the blood level of salicylat.es in di~betics treated with aspirin (Reid, Macdougall and Andrews, 1957). The higher concentration was adopted by this l a b o r a t o r y in order to s t u d y the more extreme effcct.s of acute dosage. I t is also of value in t h a t it fails into line wit}~ 0m blood concentration resulting from the injection experiments. This latter dosage is obviously excessive, the animals appearing drowsy for several hours following each injection, b u t eventual recovery is complete. E v e n a t this high conccnt, ration, however, there is no evidence of a n y degree of inhibition of the lactic acid dehydrogenase activity of the.retina (Table IV), or of' a n y interference with the isoenzyme p a t t e r n (see 1R.esults). Injection of salieylate stim ulates the ability of the tissue to produce lactic acid in vitro and result.s in an increase in the total lactic acid concentration of the retina in vivo (see Results and Table I). U n d e r aerobic conditions, the incubation of the retina with the high concentration of salicylate induces a marked st.imulation of lactic acid production. Although some of these observations are cssent.ially p r e l i m i n a r y and based on relati~'ely few det.erminations, in view of the constancy of the evidence obtained through a v a r i e t y of a p p r o a c h e s coupled with the total l~ck of contradictory evidence, there seems little d o u b t t h a t salicylates stimulate retinal lactic acid production. Such evidence is inconapatible with the concept t h a t a n y beneficial effects of salicylate on diabetic r e t i n o p a t h y might be mediated through a lowering of the local lactic ac.ir3 concentration, i~'ur~hcrmore, Table I I shows tlutt incubation of the isolated retina with salicylate u n d e r aerobic co~{ditions stimulates the glucose uptM
SOD/U.~[ NAI, ICYL2, TI~ A N D i~I,,'TINA[, ME'FABOI, IS.M
,llir.!
wa.s h m n b a t e d in t h e ln csence of s,'t]iey]at:e in a plmsl)ha~e buffer. Jl; would be of intm'es(, to repea~ t.hc presen~ e x p e r i m e n t s in a p h o s p h a t e buffer. N e v e r t h e l e s s , M a n c h e s t e r et al. ([958) e m p h a s i z e tim fact, that; a IJicarbonate buffered m e d i u m resembles m o r e closely t h e e x t r a c e l l u l a r fluid a n d t h a t it is c e r t a i n l y more s u i t a b l e for tissues e x h i b i t i n g a high r a t e of glycolysis. ] t should also be borne in mind that; all;hough t h e r e is e v i d e n c e for a n increased m e t a b o l i c a c t i v i t y in tim salieyhttc t,reatcd r e t i n a , t h e associated u n c o u p l i n g of o x i d a t i v e l ) h o s p h o r y l a t i o n will intcrtbre w i t h e n e r g y m e t a b o l i s m . I t is n o t easy, therefore, to p r e d i c t t h e u l t i m a t e effects on t h e tissue. T h e r e is no r e a d y e x p h t n a t i o n for the m a r k e d i n h i b i t o r y effect of s a l i e y l a t e on t.he m e t a b o l i s m , o f t h e r e t i n a i n c u b a t e d u n d e r m m e r o b i c c o n d i t i o n s . O b v i o u s l y such cond i t i o n s would n u l l i f y t h e effects of s a l i e y l a t c on oxidal;ive p h o s p h o r y l a t i o n , but, this does n o t explain tim r e d u c t i o n in r a t e of glucose u p t a k e a n d lactic acid pro~luction. C e r t a i n l y , t h e inhibit, ion o n l y becomes significant a t t h e higher content,rat, ion, a n d one m a y a ssmne t,h a t some n o n - p h y s i o l o g i c a l p e r m a n e n t d a m a g e results a n d t.lu~t u n d e r a e r o h i c e o n d i t i n n s some dem'ee of p r o t e c t i o n is atTorded. Thi,~ s t r i k i n g result, a n d its i m p l i c a t i o n s , will be e x a m i n e d f u r b h e r . Air,h o u g h the results r e p o r t e d here east, d o u b t o,~ the suggestion t h a t s a l i c y l a t e s m