15. 11. 1975
1365
Specialia
t h a t t h e a m o u n t s of food e a t e n c o r r e s p o n d e d t o t h e dose of g i v e n drug. A f t e r 21 d a y s t h e a n i m a l s were killed, t h e a d r e n a l s s e m i n a l vesicles a n d spleen were r e m o v e d , cleaned and weighed on a torsion balance. Organ weights were e x p r e s s e d in b o t h a b s o l u t e a n d r e l a t i v e v a l u e s (mg/ l o o t b. wt.) T h e r e s u l t s were e v a l u a t e d s t a t i s t i c a l l y b y m e a n s of t h e D u n c a n ' s t e s t v. T h e r e s u l t s a t e g i v e n in t h e Table. S i g n i f i c a n t c h a n g e s were o b s e r v e d in t h e s e m i n a l vesicles, w h i c h are a h i g h l y a n d r o g e n - d e p e n d e n t tissues. T h e a d m i n i s t r a t i o n of c y p r o t e r o n e a n d c y p r o t e r o n e a c e t a t e to i n t a c t mice caused a s i g n i f i c a n t d e c r e a s e c o m p a r e d w i t h controls. P r e d n i s o n e , o n t h e c o n t r a r y , s t i m u l a t e d t h e g r o w t h of t h e accessory s e x u a l glaI~ds. T h e s t a t i s t i c a l l y s i g n i f i c a n t decrease in a d r e n a l w e i g h t a f t e r all 3 s t e r o i d s was in a g r e e m e n t w i t h o b s e r v a t i o n s in rats, c y p r o t e r o n e a c e t a t e b e i n g a p p r o x i m a t e l y e q u i v a l e n t t o p r e d n i s o n e 4,6. C y p r o t e r o n e e x e r t s a s i m i l a r a c t i o n b u t to a s m a l l e r e x t e n t . A d r a m a t i c decrease of t h e w e i g h t of spleen was c a u s e d b y c y p r o t e r o n e a c e t a t e a n d p r e d n i s o n e , a n d less p r o n o u n c e d b u t still s i g n i f i c a n t l y b y c y p r o t e r o n e . T h e d r y w e i g h t of spleen c o r r e s p o n d s to t h e e x t e n t of t h e decrease in w e t w e i g h t of spleen in t h e e x p e r i m e n t a l animals. T h e r e is a w e l l - k n o w n decrease in t h y m u s w e i g h t a f t e r corticoids 8, 9 in t h e spleen; t h e loss of w e i g h t following cortisone t r e a t m e n t is n o t as p r o n o u n c e d as in t h e t h y m u s b u t follows t h e s a m e p a t t e r n ~ , 9. T h e c o r t i c o s t e r o i d - l i k e effect h a s b e e n d e s c r i b e d in some g e s t a g e n s 10, n. T h o u g h c y p r o t e r o n e a c e t a t e corticoid-like effect could b e conn e c t e d w i t h its gestagenic properties, t h i s is n o t v a l i d for
t h e c y p r o t e r o n e effect: c y p r o t e r o n e h a s n o g e s t a g e n i c effectL W h e n c o m p a r e d w i t h p r e d n i s o n e , c y p r o t e r o n e a c e t a t e h a s a p p r o x i m a t e l y ~/5 of t h e corticoid p o t e n c y of p r e d n i s o n e , e x p r e s s e d b y t h e decrease of t h e s p l e e n w e i g h t ; a n d so i t c o r r e s p o n d s in its efficiency t o cortisol. If c y p r o t e r o n e a c e t a t e were to e x e r t t h e corticoid-like effect also in o t h e r p a r a m e t e r s , t h e c o m b i n a t i o n Of a n t i a n d r o g e n i c p r o p e r t i e s a n d corticoid a c t i o n w o u l d m a k e t h i s d r u g s u i t a b l e for t h e t r e a t m e n t ol virilizing adrenal hyperplasia.
Summary. C y p r o t e r o n e a n d c y p r o t e r o n e a c e t a t e e x e r t t h e corticoid-like effect o n t h e a d r e n a l a n d spleen w e i g h t in t h e mice. W h e n c o m p a r e d w i t h p r e d n i s o n e , c y p r o t e r o n e a c e t a t e h a s a p p r o x i m a t e l y 1/5 of t h e corticoid p o t e n c y of p r e d n i s o n e , e x p r e s s e d b y t h e decrease of t h e spleen weight. P. D. BROULIK a n d L. STARKA
I I I r d Medical Clinic, Faculty o/General Medicine, Charles University, U nemonice 1, Praha 2 (Czechoslovakia), 76 June 7975. D. DUNCAN, B i o m e t r i c s l l , 1 (1955). 8 D. MAOR, 1~. EYLAN and P. ALEXANDER, A c t a endocr., Copenh. 74,
201 (1975). 9 j . WE~'~OOT~, Radiother. Res. 8, 307 (1958). ~0 F. CA~tANNI, F. MASSARA and G. MOLINATTI, Acta endocr. Copenh.
