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The effect of analogues of thyroxine and 2,4-dinitrophenol on the swelling of mitochondria
VOL. 36 (1959)
BIOCHIMICA ET BIOPHYSICA ACTA
499
T H E E F F E C T OF ANALOGUES OF T H Y R O X I N E AND 2,4-DINITROPHENOL ON T H E S W E L L I N G OF MITOCHONDRIA W. V. S H A W , T. J. L A N N O N AND D. F. T A P L E Y
Department o/Medicine, Columbia University College o] Physicians and Surgeons, and the Presbyterian Hospital, New York, N.Y. (U.S.A.) (Received M a r c h i i t h , 1959)
SUMMARY
Studies with analogues of thyroxine suggest that the ability of thyroxine to cause mitochondrial swelling is dependent upon the presence of the iodo-substituted diphenyl ether, but independent of the phenolic hydroxyl or the composition of the side chain. Three known "peripheral antagonists" of thyroxine have been shown to antagonize thyroxine-induced swelling. The ability of 2,4-dinitrophenol to prevent mitochondrial swelling appears to depend upon the free hydroxyl and the presence of appropriate electro-negative substituents in the ortho- and para-positions.
INTRODUCTION
Several lines of investigation have suggested that thyroxine and some of its analogues alter the morphology of mitochondria. Mitochondria in the livers of thyrotoxic rats have been shown to be swollen and distorted1, 2, and when isolated and suspended in sucrose these mitochondria swell to a greater extent than do those from the livers of normal animals1, a. It has also been demonstrated that the addition in vitro of thyroxine and certain of its analogues to suspensions of mitochondria from normal animals produces pronounced swelling8-5. Although both thyroxine and 2,4-dinitrophenol (DNP) "uncouple" oxidative phosphorylation in intact mitochondria, it seems unlikely that their mechanism of action is the same3, 5. Neither thyroxine nor its analogues "uncouple" the oxidative phosphorylation which occurs in sub-mitochondrial particles obtained by digitonin treatment or ultrasonic disruption, while DNP is effective under such conditionsS, 7. Furthermore, while thyroxine promotes the swelling of rat-liver mitochondria, 2,4-dinitrophenol provides protection against swelling when used in concentrations which "uncouple" oxidative phosphorylation3. The effect of analogues of thyroxine and of DNP on mitochondrial swelling has been studied in an attempt to determine the structural features of the parent compounds essential to their activity. The results are compared with other known in vivo and in vitro actions of these compounds. In addition, the effects of certain known "peripheral antagonists" of thyroxine on the thyroxine-induced swelling are described. Re]erences p. 504.
500
W . V . SHAW, T. j. LANNON, D. F. TAPLEY
VOL. 3 6
(1959)
MATERIALS AND METHODS
Mitochondria were prepared from the livers of Wistar strain male rats as described elsewherea. o.ooi M Ethylenediaminetetraacetate (EDTA) was present in the sucrose solution during the initial homogenization and in the first of 3 washes. The final pellet was suspended in 0.44 M sucrose so that mitochondria from I g of fresh liver were suspended in I.O ml. The final stock suspension was packed in ice and used within IO min. Swelling was measured spectrophotometrically at room temp. (approx. 23 °) as described elsewhere 3. That this is an accurate indication of volume changes has recently been verified by TEDESCHI AND HARRISs. The final test system consisted of 4.0 ml of 0.3 M sucrose containing o.02 M tris-hydroxymethyl-aminomethane(tris) buffer of pH 7.4. Sufficient mitochondria (usually 0.04-0.05 ml of the stock suspension) were added to give an initial O.D. of approx. 0.5. All test compounds were dissolved in o.ooi N KOH immediately before use and incorporated in the final test system at a concn, of 2" lO -5 M. RESULTS
Standardization o[ the method Preliminary studies resulted in findings similar to those reported in detail elsewhere3. The control rate of swelling was such that either enhancement or inhibition of swelling could be readily detected. Fig. I shows a typical experiment with thyroxine and DNP. As can be seen, DNP provided protection against endogenous swelling as well as against that produced by thyroxine. Ability to promote swelling under the standard test conditions has been graded as: no effect (o); less swelling than that produced with thyroxine ( + ) ; and an enhancement of swelling equal in magnitude to that produced by an equimolar amount of thyroxine ( + +). The abilty of compounds to protect against swelling has likewise been graded as: no effect (o) ; less protection against endogenous and thyroxine-induced swelling than that provided by DNP (+) ; and protection against swelling equal to that produced by DNP ( + +). 0.5
+ THYROXINE
0.4
o ROL 0. 3
XINE
o
TIME
20
1o
IN M I N U T E S
Fig. z. Effect of L-thyroxine and 2,4-dinitrophenol on swelling of m i t o c h o n d r i a suspended in o. 3 M sucrose and o.o2 M tris, p H 7-4. Conch. of b o t h c o m p o u n d s 2. IO -~ M.
Analogues o/thyroxine Since the absolute amount of swelling induced by thyroxine appeared to vary slightly with different preparations of mitochondria, a control containing thyroxine Re#rences p. 5o 4.