i g h t aid d i a b e t i c r e t i n o p a t h y by lowering t h e localized c o n c e n t r a t i o n of htctic acid in t,he retina, it. is of i n t e r e s t t h a t s a l i c y l a t c s acee}eratc t h e e n t r y of glucose into t h e retina: bhis tissue is t h o u g h t to bc inscnsit;ive in t h i s respect~ to insulin. ACKNO W LED(]MENTS The authors exprcss their gratitude to Profi~'ssor Norman Ashton for his continued interest, and support. They also wish to tJmnk Mr. R a l p h Kissun for his excellent t.echnic,al assistance, attd Lhe Royal Nat, ionM lnst:itute for the Blind for the tinancial assistance that, made this project, possible. R-E F E }~.E N C I~S ]}~ker, B. 1~. (l~3B0). J. mednl "pharm. Chem. 2, 633. ]3~ker, B. ]~. (1901). d. Am. chem. See. 83, :}714. Z~aker, B. P~. [1962). J. mednl 2~harm. Chcm. S, 654. ]~arker, S. ]3. ~md Summerson, W. 1t. (1941). ,1. biol. C]~em. 138, 535. .Berger, J,. and ]3roida, ]). (1960). Sigm~ ~'eehnical ]3ullebin. No. 500. l}ruce, II. ~1. and Parkes, A. S. (1946). J. Hyg., Cam& ,$4, 501. Gilgore, S. G. and Rupp, J. J. (19131). ~Ielaboli~m 10, 419. Gr~tymore, C. aN. (1964). Nature, Lend. 201, 015. Iquggins, A. K. ~tnd Smith,. hi. J. I~/. (1963). !liochem. J. 89, 112P. Imre, G. (1964). ]Jr. J. Ophthal. 48, 75. Keen, I[. and Chlouverakis, C. (1965). in lgiochemistry of the Retina, ed. by C. N. Gr~*ymore, p. 12,3. Aeademic Press, London and New York. Lynen, ]"., I-Iartmann, G., Nett~r, K. F. ~t~d Scb.Llc~gr~t[: A. (1959). In Begu[z~llon of Cell Metabolis~n, Ciba Symposium, p. 256. Ed. by Wo[stenholmo and O'Connor. Churchill, London. Manchester, K. L., Randle, P. J. and Smitl~, G. ]-I. (1958). Br. ,ned. J. 1, 1028. l~larks, V. (1959}. Clinics chim. Acta 4, 395. Reid, J., MacdougM1, A. [. ~tnd Andrews, M. ~I. (1957). Br. ~ned. J. 2, 1071. Smith, ~1. J. H. (1959). J. 19har~nacol. II, 705. Smith, M. J. tI. and Jeffrey, S. W. (19513). Biochem. J. 64, 589.
T h e E f f e c t s o f S o d i u m S a l i c y l a t e o n the N I e t a b o l i s m o f the R e t i n a C~VE ~N. G ~ Y ~ o ~
AN~ :H~.'ATH~R BI~OWN
Depa.rt~nent of ~athology, Institute of 01~hthalmolocjy, University of London, J.udd Street, London., E'~ujla~ul (Received 30 A'ugust, J965) C e r t a i n a s p e c t s o f t h e ett'ecbs o f ~ a l i c y l a t ~ o n r e t i n a l m e t a b o l i s m a r c c o n s i d e r e d . I n ' v i v o a d m i n i s t r a t i o n o f s o d i u m s a l i c y l a t e i n d u c e s an i n c r e a s e in t h e a b s o l u t e level o f la~:tie a c i d in t h e r e t i n a . S u c h t r e a t m e n t is also s h o w n t o r e s u l t in a s l i g h t s t i m u l a t o r y e f f e c t o n l~ctic a c i d p r o d u c t i o u b y t h e s u b s e q u e n t l y e x c i s e d r e t i n a , bug t o h a v e n o s i g n i f i c ; r n t e f f e c t o n t h e r a t e o f g l u c o s e u p t a k e . Ineub~Ltion o f t h e n o r m a l r e t i n a , w i t h o r w i t h o u t s a l i c y l a t e , r e v e a l s t h a t u n d e r a e r o b i c eozlditio~ls b o t h g l u c o s e u p t a k e a n d lact, ic a c i d pcoduet, ion a r c s t h u u l a t ~ d ; 2,4 d i n i t r o p h e n o l i m i t a t e s t h i s e f f e c t . U n d e r a n a e r o b i c c o n d i t i o n s , b o t h t h e s e a c t i v i t i e s a r e inhibit.ed. T h e r e is n o a p p a r e n t e f f e c t o f s a l i c y h t t o o n t o ~ a l lacbie a c i d d e h y d r o g e n a s e a c t i v i t y or on ~he isoe-rlz.Vme p a t t e r n . I t is conelude, d ~b~b t h e b e n e ~ c i a l eft'cots o f s a l i e y l a t e s o n d i a b e t i c retinop~Lthy c a n n o t be e x p l a i n e d i~ t e r m s o f a l o w e r i n g o f l a c t i c a c i d c o n t e n t . r a t i o n , as h a s b e e n s u g g e s t e d . T h e s i g n i f i c a n c e o{" She i n c r e a s e d u p t a k e o f g l u c o s e is d i s c u s s e d .