43, 447 (1963). n G. FA~ETE and S. SZEBERENYI, Steroids 6, 159 (1965).
Effect of C o r t e x o l o n e o n t h e F e e d b a c k A c t i o n of D e x a m e t h a s o n e C o r t e x o l o n e (pregn-4-ene-17cq21 diol-3,20 dione) was s h o w n t o a c t as a n a n t i g l u c o c o r t i c o i d in t h e t h y m u s ~ . I t was f o u n d to b i n d to a n d to displace t h e biologically a c t i v e glucocorticoid f r o m c y t o p l a s m i c c o r t i c o s t e r o i d r e c e p t o r s of t h e t h y m u s ~0,a a n d in some regions of t h e r a t b r a i n a. Specific glucocorticoid r e c e p t o r s h a v e b e e n d e m o n s t r a t e d in v a r i o u s areas of t h e c e n t r a l n e r v o u s system 5 8 I n t h e p r e s e n t s t u d y , t h e role of t h e s e c y t o p l a s m i c r e c e p t o r s in t h e glucocorticoid f e e d b a c k a c t i o n was i n v e s t i g a t e d b y t e s t i n g t h e effect of c o r t e x o l o n e o n t h e f e e d b a c k a c t i o n of d e x a m e t h a s o n e . Materials and methods. Male a l b i n o r a t s of t h e C F E strain, m a i n t a i n e d o n a s t a n d a r d d i e t w i t h free access to water, a c c l i m a t i z e d to a n i m a l r o o m c o n d i t i o n s of u n i f o r m t e m p e r a t u r e (24 4- 1 ~ a n d c o n t r o l l e d r e l a t i v e h u m i d i t y ( 5 0 - 7 5 % ) were used. T h e y were i n j e c t e d s.c. w i t h e i t h e r
1 m g of c o r t e x o l o n e / 1 0 0 g ( K o c h - L i g h t L a b o r a t o r i e s dissolved in 0.5 m l of s u n f l o w e r oil (Groups 2 a n d 4 in t h e Table), or 0.5 m l oil/100 g (Groups l a n d 3 in t h e Table). 1 I(. M. MOSHER, D. A. YOUNG and A. MUNCK, J. biol. Chem. 246, 654 (1971). A. 5~UNCK and T. BRINCK-JOHNSEN, J. biol. Chem. 243, 5556
(196s). a N. KAISER, R. J. MILtfOLLAND, R. W. TURNEL and F. ROSEN', Biochem. biophys. Res. Comnmn. 49, 516 (1972). a Zs. Acs, u n p u b l i s h e d data. .5 B. S. McEwI~N, J. M. W*=iss and L. S. SCUWARTZ, B r a i u Res. 16,
227 (1969). , B. I. GROSSBR,W. STEVENS, F. W. BRU~NGERand D. J. REED, J. Neurochem. 18, 1725 (1971). 7 H. KmTZLBY, J. Neuroehem. 19, 2737 (1972). s E. STARK, Zs. Acs, M. PALKOVITS and G. FOLLY, Acta Physiol. Aead. Sci. hung. in press (1974).
Effect of cortexolone (1 rag/100 g) on the stress-reaction of r a t s t r e a t e d w i t h 50 ~tg/100 g d e x a m e t h a s o n e Group 1
Group 2
Group 3
Group 4
Subcutaneous injection
oil
cortexolone
oil
cortexolone
Intraperitoneal injection Restingprestress level
saline 12.61 ~ 1.74 (9)
saline 9.98 -L 2.08 (10)
dexamethasone 3.22 zt= 0.72 ~ (10)
dexamethasone 4.48 ~= 1.46~ (10)
Increment induced by
16.03 =t= 3.86
14.90 =~ 2.18
6.55 4- 1.56 9
14.69 ~ 1.90
histamine stress
(9)
(10)
(10)
(10)
Mean ~ S E ~ in ~xg corticosterone[100 ml plasma. ~significantly different (p ~ 0.01) from its respective control group e v a l u a t e d b y the m e t h o d of analysis of v a r i a n c e for two-way layout. N u m b e r s in parentheses denote n u m b e r of d e t e r m i n a t i o n s .