VOL. 36 (I959)
SWELLING OF MITOCHONDRIA
50I
was used w i t h each e x p e r i m e n t . E v e r y analogue was t e s t e d alone a n d in the presence of t h y r o x i n e . E a r l i e r studies 3 h a d shown D-thyroxine a n d L-thyroxine to be e q u a l l y effective in causing swelling. Hence, all c o m p o u n d s w i t h t h e DL-configuration were t e s t e d a t the same c o n c e n t r a t i o n as the control L-thyroxine. I n no instance was the effect of t h y r o x i n e plus an analogue g r e a t e r t h a n t h a t of t h y r o x i n e alone. A l t h o u g h a n u m b e r of analogues w i t h a d i p h e n y l ether configuration failed to produce a n y swelling, none p r o v i d e d p r o t e c t i o n against e i t h e r endogenous or t h y r o x i n e - i n d u c e d swelling. T h e results o b t a i n e d w i t h the analogues of t h y r o x i n e are recorded in Table I a n d w a r r a n t the following observations. The presence of 2 iodine a t o m s in a single ring of an analogue possessing an alanine side-chain (e.g., 3,5-diiodothyronine) d i d n o t lead to an a b i l i t y to cause swelling. However, when I iodine a t o m was s u b s t i t u t e d in each of t h e 2 rings (e.g., 3,3'-diiodothyronine) the resulting c o m p o u n d d i d cause swelling. I n contrast, in those c o m p o u n d s w i t h propionic or acetic acid side-chains, the s u b s t i t u t i o n of 2 iodine a t o m s in a single ring (e.g., 3,5-diiodothyropropionic acid) was sufficient to p r o d u c e swelling. I t should be n o t e d t h a t i o d o - s u b s t i t u t i o n s were more effective t h a n bromo-, chloro-, or m e t h y l substitutions. A l t e r a t i o n s in the sidechain p e r se d i d not significantly alter a c t i v i t y ; thus, t e t r a i o d o - s u b s t i t u t e d c o m p o u n d s w i t h alanine, propionic, acetic a n d formic acid side chains were all e q u a l l y effective. Similarly, m e t h y l a t i o n of the phenolic h y d r o x y l d i d not alter a c t i v i t y (3,5,3'-triiodoTABLE I EFFECT
OF A N A L O G U E S OF T H Y R O X I N E ON MITOCI-IONDRIAL S W E L L I N G
Results are tabulated as : no effect (o) ; less swelling than that produced by thyroxine (+) ; swelling equal to that produced by thyroxine ( + W). Final concn, of all compounds was 2.IO -~ M. Diphenyl ether substitutions 3' 3 4" 5'
2. 3. 4. 5.
HOHOHOCH30HO-
6. 7. 8. 9. IO. I I. 12. 13. 14. 15. 16.
HOHOCH30HOHOHOHOHOHOCH30CH30-
I.
Side chain VL--
--t~.~b~l~tJt~
-CH~COOH
o o o
+
+ +
-COOH
-.-COOCH,
5
None 3-monoiodo3,5-diiodo3,5-diiodo3,5-diiodo-3',5'dimethyl3,3'-diiodo3,5,3'-triiodo3,5,3'-triiodo3,3',5'-triiodo3-iodo-3',5 '-dibromo3,5,3',5'-tetraiodo3,5,3',5'-tetrabromo3,5,3',5'-tetrachloro3-amino3,5,3'-triamino3,5-dinitro-
+ + + + + + + + + + +* +
+ + + + + +
+ + + +
+ + + +
+ +
+ +
++
o
* Provided through the courtesy of Dr. B. A. HEMS of Glaxo Laboratories, Ltd. All other compounds (except thyronine, 3-monoiodothyronine and 3,5-diiodothyronine) were provided through the courtesy of Drs. R. L. KRoc and A. W. RUDDY Of the Warner-Lambert Research Institute. Re]erences p. 504.
502
W.V.
S H A W , T. J . L A N N O N , D. F. T A P L E Y
VOL. 3 6
(1959)
t h y r o n i n e a n d 4 ' - m e t h o x y - 3 , 5 , 3 ' - t r i i o d o t h y r o n i n e w e r e e q u a l l y a c t i v e ) . A n u m b e r of c o m p o u n d s w i t h o u t t h e d i p h e n y l e t h e r s t r u c t u r e (tyrosine, p - h y d r o x y p h e n y l p r o pionic acid, 3 , 5 - d i i o d o t y r o s i n e a n d 3 , 5 - d i n i t r o t y r o s i n e ) w e r e w i t h o u t effect at a concn, of 2. lO -5 M . T h r e e " p e r i p h e r a l a n t a g o n i s t s " ~ , 1° of t h y r o x i n e ( 2 , 6 - d i i o d o p h e n o x y a c e t i c a c i d ; 2 , 4 , 6 - t r i i o d o p h e n o x y a c e t i c a c i d ( b o t h c o m p o u n d s p r o v i d e d t h r o u g h t h e c o u r t e s y of P r o f e s s o r S. B. BARKER, U n i v e r s i t y of A l a b a m a ) ; a n d N - b u t y l - 4 - h y d r o x y - 3 , 5d i i o d o b e n z o i c a c i d ( p r o v i d e d t h r o u g h t h e c o u r t e s y of P r o f e s s o r N. F. MACLAGAN, U n i v e r s i t y of L o n d o n ) w e r e f o u n d to a n t a g o n i z e t h e t h y r o x i n e - i n d u c e d sweUing. U n e s t e r i f i e d 4 - h y d r o x y - 3 , 5 - d i i o d o b e n z o i c acid, w h i c h is n o t a p e r i p h e r a l a n t a g o n i s t in vivo 9, d i d n o t p r e v e n t t h y r o x i n e - i n d u c e d swelling.