I. Introduction
K e e n a n d Chlouverakis (1965) d r a w a t t e n t i o n to the possible connection between raised concentrations of lactate a n d v~scular elmnges in the retina. T h e y r e f e r to conflicting views regarding t~}e effects of lacti~te on small blood vessels a n d suggest ~ha~ there m a y be regional c{~rerences in the ~ aso-mo~or effects of lactate. As r e g a r d s bhe retina, those a u t h o r s quote the re.cent claim by I m r e (1964) to h a v e induced i n t r a v i t r e a l new vessel formation b y injection of lactic acid into the vitreous of the kitten. The reeenr finding from this l a b o r a t o r y ( G r k y ~ o r e , 1964) of a m a r k e d increase ia one of the isoenzymes of lactic acid dehydrogcnase during the vase-proliferative phase of e x p e r i m e n t a l ~etrolental fibroplasi~ is cited as corroborative evidence. K e e n a n d Chlouverakis speculate t h a t the clinical effects of salicylates on diabetic retinopa~hy migh~ be explicable in t e r m s of reducing the lactic acid c o n t e n t of the re~ina. On the basis of evidence f r o m other tissues ~hey suggest ~hat this redtmtion m i g h t be accomplished' b y lowering the blood glucose (~ilgore and 12upp, "1961), increaskng the r a t e of oxidation of p y r u v a t e by uncouplh~g oxidative phosphorylation (Smith, 1959}, and b y directly inhibiting lactic acid dehydrogenase (Baker, 1960, 1961, 1962). On the o~Imr har~d, Ashton a n d I:[enkind (personal c o m m u n i c a t i o n ) have not as y e t been able to confirm I m r e ' s observation, although only a small n u m b e r of p r e l i m i n a r y experiments h a v e s o f a r been a t t e m p t e d . Ii'urthermore, lactic acid levels h a v e been d e t e r m i n e d on rapidly frozen retinas of normal a n d aIloxan-diabetic r a t s a n d n.o Significant diffel"enees found (Gr;Lb'more and Towlsor., unpublished results). I n view of t h e circumstantial n a t u r e of much of the evidence relating to salicylates, lactic acid and vascular changes, it was t h o u g h t to be of value to investigate, directly, the effects of salicylate on certain p a r a m e t e r s of the. metabolism of the retina. 364
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2. M e t h o d s A ~lima Is T h e a n i m a l s u s e d in t h i s i~lvesbigation were a l b i n o r a t s a g e d 3 - 4 m o n t h s , of b o t h sexes a n d w e i g h i n g b e t w e e n 180 a n d 220 g. They were m a i n t a i n e d on a s t a n d a r d d i e t used in this l a b o r a t o r y ( d i e t ,t 1, Bru~cc a n d P a r k e s , 1946) a n d w e r e M l o w e d food a n d w a t e r a d lib.