Specialia
1366
2 h later, t h e a n i m a l s received i.p. e i t h e r 50 ~zg of d e x a m e t h a s o n e / 1 0 0 g (Oradexon, O r g a n o n ) (Groups 3 a n d 4), or 0.2 m l of physiological saline/100 g (Groups 1 a n d 2). 2 h a f t e r t h e i.p. injection, 1 m l of b l o o d was w i t h d r a w n f r o m t h e s a p h e n o u s vein, u n d e r l i g h t e t h e r a n a e s t h e s i a , w i t h i n 2 m i n of r e m o v i n g t h e a n i m a l f r o m its cage. H i s t a m i n e (1 rng/100 g) was i n j e c t e d i.p., followed b y b l o o d w i t h d r a w a l f r o m t h e a b d o m i n a l a o r t a 30 m i n later. P r e - a n d a f t e r - s t r e s s p l a s m a c o r t i c o s t e r o n e was d e t e r m i n e d b y t h e c o m p e t i t i v e p r o t e i n - b i n d i n g m e t h o d 9. C o r t e x o l o n e d i d n o t i n t e r f e r e w i t h c o r t i c o s t e r o n e d e t e r m i n a t i o n , since 3 h a f t e r a d m i n i s t e r i n g s.c. t h e a b o v e dose of c o r t e x o l o n e t o r a t s a d r e n a l e c t o m i z e d 24 h earlier p l a s m a c o r t i c o s t e r o n e e q u a l l e d t h a t in t h e a d r e n a l e c t o m i z e d c o n t r o l s (0.57 0.08 ~g/100 m l v.s. 0.54 d2 0.05 ~g/100 m l ; n u m b e r of d e t e r m i n a t i o n s is 9 in b o t h groups). Results and discussion. D e x a m e t h a s o n e a d m i n i s t r a t i o n lowered t h e r e s t i n g p l a s m a c o r t i c o s t e r o n e level (Table). A f t e r h i s t a m i n e , c o r t i c o s t e r o n e level in t h e d e x a m e t h a s o n e - t r e a t e d r a t s rose, t h e i n c r e m e n t b e i n g signifi c a n t l y s m a l l e r t h a n in t h e c o n t r o l s (Table). C o r t e x o l o n e e x e r t e d n o effect o n t h e stress r e a c t i o n of r a t s (Group 2 in t h e Table). T h i s f i n d i n g seems to b e a t v a r i a n c e w i t h t h a t of JoNEs et al. 19, a difference p r o b a b l y d u e t o t h e f a c t t h a t we a d m i n i s t e r e d c o r t e x o l o n e in 10 t i m e s lower doses t h a n t h e y did. Cortexolone administration prior to dexamethasone did n o t p r e v e n t t h e d e c r e a s e in t h e r e s t i n g c o r t i c o s t e r o n e level (Group 4 in t h e Table), b u t i n t h e s e a n i m a l s h i s t a m i n e stress i n d u c e d a n i n c r e m e n t in p l a s m a c o r t i c o s t e r o n e s i m i l a r t o t h a t in t h e v e h i c l e - t r e a t e d c o n t r o l s (v.s. G r o u p 4 a n d G r o u p 1 in t h e Table). O u r i n t e r p r e t a t i o n of t h e s e d a t a is t h a t c o r t e x o l o n e p r o b a b l y displaces d e x a m e t h a s o n e f r o m t h e h y p o t h a l a m i c a n d / o r e x t r a h y p o t h a l a m i c c o r t i c o s t e r o i d recept o r sites, a n d t h u s p r e v e n t s d e x a m e t h a s o n e f r o m i n h i b i t i n g
E f f e c t of P r o g e s t e r o n e
on Human
EXPERIENTIA31/11
s t r e s s - i n d u c e d A C T H release. T h e d a t a f a v o r t h e a s s u m p tion that the corticosteroid feedback action depends on t h e b i n d i n g of t h e h o r m o n e t o specific r e c e p t o r s in t h e t a r g e t cells, t h e m e c h a n i s m Of f e e d b a c k a c t i o n b e i n g in t h i s r e s p e c t s i m i l a r t o t h a t of o t h e r w e l l - k n o w n c o r t i c o s t e r o i d effects. I t was s u g g e s t e d n, 12 t h a t t h e m e c h a nisms controlling ACTH-release under non-stress and stress c o n d i t i o n s are f u n c t i o n a l l y dissociable. O u r pres e n t d a t a seem t o s u p p o r t t h i s a s s u m p t i o n b y i n d i c a t i n g t h a t t h e site a n d / o r t h e m e c h a n i s m of d e x a m e t h a s o n e s u p p r e s s i o n of r e s t i n g A C T H s e c r e t i o n are d i s t i n c t f r o m t h o s e d e p r e s s i n g stress i n d u c e d A C T H release.