Analogues o/2,4-dinitrophenol T h e a b i l i t y of D N P t o p r e v e n t m i t o c h o n d r i a l swelling is well d o c u m e n t e d 3 , n. T a b l e I I s h o w s t h e effect on swelling of a n u m b e r of its a n a l o g u e s . CROSS, TAGGART, TABLE II JglrFECT OF ANALOGUES OF 2,4-DINITROPHENOL ON MITOCHONDRIAL SWELLING
Results are tabulated as: no effect (o); protection against swelling less than that afforded by DNP (+) ; protection equal to that of DNP ( + +). Final conch, of all compounds was 2. io -~ M. Position x
2
3
4
5
HO HO HO HO HO HO HO HO HO HO CHsO C2H50 HO
NO, NO, NO~ NO~ NO~ NH 2 NO 2 NO~ NO 2
NO, NO 2 NO~ NO 2 NO~ NO~ NOz NO~ NO 2 C1
HO
C1
NO 2
C1
HO HO H~N COOH COOH COOH COOH HO HO HO HO HO HO HO
NO~ NO~ NO~ NO 2 I
HO
I I Br
I
NO, NO 2
0
+ +
C6H 5
+ +
Cyclohexyl NO 2
+ + o +
o o NO~
NO 2 NO~ NO¢ NO,
COOH
I
Br NH 2 C1
o o o + + +
Br NH~ C1
A b i l i t y to uncouple oxidative phosphorylation*
CH 3
I
C1
Inhibition of swelling 6
C1
C1
+ + + + o
o o o o o o o o o o o o +
0 0 0
+ + + +
+++ +++ +++ +++ o 0 o 0
+ +++ + +++ 0 o 0 0
* The data of CRoss et al. 1~ have been tabulated here for convenience. The ability of DNP analogues to "uncouple" oxidative phosphorylation was tested with a "cyclophorase" preparation and graded from o to + + W + . Consult the original article for details.
Re/erences p. 504.
VOL. 36 (1959)
SWELLING OF MITOCHONDRIA
503
Covo AND GREENTM tested the ability of compounds related to phenol to "uncouple" oxidative phosphorylation in kidney "cyclophorase" preparations; their results are tabulated in order to demonstrate the parallel findings. The presence of a free phenolic hydroxyl and appropriate electronegative substitutions in the ortho- and para-positions were necessary for full activity, p-nitrophenol and 2-amino-4-nitrophenol provided some protection, but less than that of DNP. The presence of two ortho-chloro-groups appeared to reinforce the effect of a single p-nitro substitution. Introduction of phenyl, cyclohexyl or methyl groups into the 6-position of DNP did not alter activity, while the presence of a carboxylic acid group abolished the protection against swelling. The inactivity of dinitroanisole, dinitrophenetole and dinitrobenzene indicate the importance of a free phenolic hydroxyl.
DISCUSSION Thyronine derivatives lacking a halogen substitution in either ring of the diphenyl ether have been shown to possess little or no thyroxine-like activity in vivo TM. A similar structural requirement is apparent in those analogues which produce mitochondrial swelling. Both in vivo and in vitro, an iodo-substitution is more effective than an equivalent chloro- or bromo-substitution. Physiological activity in vivo is profoundly altered by minor changes in the sidechain of thyroxine. For example, in the intact animal D-thyroxine has only one-tenth to one-third the potency of L-thyroxine13. On the other hand, the ability of thyroxine to cause swelling of mitochondria is apparently unrelated to the composition of the side-chain; D-thyroxine, as well as the propionic, acetic and formic acid analogues of thyroxine are as effective as L-thyroxine. It has recently been demonstrated in this laboratory that analogues of thyroxine which differ only in the composition of their side-chains are distributed to various tissues in vivo in quite different concentrations 14. Thus, the concentration of L-thyroxine in rat muscle is as much as 6 times that of D-thyroxine; in brain the difference is up to i4-fold. Moreover, the uptake of analogues of thyroxine by various tissues in vitro varies with alterations in the side-chain 15. Thus, differences in the physiological activity of various analogues noted in vivo may well be dependent, at least in part, on differences in their distribution. Analogues which are less potent in vivo might be more effective if they reached the peripheral site of action in sufficient concentration. Although methylation of the phenolic hydroxyl of triiodothyronine does not alter ability to cause swelling, it does decrease activity in vivo 15. Altering this part of the molecule has been shown to affect distribution in the whole animal, as illustrated by the very different distributions of thyroxine and triiodothyronine in the rat TM. It would seem possible, then, that methylation of the phenolic hydroxyl might affect distribution, and that this may be the basis for the inactivity in vivo of the methylated compound. A difference in structural requirements is also apparent in regard to two functions of thyroxine related to the mitochondria themselves. The binding of thyroxine to isolated rat-liver mitochondria has been found to be dependent largely upon the phenolic ring and to a lesser extent upon the composition of the side-chain 17. On the other hand, the ability of thyroxine to cause swelling of the mitochondria is Re#rences p. 504.