1.njectious S~tlicylate w a s a d m i n i s t e r e d as t w o d o s e s , . s e p a r a t e d b y a t i m e i n t e r v a l o f I 5 hr, o f 450 m g o f s o d i u m s ~ i c y t a t e p e r kg of b o d y w e i g h t . T h e s a l i e y l a t c w a s m a d e u p in sMine at a eonecnt, rt~tion s u c h t h a t e a c h i n j e c t i o n was a p p r o x i m a t e l y 1 "0 m l ; t h e a n i m a l s w e r e killed 1 h r a f t e r t h e second i n j e c t i o n (see H u g g i n s a n d S m i t h , ]963). Preparation of. tissue Anim~tls were killed I)v c e r v i c a l d i s l o c a t i o n u n d e r l i g h t a n a e s t h e s i a a n d t h e r e t i n a s r e n m v e d wit~hitt 1-2 rain of d e a t h by c o n v e n t i o n a l t e c h n i q u e s . L i q u i d n i t r o g e n f r e e z i n g o f t h e w h o l e e y e w a s e m p l o y e d ii~ c e r t a i n of t h e d e t e r m i n a t i o n s of t o t a l lactic a c i d in o r d e r to m i n i m i z e post, m o r t c m c h a n g e s : Inc~dmt.ions All i n c u b a t i o n s were c a r r i e d o u t in f f r e b s - R i n g e r b i c a r b o n a t e buffer f o r t i f i e d w i t h 100 m g °,/o glucose. G r o u p s o f 4 r e t i n a s wcre i n c u b a t e d for 1 hr a t 37°C ia 3 . 0 ml of t h i s m e d i u m , u s i n g a c o n v e n t i o n a l W a r b u r g a p p a r a t u s . T h e gas p h a s e s w e r e tit.her 9 5 % N ~ 5o/ CO,, or o~,o~ .,o/o O2-5~/~ COo, a c c o r d i n g to choice. S a l i c y l a t e , w h e r e i n c l u d e d in t h e m e d i u m , w a s p r e s e n t in a final c o n c e n t r a t i o n of 5 × l 0 -a ~1 or 25 x 10 -a ~I as s p e c i f i e d in the t e x t a n d tables. C,lucose uptake ik p a i r of flasks was e m p l o y e d for e a c h d e t e r m i n a t i o n , o n e of w h i c h w a s s t o p p e d a f t e r e q u i l i b r i u m it1 o r d e r to p r o v i d e a n initiM r e a d i n g , t h e o t h e r o f w h i c h w a s a l l o w e d to i n c u b a t e for t.he p r e s c r i b e d 60 rain. 0-2 ml a l i q u o t s w e r e r e m o v e d f r o m t h e " i n i t i a l " a n d "fi~ml" flasks, a n d t h e i r g l u c o s e c o n t e n t d e t e r m i n e d b y t h e g l u c o s e o x i d a s e m e t h o d (see M a r k s , 1959): G l u c o s e u p t a k e was c a l c u l a t e d b y difference. Lactic acid production 1-0 ml a.liquots f r o m t h e s a m e s y s t e m as u s e d for m e a s u r i n g g l u c o s e u p t a k e ware a s s a y e d for htetie acid b y t h e m e t h o d of B a r k e r ~ n d S u m m e r s o n (1941), I n t h e s e d e t e r m i n e d , ions, initial s a m p l e s w e r e n o t t a k e n , as p r e l i m i n a r y e x p e r i m e n t s s h o w e d t h a t t h e initial r e a d i n g at e q u i l i b r i u m was negligible. Total activity of lactic avid dehydrogenase A f t e r i n c u b a t i o n , t h e f o u r r e t i n a s w e r e h o m o g e n i z e d in t h e i r o w n m e d i u m a n d c e n t r i f u g e d . T h e v o l u m e of t h e s u p e r n a t a n t w a s n o t e d , a n d a n 0-1 a l i q u o t w a s r e m o v e d a n d d i l u t e d t o 10 ml w i t h d e - i o n i z e d w a t e r . 0-g rnl of t h i s d i l u e n t w a s a s s a y e d b y t h e S i g m a p r o c e d u r e - ( s e e B e t t e r a n d B r o i d a , 1960). Lactic acid dehydroyenase isoenzyme patterns T h e s e were c a r r i e d o u t on t h e s u p e r n a t a n t as g e s c r i b e d p r e v i o u s l y ( G r a y m o r e ; 1964). Total lactic acid content W h e n t h e f r e e z i n g t e c h n i q u e w a s n o t e m p l o y e d , r e t i n a s were e x c i s e d u n d e r 1 0 % t r i c M o r o a c c t i c acid (TOA). A f t e r h o m o g e n i z a t i o n in 1 0 % T C A f o l l o w e d by cengrifugatioff, t h e s u p e r n a t a n t w a s a s s a y e d for l a c t i c acid by t h e m e t h o d of B a r k e r a n d S u m m e r s o n (1941).