Summary. C o r t e x o l o n e i n a dose of 1 mg/100 g b o d y wt., a d m i n i s t e r e d t o r a t s p r i o r t o d e x a m e t h a s o n e , pre-: vented dexamethasone from suppressing stress-induced A C T H - r e l e a s e w i t h o u t i n t e r f e r i n g w i t h t h e effect of d e x a m e t h a s o n e on t h e r e s t i n g p l a s m a c o r t i c o s t e r o n e level. ZSUZSANNA fikCS and E. STARK13
Institute o/Experimental Medicine, Hungarian Academy o/Sciences, -P.O. Box 67, H - I J 5 0 Budapest (Hungary), 23 June 7975.
9 B. E. P. MURPHY,J. elin. Endocr. Metab. 27, 973 (1967). 10 M. T. JONES, E. 1V[.TIPTAFT, t 2. R. BRUStt, D. A. N. FERGUSONand R. L. B. NEAME,J. Endocr. 60, 223 (1974). 11 E. ZIMMERMANand V. CRITCHLOW,Am. J. Physiol. 216, 148 (1969). IS j. R. HODGES, Progr. Brain Res. 32, 12 (1970). 18 Acknowledgements. Authors wish to thank Mr. G. FOLLY for the mathematical analyses and Mrs. M. PONGR$,CZfor her valuable technical assistance.
Corticosteroid 21-Hydroxylation
T h e b i o c h e m i c a l b a s i s of t h e g e n e t i c a l l y i n h e r i t e d m e t a b o l i c d i s o r d e r s k n o w n as t h e a d r e n o g e n i t a l s y n d r o m e 1, 2 is as y e t n o t fully u n d e r s t o o d . W h i l e t w o forms, t h e 'salt-losing' a n d t h e ' n o n - s a l t - l o s i n g ' varieties, are k n o w n , b o t h of w h i c h a p p e a r t o r e s u l t f r o m d e f e c t i v e a d r e n a l 2 1 - h y d r o x y l a t i o n c a p a b i l i t y , i t is n o t y e t k n o w n w h e t h e r t h e s e r e p r e s e n t a single or t w o genetic defects 3, ~. R e s e a r c h e s o n t h e s e possibilities h a v e r e c e n t l y b e e n r e v i e w e d elsewhere 5. T h e p r e s e n t a c c o u n t r e p o r t s t h e r e s u l t s of e x p e r i m e n t s t o i n v e s t i g a t e w h e t h e r i n d e p e n d e n t a c t i v e sites for t h e 2 1 - h y d r o x y l a t i o n 6 of p r o g e s t e r o n e t o 1 1 - d e o x y c o r t i c o s t e r o n e (DOC) a n d of 17 e - h y d r o x y progesterone to 11-deoxycortisol (Reichstein's compound S) are p r e s e n t in h u m a n a d r e n o c o r t i c a l m i c r o s o m a l p r o t e i n . T h i s i n v e s t i g a t i o n f o r m e d p a r t of a c o m m u n i c a t i o n ~ p r e s e n t e d a t t h e m e e t i n g of t h e S o u t h e r n S o c i e t y for P e d i a t r i c R e s e a r c h in 1974. T w o n o r m a l h u m a n a d r e n a l s were o b t a i n e d f r e s h p o s t m o r t e m a n d t h e e x t e r n a l f a t a n d c o n n e c t i v e t i s s u e were t r i m m e d off. T h e w e i g h t of t h e 2 g l a n d s was 7.07 g. T h e y were c u t i n t o s m a l l pieces in a b o u t 100 m l cold 0.25 M sucrose. T h e s u s p e n s i o n was h o m o g e n i z e d u s i n g a m o t o r d r i v e n glass-teflon h o m o g e n i z e r a n d c e n t r i f u g e d a t 600 g for 10 m i n t o r e m o v e n u c l e a r c o m p o n e n t s , u n b r o k e n cells a n d u n w a n t e d d e b r i s s. T h e s u p e r n a t a n t w a s c e n t r i fuged a t 8,720 g for 10 m i n t o s e p a r a t e t h e h e a v y m i t o c h o n d r i a a n d t w i c e a t 26,700 g for 30 rain t o r e m o v e l i g h t m i t o c h o n d r i a . T h e m i c r o s o m e s were i s o l a t e d f r o m t h e
s u p e r n a t a n t b y c e n t r i f u g a t i o n a t 78,500 g for 90 m i n . T h e y were g e n t l y h a n d - h o m o g e n i z e d in 1-2 m l Tris-HC1, 50 r a M , p H 7.45 c o n t a i n i n g 3 m M MgC12 a n d t h e p r o t e i n concentration determined by the biuret method. Determ i n a t i o n of t h e c y t o c h r o m e P 4 5 0 c o n t e n t of t h e m i c r o s o m a l p r o t e i n was p e r f o r m e d f r o m t h e o p t i c a l e x t i n c t i o n a t 450 n m a f t e r t r e a t m e n t w i t h d i t h i o n i t e a n d c a r b o n m o n o x i d e 9. I n c u b a t i o n s were p e r f o r m e d for 5 m i n w i t h a g i t a t i o n in a D u b n o f f i n c u b a t o r m a i n t a i n e d a t 37~ The reaction m i x t u r e s c o n t a i n e d m i c r o s o m a l e n z y m e (1.7 m g p r o t e i n ) ; t r i s o d i u m isocitrate, 11 v m o l e s ( S i g m a ) ; p i g h e a r t iso-