504
W . V . SHAW, T. J. LANNON, D. F. TAPLEY
VOL. 2 6
(1959)
apparently independent of the phenolic hydroxyl or the composition of the side-chain, but is dependent upon the iodo-substituted diphenyl ether. It is of interest that the 3 "peripheral antagonists" of thyroxine which have been studied antagonize thyroxine-induced swelling. Simple competition by such compounds for thyroxine binding sites on the mitochondria appears improbable since only one of them (2,4,6-diiodophenoxy-acetic acid) is an effective competitor 17 at a concn, of 2. IO-s M. The results obtained with the analogues of 2,4-dinitrophenol are remarkably similar to those obtained with other in vitro systems. All the phenols which prevent mitochondrial swelling likewise increase respiration and block cell division in Arbacia eggslS, 10. The correlation between the present findings and those of CROSS,TAGGART, Covo AND GREEN12, who investigated the ability of analogues of DNP to uncouple oxidative phosphorylation in kidney "cyclophorase" preparations has been noted in Table II. Although compounds with a phenolic pK' in the range of 3-5-4.5 appear to be maximally active in all systems, there is no direct relationship between pK' and biological activity. The inactivity of picric acid with respect to mitochondria and in the other systems may be related to its much greater dissociation (pK' 0.8) and poor lipid solubility TM. It is possible that the inhibition of swelling caused by DNP may be related in some way to its firm binding to mitochondria *°. The structural requirements observed may thus represent the minimal requirements for mitochondrial penetration or binding. ACKNOWLEDGEMENTS
This work has been supported by U.S. Public Health Service Grant Number A-I5O6, and by a grant from the Rockefeller Foundation to Dr. J. V. TAGGART.
REFERENCES 1 H. AEBI AND I. ABELIN, Biochem. Z., 324 (1953) 364. 8 H. SCHUL2, H. Low, L. ERNSTER AND F. S. SJOSTRAND, Proc. Stockholm Con[. Electron Microscopy, A c a d e m i c P r e s s , Inc., New York, 1957, p. 134. 8 D. F. TAPLEY, J. Biol. Chem., 222 (1956) 325. 4 F. DICKENS AND D. SALMONY, Biochem. J., 64 (1956) 645. s D. F. TAPLE¥ AND C. COOPER, Nature, 178 (1956) 1119. e D. F. TAPLEY AND C. COOPER, J. Biol. Chem., 222 (1956) 341. W. W. KIELLEY AND J. R. BRONK, Biochim. Biophys. Acta, 23 (1957) 448. 8 H. TEDESCHI AND D. U. HARRIS, Biochim. Biophys. Acta, 28 (I958~ 392. 9 M. M. SHEAHAN, J. H. WILKINSON AND N. F. MACLAGEN, Biochem. J., 48 (1951) 188. lo H. M. KLITGAARD, H. B. DIRKS JR., S. B. BARKER, S. C. WANG AND S. WAWZONEK, Endocrinology, 48 (1951) 525 . 11 j . B. CHAPPELL AND G. D. GREVlLLE, Nature, 182 (1958) 813. 18 R. J. CROSS, J. V. TAGGARr, G. A. Covo AND D. E. GREEN, J. Biol. Chem., 177 (1949) 655. 13 H. A. SELENKOW AND S. P. ASPER, Physiol. Rev., 35 (1955) 426. 14 D. F. TAPLEY, F. F. DAVIDOFF, W. B. I-IATFIELD AND J. E. R o s s , Am. J. Physiol., in t h e press. 15 S. B. BARKER, C. E. I~IELY, H. M. KLITGAARD, H. B. DIRKS JR., S. C. WANG AND S. WAWZONEK, Endocrinology, 48 (1951) 7 o. le p. VAN ARSD~L JR., J. R. HOGNESS, R. H. WILLIAMS AND N. ELGEE, Endocrinology, 55 (1954) 332. 17 D. V. TAPLEY AND N. BASSO, Biochim. Biophys. Acta, 36 (1959) 486. 18 G. H. A. CLOWES ANn M. E. KRAHL, J. Gen. Physiol., 2o (1936) 145. 19 G. H. A. CLOWES, Ann. N . Y . ,4cad. Sci., 51 (1951) 14o9. 80 n . KAMIN AND P. HANDLER, Federation Proc., 17 (1958) 252.