36#5
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3. Results
Injection of sal@flate Table I shows the r a t e s of glucose u p t a k e and lactic acid production, under aerobic T a ~L~. I
F,ffect of the 'in vivo administration of 900 ~g of sodium salicylate Ter k 9 bo~q weight, a.s a diviAed dose, on the ghtcose uptake and lactic acid outTut of the isolated reti'~a incubated in a bicarbo~mte buffer under aerobic conditions Exporimont no. i 2
3
Animal
Glucose u p t a k e (/~g/hr per retina)
L a c t i c acid p r o d u c t i o n (t~g/hr per retina)
Control Experimental
268
2(58
20,t 207
Control Experimental
272 282
25S
Con trol /~xpe~imental
2,t 7 242
207 2.t2
296
]gaoh figure represents the average of 3 tlasks.
conditions, of retinas from animals t h a t had been pro-treated with sodium salicylatc. Glucose u p t a k e is not affected, although there is a tende~my for lactic acid production to be stimulated. The validity of this latter finding is endorsed by the determinations of total lactic acid in retinas f¢om normal and salicylate treated animals. When the results are expressed on the basis of an entire retina., two experiments showed t h a t the total content was increased by 59~/o and 50~/o, respectively, following t r e a t m e n t with salieylate. When expressed in terms of per mg protein these figures became 35~/o and 33~/o. The discrepancy in values according to the method of expression undoubtedly results from the difficulties inherent in the isolation, preservation and assay of the tissue, b u t the constancy and magnitude of the i n crease would suggest t h a t this increase is r e a l Duplicate estimations were carried out in these dhterminations.
In vitro experiments Table I I shows the effects of two differex~t concentrations of salicylate on the TAnuz I!
Effects of salicyIat~, in vitro, on the glucose uptake and lactic acid production of the retina i~zcubated ez'ther anaerobically or aerobically Salioylate 5 × I0 -a.~i Anaerobic Aerobic Giucov~ upt.ako /zg/hr p~r r~tina
Control ExImriment
L a c t i c acid
production t~g/hr p e r retina
Control E~x p e r m ~ c n t
S~licylato 25 × 10 -a~I Anaerobic Aerobic
410 _+ 44.6 (6) 374 + 40-4 (6)
(15) 287.4 _ 17.0 (15)
475.5 +- 9-2 (S) 272-4 +- 25.3 (9)
263-5 +- 24.1 (6) 293 _-4-33-6 (6)
401-1 ± 17-2 (6) 380-5 +- 25.6 (6)
190-2 ± 8-4 (12) 221-9 ± 13-S (12)
3 9 7 - 4 + 28-3 (8) 218.8 ± 28"3 (8)
213-0 ± 5-5 ({i) 285.0 ± 21-3 (6)
2 2 5 . 7 4- 15-0
Tho n u m b e r o f d e t e r m i n a t i o n s ar~-sbown in parenthesis.
SOl)lUll
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A~NI) H E T I N A L
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glucose u p t a k e and lactic acid production of retinas incubated either aerobically or amterobicully. Under.aerobic conditions both these parameters are increased, bhe most striking stimulation occurring in lactic acid p r o d u c t i o n in the presence of the higher concc~atration of salicylate (3'/~/o). This situt~tion is reversed under anaerobic conditions, b o t k ghtcosc.'4pt,ake and lactic acid p r o d u c t i o n being inhibited by salicylate. At~ the higher concentration, this amount, s to an inhibition of 43°/0 and ,15°/oi respectively. Table l l I shows She results of two series of experiments in which retintts 'I'ABL~ . I l l
Stimulatory effect o f 0.5 × 10 -J ~t 2,4 dinitro2)henol and/or 5 × 10 -~ M salicylate on the aerobic metabolism of the retina (?;?ucose Salicvlat¢~ 2,,1 1 ) i n i t . r o p h c n o l Salieylate -" 2,-1 1) i n i t r o p h c n o l
ul)take ( % )
l . a c t i e aci(i production (%)
63.4 125-3
,51 "2 ! 10.2
72-ti
74 "8
Rcsl~lts a r e e x p r e s s e d a s percentage s t i m u l a t i o n o f t h e n o r m a l a c t i v i t y a n d r e p r e s e n t t i m means Of two
runs.