1 I{. S. HOLT and D. N. EAItCE, Basic Concepts o] Inborn Errors and Dejects o] Steroid Biosynthesis (E. and S. Livingstone, Edinburgh 1966). 2 D. B. VILLEE, Human Endocrinology, a Developmental Approach (Saunders, Philadelphia 1975). 8 F. C. BARTTER, Arch. Dis. Childhood 44, 138 (1969). G. T. BRYAN, B. KLIMAN and 12. C. BARTTER, J. elin. Invest. 44, 957 (1965). R, H. WlCKRA~tASlNGHE,Enzyme 19, 348 (1975). s K. J. RYAN and L. L. ENOEL, J. Am. chem. Soe. 78, 2654 (1956). E. B. NELSON, R. H. WICKRAMASINGHEand G. T. BRYAN, Clin. Res. 22, 79A (1974). s R. H. WICKRAMASINGHE,Int. j. Peptide Protein Res. 6, 187 (1974). 9 T. OMURAand R. SATO, J. biol. Chem. 239, 2379 (1964).
1365
Specialia
t h a t t h e a m o u n t s of food e a t e n c o r r e s p o n d e d t o t h e dose of g i v e n drug. A f t e r 21 d a y s t h e a n i m a l s were killed, t h e a d r e n a l s s e m i n a l vesicles a n d spleen were r e m o v e d , cleaned and weighed on a torsion balance. Organ weights were e x p r e s s e d in b o t h a b s o l u t e a n d r e l a t i v e v a l u e s (mg/ l o o t b. wt.) T h e r e s u l t s were e v a l u a t e d s t a t i s t i c a l l y b y m e a n s of t h e D u n c a n ' s t e s t v. T h e r e s u l t s a t e g i v e n in t h e Table. S i g n i f i c a n t c h a n g e s were o b s e r v e d in t h e s e m i n a l vesicles, w h i c h are a h i g h l y a n d r o g e n - d e p e n d e n t tissues. T h e a d m i n i s t r a t i o n of c y p r o t e r o n e a n d c y p r o t e r o n e a c e t a t e to i n t a c t mice caused a s i g n i f i c a n t d e c r e a s e c o m p a r e d w i t h controls. P r e d n i s o n e , o n t h e c o n t r a r y , s t i m u l a t e d t h e g r o w t h of t h e accessory s e x u a l glaI~ds. T h e s t a t i s t i c a l l y s i g n i f i c a n t decrease in a d r e n a l w e i g h t a f t e r all 3 s t e r o i d s was in a g r e e m e n t w i t h o b s e r v a t i o n s in rats, c y p r o t e r o n e a c e t a t e b e i n g a p p r o x i m a t e l y e q u i v a l e n t t o p r e d n i s o n e 4,6. C y p r o t e r o n e e x e r t s a s i m i l a r a c t i o n b u t to a s m a l l e r e x t e n t . A d r a m a t i c decrease of t h e w e i g h t of spleen was c a u s e d b y c y p r o t e r o n e a c e t a t e a n d p r e d n i s o n e , a n d less p r o n o u n c e d b u t still s i g n i f i c a n t l y b y c y p r o t e r o n e . T h e d r y w e i g h t of spleen c o r r e s p o n d s to t h e e x t e n t of t h e decrease in w e t w e i g h t of spleen in t h e e x p e r i m e n t a l animals. T h e r e is a w e l l - k n o w n decrease in t h y m u s w e i g h t a f t e r corticoids 8, 9 in t h e spleen; t h e loss of w e i g h t following cortisone t r e a t m e n t is n o t as p r o n o u n c e d as in t h e t h y m u s b u t follows t h e s a m e p a t t e r n ~ , 9. T h e c o r t i c o s t e r o i d - l i k e effect h a s b e e n d e s c r i b e d in some g e s t a g e n s 10, n. T h o u g h c y p r o t e r o n e a c e t a t e corticoid-like effect could b e conn e c t e d w i t h its gestagenic properties, t h i s is n o t v a l i d for
t h e c y p r o t e r o n e effect: c y p r o t e r o n e h a s n o g e s t a g e n i c effectL W h e n c o m p a r e d w i t h p r e d n i s o n e , c y p r o t e r o n e a c e t a t e h a s a p p r o x i m a t e l y ~/5 of t h e corticoid p o t e n c y of p r e d n i s o n e , e x p r e s s e d b y t h e decrease of t h e s p l e e n w e i g h t ; a n d so i t c o r r e s p o n d s in its efficiency t o cortisol. If c y p r o t e r o n e a c e t a t e were to e x e r t t h e corticoid-like effect also in o t h e r p a r a m e t e r s , t h e c o m b i n a t i o n Of a n t i a n d r o g e n i c p r o p e r t i e s a n d corticoid a c t i o n w o u l d m a k e t h i s d r u g s u i t a b l e for t h e t r e a t m e n t ol virilizing adrenal hyperplasia.