BIOCHIMICA ET BIOPHYSICA ACTA
499
T H E E F F E C T OF ANALOGUES OF T H Y R O X I N E AND 2,4-DINITROPHENOL ON T H E S W E L L I N G OF MITOCHONDRIA W. V. S H A W , T. J. L A N N O N AND D. F. T A P L E Y
Department o/Medicine, Columbia University College o] Physicians and Surgeons, and the Presbyterian Hospital, New York, N.Y. (U.S.A.) (Received M a r c h i i t h , 1959)
SUMMARY
Studies with analogues of thyroxine suggest that the ability of thyroxine to cause mitochondrial swelling is dependent upon the presence of the iodo-substituted diphenyl ether, but independent of the phenolic hydroxyl or the composition of the side chain. Three known "peripheral antagonists" of thyroxine have been shown to antagonize thyroxine-induced swelling. The ability of 2,4-dinitrophenol to prevent mitochondrial swelling appears to depend upon the free hydroxyl and the presence of appropriate electro-negative substituents in the ortho- and para-positions.
INTRODUCTION
Several lines of investigation have suggested that thyroxine and some of its analogues alter the morphology of mitochondria. Mitochondria in the livers of thyrotoxic rats have been shown to be swollen and distorted1, 2, and when isolated and suspended in sucrose these mitochondria swell to a greater extent than do those from the livers of normal animals1, a. It has also been demonstrated that the addition in vitro of thyroxine and certain of its analogues to suspensions of mitochondria from normal animals produces pronounced swelling8-5. Although both thyroxine and 2,4-dinitrophenol (DNP) "uncouple" oxidative phosphorylation in intact mitochondria, it seems unlikely that their mechanism of action is the same3, 5. Neither thyroxine nor its analogues "uncouple" the oxidative phosphorylation which occurs in sub-mitochondrial particles obtained by digitonin treatment or ultrasonic disruption, while DNP is effective under such conditionsS, 7. Furthermore, while thyroxine promotes the swelling of rat-liver mitochondria, 2,4-dinitrophenol provides protection against swelling when used in concentrations which "uncouple" oxidative phosphorylation3. The effect of analogues of thyroxine and of DNP on mitochondrial swelling has been studied in an attempt to determine the structural features of the parent compounds essential to their activity. The results are compared with other known in vivo and in vitro actions of these compounds. In addition, the effects of certain known "peripheral antagonists" of thyroxine on the thyroxine-induced swelling are described. Re]erences p. 504.
500
W . V . SHAW, T. j. LANNON, D. F. TAPLEY
VOL. 3 6
(1959)
MATERIALS AND METHODS
Mitochondria were prepared from the livers of Wistar strain male rats as described elsewherea. o.ooi M Ethylenediaminetetraacetate (EDTA) was present in the sucrose solution during the initial homogenization and in the first of 3 washes. The final pellet was suspended in 0.44 M sucrose so that mitochondria from I g of fresh liver were suspended in I.O ml. The final stock suspension was packed in ice and used within IO min. Swelling was measured spectrophotometrically at room temp. (approx. 23 °) as described elsewhere 3. That this is an accurate indication of volume changes has recently been verified by TEDESCHI AND HARRISs. The final test system consisted of 4.0 ml of 0.3 M sucrose containing o.02 M tris-hydroxymethyl-aminomethane(tris) buffer of pH 7.4. Sufficient mitochondria (usually 0.04-0.05 ml of the stock suspension) were added to give an initial O.D. of approx. 0.5. All test compounds were dissolved in o.ooi N KOH immediately before use and incorporated in the final test system at a concn, of 2" lO -5 M. RESULTS
Standardization o[ the method Preliminary studies resulted in findings similar to those reported in detail elsewhere3. The control rate of swelling was such that either enhancement or inhibition of swelling could be readily detected. Fig. I shows a typical experiment with thyroxine and DNP. As can be seen, DNP provided protection against endogenous swelling as well as against that produced by thyroxine. Ability to promote swelling under the standard test conditions has been graded as: no effect (o); less swelling than that produced with thyroxine ( + ) ; and an enhancement of swelling equal in magnitude to that produced by an equimolar amount of thyroxine ( + +). The abilty of compounds to protect against swelling has likewise been graded as: no effect (o) ; less protection against endogenous and thyroxine-induced swelling than that provided by DNP (+) ; and protection against swelling equal to that produced by DNP ( + +). 0.5
+ THYROXINE
0.4
o ROL 0. 3
XINE
o
TIME
20
1o
IN M I N U T E S
Fig. z. Effect of L-thyroxine and 2,4-dinitrophenol on swelling of m i t o c h o n d r i a suspended in o. 3 M sucrose and o.o2 M tris, p H 7-4. Conch. of b o t h c o m p o u n d s 2. IO -~ M.
Analogues o/thyroxine Since the absolute amount of swelling induced by thyroxine appeared to vary slightly with different preparations of mitochondria, a control containing thyroxine Re#rences p. 5o 4.