were i~mubated aerobically in the presence of salieylate a n d / o r 2,4 d i n i t r o p h e n o l (DNP), and t.he metabolic activities or" these tissues compared wi~h t h a t of the norm~d retina incubated alone. The results of the determinatio~s are expressed as the pcrcent flge s t i m u l a t i o a of the normal control level. 2,4 D N P stimulates the a c t i v i t y of the retina to an even more marked degq'ec t h a n salicylate. The addition of ] ) N t ) to a system a l r e a d y containing salicylate induces a slight increase in the stimulator), effect of the kttter. Table IV shows t h e in vitro effect of salicylatc on the T,~ ~LS I V
E ~ c t of saIi.cylate in vitro on total lactic atilt dehydrogen.ase activity in rat retina '.Pre~, t,m e n t
}';× p c r i m c n t a l
Normal
25 )." l 0 - a 5.~ Sodium salieylate Anaerobic
:2110) 2 3 3 0 ~ 2283 2-t07)
2113~ 2218~ 231,1
5 ~< 10 - z .-,t Sodium salicylate Aerobic
22,q3"~ 235S~ 2333)
OO2l)
21}12)
2750)
R e s u l t s e x p r e s s e d a s :B.B. u n i t s ] r e t i n a .
total lactic acid dehydrogenase of the retina. The results are expressed as B.J3. units, each uni~ being equivalen~ to t h a t amounb of enzyme t h a t would cause a decreaze in optical d e n s i t y a t 340 m~t of 0-00I per rain i~x a reaction mixture of 3.0 ml (see Berger aIld Broida, 1960). There is no evidence of ally change. The isoenzyme p a t t e r n s did n o t a p p e a r to differ significantly from n o r m a l
36B
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N. G I l A Y , ' I I O I I F - " A N D
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4. Discussion
The lower concentration of salicylate employed iu the in vitro investigations is recommended by Manchester, I{andle and Smith (1958) as being c o m m e n s u r a t e with the blood level of salicylat.es in di~betics treated with aspirin (Reid, Macdougall and Andrews, 1957). The higher concentration was adopted by this l a b o r a t o r y in order to s t u d y the more extreme effcct.s of acute dosage. I t is also of value in t h a t it fails into line wit}~ 0m blood concentration resulting from the injection experiments. This latter dosage is obviously excessive, the animals appearing drowsy for several hours following each injection, b u t eventual recovery is complete. E v e n a t this high conccnt, ration, however, there is no evidence of a n y degree of inhibition of the lactic acid dehydrogenase activity of the.retina (Table IV), or of' a n y interference with the isoenzyme p a t t e r n (see 1R.esults). Injection of salieylate stim ulates the ability of the tissue to produce lactic acid in vitro and result.s in an increase in the total lactic acid concentration of the retina in vivo (see Results and Table I). U n d e r aerobic conditions, the incubation of the retina with the high concentration of salicylate induces a marked st.imulation of lactic acid production. Although some of these observations are cssent.ially p r e l i m i n a r y and based on relati~'ely few det.erminations, in view of the constancy of the evidence obtained through a v a r i e t y of a p p r o a c h e s coupled with the total l~ck of contradictory evidence, there seems little d o u b t t h a t salicylates stimulate retinal lactic acid production. Such evidence is inconapatible with the concept t h a t a n y beneficial effects of salicylate on diabetic r e t i n o p a t h y might be mediated through a lowering of the local lactic ac.ir3 concentration, i~'ur~hcrmore, Table I I shows tlutt incubation of the isolated retina with salicylate u n d e r aerobic co~{ditions stimulates the glucose uptM