Summary. C y p r o t e r o n e a n d c y p r o t e r o n e a c e t a t e e x e r t t h e corticoid-like effect o n t h e a d r e n a l a n d spleen w e i g h t in t h e mice. W h e n c o m p a r e d w i t h p r e d n i s o n e , c y p r o t e r o n e a c e t a t e h a s a p p r o x i m a t e l y 1/5 of t h e corticoid p o t e n c y of p r e d n i s o n e , e x p r e s s e d b y t h e decrease of t h e spleen weight. P. D. BROULIK a n d L. STARKA
I I I r d Medical Clinic, Faculty o/General Medicine, Charles University, U nemonice 1, Praha 2 (Czechoslovakia), 76 June 7975. D. DUNCAN, B i o m e t r i c s l l , 1 (1955). 8 D. MAOR, 1~. EYLAN and P. ALEXANDER, A c t a endocr., Copenh. 74,
201 (1975). 9 j . WE~'~OOT~, Radiother. Res. 8, 307 (1958). ~0 F. CA~tANNI, F. MASSARA and G. MOLINATTI, Acta endocr. Copenh.
43, 447 (1963). n G. FA~ETE and S. SZEBERENYI, Steroids 6, 159 (1965).
Effect of C o r t e x o l o n e o n t h e F e e d b a c k A c t i o n of D e x a m e t h a s o n e C o r t e x o l o n e (pregn-4-ene-17cq21 diol-3,20 dione) was s h o w n t o a c t as a n a n t i g l u c o c o r t i c o i d in t h e t h y m u s ~ . I t was f o u n d to b i n d to a n d to displace t h e biologically a c t i v e glucocorticoid f r o m c y t o p l a s m i c c o r t i c o s t e r o i d r e c e p t o r s of t h e t h y m u s ~0,a a n d in some regions of t h e r a t b r a i n a. Specific glucocorticoid r e c e p t o r s h a v e b e e n d e m o n s t r a t e d in v a r i o u s areas of t h e c e n t r a l n e r v o u s system 5 8 I n t h e p r e s e n t s t u d y , t h e role of t h e s e c y t o p l a s m i c r e c e p t o r s in t h e glucocorticoid f e e d b a c k a c t i o n was i n v e s t i g a t e d b y t e s t i n g t h e effect of c o r t e x o l o n e o n t h e f e e d b a c k a c t i o n of d e x a m e t h a s o n e . Materials and methods. Male a l b i n o r a t s of t h e C F E strain, m a i n t a i n e d o n a s t a n d a r d d i e t w i t h free access to water, a c c l i m a t i z e d to a n i m a l r o o m c o n d i t i o n s of u n i f o r m t e m p e r a t u r e (24 4- 1 ~ a n d c o n t r o l l e d r e l a t i v e h u m i d i t y ( 5 0 - 7 5 % ) were used. T h e y were i n j e c t e d s.c. w i t h e i t h e r
1 m g of c o r t e x o l o n e / 1 0 0 g ( K o c h - L i g h t L a b o r a t o r i e s dissolved in 0.5 m l of s u n f l o w e r oil (Groups 2 a n d 4 in t h e Table), or 0.5 m l oil/100 g (Groups l a n d 3 in t h e Table). 1 I(. M. MOSHER, D. A. YOUNG and A. MUNCK, J. biol. Chem. 246, 654 (1971). A. 5~UNCK and T. BRINCK-JOHNSEN, J. biol. Chem. 243, 5556
(196s). a N. KAISER, R. J. MILtfOLLAND, R. W. TURNEL and F. ROSEN', Biochem. biophys. Res. Comnmn. 49, 516 (1972). a Zs. Acs, u n p u b l i s h e d data. .5 B. S. McEwI~N, J. M. W*=iss and L. S. SCUWARTZ, B r a i u Res. 16,
227 (1969). , B. I. GROSSBR,W. STEVENS, F. W. BRU~NGERand D. J. REED, J. Neurochem. 18, 1725 (1971). 7 H. KmTZLBY, J. Neuroehem. 19, 2737 (1972). s E. STARK, Zs. Acs, M. PALKOVITS and G. FOLLY, Acta Physiol. Aead. Sci. hung. in press (1974).