VOL. 36 (I959)
SWELLING OF MITOCHONDRIA
50I
was used w i t h each e x p e r i m e n t . E v e r y analogue was t e s t e d alone a n d in the presence of t h y r o x i n e . E a r l i e r studies 3 h a d shown D-thyroxine a n d L-thyroxine to be e q u a l l y effective in causing swelling. Hence, all c o m p o u n d s w i t h t h e DL-configuration were t e s t e d a t the same c o n c e n t r a t i o n as the control L-thyroxine. I n no instance was the effect of t h y r o x i n e plus an analogue g r e a t e r t h a n t h a t of t h y r o x i n e alone. A l t h o u g h a n u m b e r of analogues w i t h a d i p h e n y l ether configuration failed to produce a n y swelling, none p r o v i d e d p r o t e c t i o n against e i t h e r endogenous or t h y r o x i n e - i n d u c e d swelling. T h e results o b t a i n e d w i t h the analogues of t h y r o x i n e are recorded in Table I a n d w a r r a n t the following observations. The presence of 2 iodine a t o m s in a single ring of an analogue possessing an alanine side-chain (e.g., 3,5-diiodothyronine) d i d n o t lead to an a b i l i t y to cause swelling. However, when I iodine a t o m was s u b s t i t u t e d in each of t h e 2 rings (e.g., 3,3'-diiodothyronine) the resulting c o m p o u n d d i d cause swelling. I n contrast, in those c o m p o u n d s w i t h propionic or acetic acid side-chains, the s u b s t i t u t i o n of 2 iodine a t o m s in a single ring (e.g., 3,5-diiodothyropropionic acid) was sufficient to p r o d u c e swelling. I t should be n o t e d t h a t i o d o - s u b s t i t u t i o n s were more effective t h a n bromo-, chloro-, or m e t h y l substitutions. A l t e r a t i o n s in the sidechain p e r se d i d not significantly alter a c t i v i t y ; thus, t e t r a i o d o - s u b s t i t u t e d c o m p o u n d s w i t h alanine, propionic, acetic a n d formic acid side chains were all e q u a l l y effective. Similarly, m e t h y l a t i o n of the phenolic h y d r o x y l d i d not alter a c t i v i t y (3,5,3'-triiodoTABLE I EFFECT
OF A N A L O G U E S OF T H Y R O X I N E ON MITOCI-IONDRIAL S W E L L I N G
Results are tabulated as : no effect (o) ; less swelling than that produced by thyroxine (+) ; swelling equal to that produced by thyroxine ( + W). Final concn, of all compounds was 2.IO -~ M. Diphenyl ether substitutions 3' 3 4" 5'
2. 3. 4. 5.
HOHOHOCH30HO-
6. 7. 8. 9. IO. I I. 12. 13. 14. 15. 16.
HOHOCH30HOHOHOHOHOHOCH30CH30-
I.
Side chain VL--
--t~.~b~l~tJt~
-CH~COOH
o o o
+
+ +
-COOH
-.-COOCH,
5
None 3-monoiodo3,5-diiodo3,5-diiodo3,5-diiodo-3',5'dimethyl3,3'-diiodo3,5,3'-triiodo3,5,3'-triiodo3,3',5'-triiodo3-iodo-3',5 '-dibromo3,5,3',5'-tetraiodo3,5,3',5'-tetrabromo3,5,3',5'-tetrachloro3-amino3,5,3'-triamino3,5-dinitro-
+ + + + + + + + + + +* +
+ + + + + +
+ + + +
+ + + +
+ +
+ +
++
o
* Provided through the courtesy of Dr. B. A. HEMS of Glaxo Laboratories, Ltd. All other compounds (except thyronine, 3-monoiodothyronine and 3,5-diiodothyronine) were provided through the courtesy of Drs. R. L. KRoc and A. W. RUDDY Of the Warner-Lambert Research Institute. Re]erences p. 504.
502
W.V.
S H A W , T. J . L A N N O N , D. F. T A P L E Y
VOL. 3 6
(1959)
t h y r o n i n e a n d 4 ' - m e t h o x y - 3 , 5 , 3 ' - t r i i o d o t h y r o n i n e w e r e e q u a l l y a c t i v e ) . A n u m b e r of c o m p o u n d s w i t h o u t t h e d i p h e n y l e t h e r s t r u c t u r e (tyrosine, p - h y d r o x y p h e n y l p r o pionic acid, 3 , 5 - d i i o d o t y r o s i n e a n d 3 , 5 - d i n i t r o t y r o s i n e ) w e r e w i t h o u t effect at a concn, of 2. lO -5 M . T h r e e " p e r i p h e r a l a n t a g o n i s t s " ~ , 1° of t h y r o x i n e ( 2 , 6 - d i i o d o p h e n o x y a c e t i c a c i d ; 2 , 4 , 6 - t r i i o d o p h e n o x y a c e t i c a c i d ( b o t h c o m p o u n d s p r o v i d e d t h r o u g h t h e c o u r t e s y of P r o f e s s o r S. B. BARKER, U n i v e r s i t y of A l a b a m a ) ; a n d N - b u t y l - 4 - h y d r o x y - 3 , 5d i i o d o b e n z o i c a c i d ( p r o v i d e d t h r o u g h t h e c o u r t e s y of P r o f e s s o r N. F. MACLAGAN, U n i v e r s i t y of L o n d o n ) w e r e f o u n d to a n t a g o n i z e t h e t h y r o x i n e - i n d u c e d sweUing. U n e s t e r i f i e d 4 - h y d r o x y - 3 , 5 - d i i o d o b e n z o i c acid, w h i c h is n o t a p e r i p h e r a l a n t a g o n i s t in vivo 9, d i d n o t p r e v e n t t h y r o x i n e - i n d u c e d swelling.