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wa.s h m n b a t e d in t h e ln csence of s,'t]iey]at:e in a plmsl)ha~e buffer. Jl; would be of intm'es(, to repea~ t.hc presen~ e x p e r i m e n t s in a p h o s p h a t e buffer. N e v e r t h e l e s s , M a n c h e s t e r et al. ([958) e m p h a s i z e tim fact, that; a IJicarbonate buffered m e d i u m resembles m o r e closely t h e e x t r a c e l l u l a r fluid a n d t h a t it is c e r t a i n l y more s u i t a b l e for tissues e x h i b i t i n g a high r a t e of glycolysis. ] t should also be borne in mind that; all;hough t h e r e is e v i d e n c e for a n increased m e t a b o l i c a c t i v i t y in tim salieyhttc t,reatcd r e t i n a , t h e associated u n c o u p l i n g of o x i d a t i v e l ) h o s p h o r y l a t i o n will intcrtbre w i t h e n e r g y m e t a b o l i s m . I t is n o t easy, therefore, to p r e d i c t t h e u l t i m a t e effects on t h e tissue. T h e r e is no r e a d y e x p h t n a t i o n for the m a r k e d i n h i b i t o r y effect of s a l i e y l a t e on t.he m e t a b o l i s m , o f t h e r e t i n a i n c u b a t e d u n d e r m m e r o b i c c o n d i t i o n s . O b v i o u s l y such cond i t i o n s would n u l l i f y t h e effects of s a l i e y l a t c on oxidal;ive p h o s p h o r y l a t i o n , but, this does n o t explain tim r e d u c t i o n in r a t e of glucose u p t a k e a n d lactic acid pro~luction. C e r t a i n l y , t h e inhibit, ion o n l y becomes significant a t t h e higher content,rat, ion, a n d one m a y a ssmne t,h a t some n o n - p h y s i o l o g i c a l p e r m a n e n t d a m a g e results a n d t.lu~t u n d e r a e r o h i c e o n d i t i n n s some dem'ee of p r o t e c t i o n is atTorded. Thi,~ s t r i k i n g result, a n d its i m p l i c a t i o n s , will be e x a m i n e d f u r b h e r . Air,h o u g h the results r e p o r t e d here east, d o u b t o,~ the suggestion t h a t s a l i c y l a t e s m i g h t aid d i a b e t i c r e t i n o p a t h y by lowering t h e localized c o n c e n t r a t i o n of htctic acid in t,he retina, it. is of i n t e r e s t t h a t s a l i c y l a t c s acee}eratc t h e e n t r y of glucose into t h e retina: bhis tissue is t h o u g h t to bc inscnsit;ive in t h i s respect~ to insulin. ACKNO W LED(]MENTS The authors exprcss their gratitude to Profi~'ssor Norman Ashton for his continued interest, and support. They also wish to tJmnk Mr. R a l p h Kissun for his excellent t.echnic,al assistance, attd Lhe Royal Nat, ionM lnst:itute for the Blind for the tinancial assistance that, made this project, possible. R-E F E }~.E N C I~S ]}~ker, B. 1~. (l~3B0). J. mednl "pharm. Chem. 2, 633. ]3~ker, B. ]~. (1901). d. Am. chem. See. 83, :}714. Z~aker, B. P~. [1962). J. mednl 2~harm. Chcm. S, 654. ]~arker, S. ]3. ~md Summerson, W. 1t. (1941). ,1. biol. C]~em. 138, 535. .Berger, J,. and ]3roida, ]). (1960). Sigm~ ~'eehnical ]3ullebin. No. 500. l}ruce, II. ~1. and Parkes, A. S. (1946). J. Hyg., Cam& ,$4, 501. Gilgore, S. G. and Rupp, J. J. (19131). ~Ielaboli~m 10, 419. Gr~tymore, C. aN. (1964). Nature, Lend. 201, 015. Iquggins, A. K. ~tnd Smith,. hi. J. I~/. (1963). !liochem. J. 89, 112P. Imre, G. (1964). ]Jr. J. Ophthal. 48, 75. Keen, I[. and Chlouverakis, C. (1965). in lgiochemistry of the Retina, ed. by C. N. Gr~*ymore, p. 12,3. Aeademic Press, London and New York. Lynen, ]"., I-Iartmann, G., Nett~r, K. F. ~t~d Scb.Llc~gr~t[: A. (1959). In Begu[z~llon of Cell Metabolis~n, Ciba Symposium, p. 256. Ed. by Wo[stenholmo and O'Connor. Churchill, London. Manchester, K. L., Randle, P. J. and Smitl~, G. ]-I. (1958). Br. ,ned. J. 1, 1028. l~larks, V. (1959}. Clinics chim. Acta 4, 395. Reid, J., MacdougM1, A. [. ~tnd Andrews, M. ~I. (1957). Br. ~ned. J. 2, 1071. Smith, ~1. J. H. (1959). J. 19har~nacol. II, 705. Smith, M. J. tI. and Jeffrey, S. W. (19513). Biochem. J. 64, 589.