Effect of cortexolone (1 rag/100 g) on the stress-reaction of r a t s t r e a t e d w i t h 50 ~tg/100 g d e x a m e t h a s o n e Group 1
Group 2
Group 3
Group 4
Subcutaneous injection
oil
cortexolone
oil
cortexolone
Intraperitoneal injection Restingprestress level
saline 12.61 ~ 1.74 (9)
saline 9.98 -L 2.08 (10)
dexamethasone 3.22 zt= 0.72 ~ (10)
dexamethasone 4.48 ~= 1.46~ (10)
Increment induced by
16.03 =t= 3.86
14.90 =~ 2.18
6.55 4- 1.56 9
14.69 ~ 1.90
histamine stress
(9)
(10)
(10)
(10)
Mean ~ S E ~ in ~xg corticosterone[100 ml plasma. ~significantly different (p ~ 0.01) from its respective control group e v a l u a t e d b y the m e t h o d of analysis of v a r i a n c e for two-way layout. N u m b e r s in parentheses denote n u m b e r of d e t e r m i n a t i o n s .
Specialia
1366
2 h later, t h e a n i m a l s received i.p. e i t h e r 50 ~zg of d e x a m e t h a s o n e / 1 0 0 g (Oradexon, O r g a n o n ) (Groups 3 a n d 4), or 0.2 m l of physiological saline/100 g (Groups 1 a n d 2). 2 h a f t e r t h e i.p. injection, 1 m l of b l o o d was w i t h d r a w n f r o m t h e s a p h e n o u s vein, u n d e r l i g h t e t h e r a n a e s t h e s i a , w i t h i n 2 m i n of r e m o v i n g t h e a n i m a l f r o m its cage. H i s t a m i n e (1 rng/100 g) was i n j e c t e d i.p., followed b y b l o o d w i t h d r a w a l f r o m t h e a b d o m i n a l a o r t a 30 m i n later. P r e - a n d a f t e r - s t r e s s p l a s m a c o r t i c o s t e r o n e was d e t e r m i n e d b y t h e c o m p e t i t i v e p r o t e i n - b i n d i n g m e t h o d 9. C o r t e x o l o n e d i d n o t i n t e r f e r e w i t h c o r t i c o s t e r o n e d e t e r m i n a t i o n , since 3 h a f t e r a d m i n i s t e r i n g s.c. t h e a b o v e dose of c o r t e x o l o n e t o r a t s a d r e n a l e c t o m i z e d 24 h earlier p l a s m a c o r t i c o s t e r o n e e q u a l l e d t h a t in t h e a d r e n a l e c t o m i z e d c o n t r o l s (0.57 0.08 ~g/100 m l v.s. 0.54 d2 0.05 ~g/100 m l ; n u m b e r of d e t e r m i n a t i o n s is 9 in b o t h groups). Results and discussion. D e x a m e t h a s o n e a d m i n i s t r a t i o n lowered t h e r e s t i n g p l a s m a c o r t i c o s t e r o n e level (Table). A f t e r h i s t a m i n e , c o r t i c o s t e r o n e level in t h e d e x a m e t h a s o n e - t r e a t e d r a t s rose, t h e i n c r e m e n t b e i n g signifi c a n t l y s m a l l e r t h a n in t h e c o n t r o l s (Table). C o r t e x o l o n e e x e r t e d n o effect o n t h e stress r e a c t i o n of r a t s (Group 2 in t h e Table). T h i s f i n d i n g seems to b e a t v a r i a n c e w i t h t h a t of JoNEs et al. 19, a difference p r o b a b l y d u e t o t h e f a c t t h a t we a d m i n i s t e r e d c o r t e x o l o n e in 10 t i m e s lower doses t h a n t h e y did. Cortexolone administration prior to dexamethasone did n o t p r e v e n t t h e d e c r e a s e in t h e r e s t i n g c o r t i c o s t e r o n e level (Group 4 in t h e Table), b u t i n t h e s e a n i m a l s h i s t a m i n e stress i n d u c e d a n i n c r e m e n t in p l a s m a c o r t i c o s t e r o n e s i m i l a r t o t h a t in t h e v e h i c l e - t r e a t e d c o n t r o l s (v.s. G r o u p 4 a n d G r o u p 1 in t h e Table). O u r i n t e r p r e t a t i o n of t h e s e d a t a is t h a t c o r t e x o l o n e p r o b a b l y displaces d e x a m e t h a s o n e f r o m t h e h y p o t h a l a m i c a n d / o r e x t r a h y p o t h a l a m i c c o r t i c o s t e r o i d recept o r sites, a n d t h u s p r e v e n t s d e x a m e t h a s o n e f r o m i n h i b i t i n g
E f f e c t of P r o g e s t e r o n e
on Human
EXPERIENTIA31/11
s t r e s s - i n d u c e d A C T H release. T h e d a t a f a v o r t h e a s s u m p tion that the corticosteroid feedback action depends on t h e b i n d i n g of t h e h o r m o n e t o specific r e c e p t o r s in t h e t a r g e t cells, t h e m e c h a n i s m Of f e e d b a c k a c t i o n b e i n g in t h i s r e s p e c t s i m i l a r t o t h a t of o t h e r w e l l - k n o w n c o r t i c o s t e r o i d effects. I t was s u g g e s t e d n, 12 t h a t t h e m e c h a nisms controlling ACTH-release under non-stress and stress c o n d i t i o n s are f u n c t i o n a l l y dissociable. O u r pres e n t d a t a seem t o s u p p o r t t h i s a s s u m p t i o n b y i n d i c a t i n g t h a t t h e site a n d / o r t h e m e c h a n i s m of d e x a m e t h a s o n e s u p p r e s s i o n of r e s t i n g A C T H s e c r e t i o n are d i s t i n c t f r o m t h o s e d e p r e s s i n g stress i n d u c e d A C T H release.