Analogues o/2,4-dinitrophenol T h e a b i l i t y of D N P t o p r e v e n t m i t o c h o n d r i a l swelling is well d o c u m e n t e d 3 , n. T a b l e I I s h o w s t h e effect on swelling of a n u m b e r of its a n a l o g u e s . CROSS, TAGGART, TABLE II JglrFECT OF ANALOGUES OF 2,4-DINITROPHENOL ON MITOCHONDRIAL SWELLING
Results are tabulated as: no effect (o); protection against swelling less than that afforded by DNP (+) ; protection equal to that of DNP ( + +). Final conch, of all compounds was 2. io -~ M. Position x
2
3
4
5
HO HO HO HO HO HO HO HO HO HO CHsO C2H50 HO
NO, NO, NO~ NO~ NO~ NH 2 NO 2 NO~ NO 2
NO, NO 2 NO~ NO 2 NO~ NO~ NOz NO~ NO 2 C1
HO
C1
NO 2
C1
HO HO H~N COOH COOH COOH COOH HO HO HO HO HO HO HO
NO~ NO~ NO~ NO 2 I
HO
I I Br
I
NO, NO 2
0
+ +
C6H 5
+ +
Cyclohexyl NO 2
+ + o +
o o NO~
NO 2 NO~ NO¢ NO,
COOH
I
Br NH 2 C1
o o o + + +
Br NH~ C1
A b i l i t y to uncouple oxidative phosphorylation*
CH 3
I
C1
Inhibition of swelling 6
C1
C1
+ + + + o
o o o o o o o o o o o o +
0 0 0
+ + + +
+++ +++ +++ +++ o 0 o 0
+ +++ + +++ 0 o 0 0
* The data of CRoss et al. 1~ have been tabulated here for convenience. The ability of DNP analogues to "uncouple" oxidative phosphorylation was tested with a "cyclophorase" preparation and graded from o to + + W + . Consult the original article for details.
Re/erences p. 504.
VOL. 36 (1959)
SWELLING OF MITOCHONDRIA
503
Covo AND GREENTM tested the ability of compounds related to phenol to "uncouple" oxidative phosphorylation in kidney "cyclophorase" preparations; their results are tabulated in order to demonstrate the parallel findings. The presence of a free phenolic hydroxyl and appropriate electronegative substitutions in the ortho- and para-positions were necessary for full activity, p-nitrophenol and 2-amino-4-nitrophenol provided some protection, but less than that of DNP. The presence of two ortho-chloro-groups appeared to reinforce the effect of a single p-nitro substitution. Introduction of phenyl, cyclohexyl or methyl groups into the 6-position of DNP did not alter activity, while the presence of a carboxylic acid group abolished the protection against swelling. The inactivity of dinitroanisole, dinitrophenetole and dinitrobenzene indicate the importance of a free phenolic hydroxyl.
DISCUSSION Thyronine derivatives lacking a halogen substitution in either ring of the diphenyl ether have been shown to possess little or no thyroxine-like activity in vivo TM. A similar structural requirement is apparent in those analogues which produce mitochondrial swelling. Both in vivo and in vitro, an iodo-substitution is more effective than an equivalent chloro- or bromo-substitution. Physiological activity in vivo is profoundly altered by minor changes in the sidechain of thyroxine. For example, in the intact animal D-thyroxine has only one-tenth to one-third the potency of L-thyroxine13. On the other hand, the ability of thyroxine to cause swelling of mitochondria is apparently unrelated to the composition of the side-chain; D-thyroxine, as well as the propionic, acetic and formic acid analogues of thyroxine are as effective as L-thyroxine. It has recently been demonstrated in this laboratory that analogues of thyroxine which differ only in the composition of their side-chains are distributed to various tissues in vivo in quite different concentrations 14. Thus, the concentration of L-thyroxine in rat muscle is as much as 6 times that of D-thyroxine; in brain the difference is up to i4-fold. Moreover, the uptake of analogues of thyroxine by various tissues in vitro varies with alterations in the side-chain 15. Thus, differences in the physiological activity of various analogues noted in vivo may well be dependent, at least in part, on differences in their distribution. Analogues which are less potent in vivo might be more effective if they reached the peripheral site of action in sufficient concentration. Although methylation of the phenolic hydroxyl of triiodothyronine does not alter ability to cause swelling, it does decrease activity in vivo 15. Altering this part of the molecule has been shown to affect distribution in the whole animal, as illustrated by the very different distributions of thyroxine and triiodothyronine in the rat TM. It would seem possible, then, that methylation of the phenolic hydroxyl might affect distribution, and that this may be the basis for the inactivity in vivo of the methylated compound. A difference in structural requirements is also apparent in regard to two functions of thyroxine related to the mitochondria themselves. The binding of thyroxine to isolated rat-liver mitochondria has been found to be dependent largely upon the phenolic ring and to a lesser extent upon the composition of the side-chain 17. On the other hand, the ability of thyroxine to cause swelling of the mitochondria is Re#rences p. 504.
504
W . V . SHAW, T. J. LANNON, D. F. TAPLEY
VOL. 2 6
(1959)
apparently independent of the phenolic hydroxyl or the composition of the side-chain, but is dependent upon the iodo-substituted diphenyl ether. It is of interest that the 3 "peripheral antagonists" of thyroxine which have been studied antagonize thyroxine-induced swelling. Simple competition by such compounds for thyroxine binding sites on the mitochondria appears improbable since only one of them (2,4,6-diiodophenoxy-acetic acid) is an effective competitor 17 at a concn, of 2. IO-s M. The results obtained with the analogues of 2,4-dinitrophenol are remarkably similar to those obtained with other in vitro systems. All the phenols which prevent mitochondrial swelling likewise increase respiration and block cell division in Arbacia eggslS, 10. The correlation between the present findings and those of CROSS,TAGGART, Covo AND GREEN12, who investigated the ability of analogues of DNP to uncouple oxidative phosphorylation in kidney "cyclophorase" preparations has been noted in Table II. Although compounds with a phenolic pK' in the range of 3-5-4.5 appear to be maximally active in all systems, there is no direct relationship between pK' and biological activity. The inactivity of picric acid with respect to mitochondria and in the other systems may be related to its much greater dissociation (pK' 0.8) and poor lipid solubility TM. It is possible that the inhibition of swelling caused by DNP may be related in some way to its firm binding to mitochondria *°. The structural requirements observed may thus represent the minimal requirements for mitochondrial penetration or binding. ACKNOWLEDGEMENTS
This work has been supported by U.S. Public Health Service Grant Number A-I5O6, and by a grant from the Rockefeller Foundation to Dr. J. V. TAGGART.
REFERENCES 1 H. AEBI AND I. ABELIN, Biochem. Z., 324 (1953) 364. 8 H. SCHUL2, H. Low, L. ERNSTER AND F. S. SJOSTRAND, Proc. Stockholm Con[. Electron Microscopy, A c a d e m i c P r e s s , Inc., New York, 1957, p. 134. 8 D. F. TAPLEY, J. Biol. Chem., 222 (1956) 325. 4 F. DICKENS AND D. SALMONY, Biochem. J., 64 (1956) 645. s D. F. TAPLE¥ AND C. COOPER, Nature, 178 (1956) 1119. e D. F. TAPLEY AND C. COOPER, J. Biol. Chem., 222 (1956) 341. W. W. KIELLEY AND J. R. BRONK, Biochim. Biophys. Acta, 23 (1957) 448. 8 H. TEDESCHI AND D. U. HARRIS, Biochim. Biophys. Acta, 28 (I958~ 392. 9 M. M. SHEAHAN, J. H. WILKINSON AND N. F. MACLAGEN, Biochem. J., 48 (1951) 188. lo H. M. KLITGAARD, H. B. DIRKS JR., S. B. BARKER, S. C. WANG AND S. WAWZONEK, Endocrinology, 48 (1951) 525 . 11 j . B. CHAPPELL AND G. D. GREVlLLE, Nature, 182 (1958) 813. 18 R. J. CROSS, J. V. TAGGARr, G. A. Covo AND D. E. GREEN, J. Biol. Chem., 177 (1949) 655. 13 H. A. SELENKOW AND S. P. ASPER, Physiol. Rev., 35 (1955) 426. 14 D. F. TAPLEY, F. F. DAVIDOFF, W. B. I-IATFIELD AND J. E. R o s s , Am. J. Physiol., in t h e press. 15 S. B. BARKER, C. E. I~IELY, H. M. KLITGAARD, H. B. DIRKS JR., S. C. WANG AND S. WAWZONEK, Endocrinology, 48 (1951) 7 o. le p. VAN ARSD~L JR., J. R. HOGNESS, R. H. WILLIAMS AND N. ELGEE, Endocrinology, 55 (1954) 332. 17 D. V. TAPLEY AND N. BASSO, Biochim. Biophys. Acta, 36 (1959) 486. 18 G. H. A. CLOWES ANn M. E. KRAHL, J. Gen. Physiol., 2o (1936) 145. 19 G. H. A. CLOWES, Ann. N . Y . ,4cad. Sci., 51 (1951) 14o9. 80 n . KAMIN AND P. HANDLER, Federation Proc., 17 (1958) 252.