Summary. C o r t e x o l o n e i n a dose of 1 mg/100 g b o d y wt., a d m i n i s t e r e d t o r a t s p r i o r t o d e x a m e t h a s o n e , pre-: vented dexamethasone from suppressing stress-induced A C T H - r e l e a s e w i t h o u t i n t e r f e r i n g w i t h t h e effect of d e x a m e t h a s o n e on t h e r e s t i n g p l a s m a c o r t i c o s t e r o n e level. ZSUZSANNA fikCS and E. STARK13
Institute o/Experimental Medicine, Hungarian Academy o/Sciences, -P.O. Box 67, H - I J 5 0 Budapest (Hungary), 23 June 7975.
9 B. E. P. MURPHY,J. elin. Endocr. Metab. 27, 973 (1967). 10 M. T. JONES, E. 1V[.TIPTAFT, t 2. R. BRUStt, D. A. N. FERGUSONand R. L. B. NEAME,J. Endocr. 60, 223 (1974). 11 E. ZIMMERMANand V. CRITCHLOW,Am. J. Physiol. 216, 148 (1969). IS j. R. HODGES, Progr. Brain Res. 32, 12 (1970). 18 Acknowledgements. Authors wish to thank Mr. G. FOLLY for the mathematical analyses and Mrs. M. PONGR$,CZfor her valuable technical assistance.
Corticosteroid 21-Hydroxylation
T h e b i o c h e m i c a l b a s i s of t h e g e n e t i c a l l y i n h e r i t e d m e t a b o l i c d i s o r d e r s k n o w n as t h e a d r e n o g e n i t a l s y n d r o m e 1, 2 is as y e t n o t fully u n d e r s t o o d . W h i l e t w o forms, t h e 'salt-losing' a n d t h e ' n o n - s a l t - l o s i n g ' varieties, are k n o w n , b o t h of w h i c h a p p e a r t o r e s u l t f r o m d e f e c t i v e a d r e n a l 2 1 - h y d r o x y l a t i o n c a p a b i l i t y , i t is n o t y e t k n o w n w h e t h e r t h e s e r e p r e s e n t a single or t w o genetic defects 3, ~. R e s e a r c h e s o n t h e s e possibilities h a v e r e c e n t l y b e e n r e v i e w e d elsewhere 5. T h e p r e s e n t a c c o u n t r e p o r t s t h e r e s u l t s of e x p e r i m e n t s t o i n v e s t i g a t e w h e t h e r i n d e p e n d e n t a c t i v e sites for t h e 2 1 - h y d r o x y l a t i o n 6 of p r o g e s t e r o n e t o 1 1 - d e o x y c o r t i c o s t e r o n e (DOC) a n d of 17 e - h y d r o x y progesterone to 11-deoxycortisol (Reichstein's compound S) are p r e s e n t in h u m a n a d r e n o c o r t i c a l m i c r o s o m a l p r o t e i n . T h i s i n v e s t i g a t i o n f o r m e d p a r t of a c o m m u n i c a t i o n ~ p r e s e n t e d a t t h e m e e t i n g of t h e S o u t h e r n S o c i e t y for P e d i a t r i c R e s e a r c h in 1974. T w o n o r m a l h u m a n a d r e n a l s were o b t a i n e d f r e s h p o s t m o r t e m a n d t h e e x t e r n a l f a t a n d c o n n e c t i v e t i s s u e were t r i m m e d off. T h e w e i g h t of t h e 2 g l a n d s was 7.07 g. T h e y were c u t i n t o s m a l l pieces in a b o u t 100 m l cold 0.25 M sucrose. T h e s u s p e n s i o n was h o m o g e n i z e d u s i n g a m o t o r d r i v e n glass-teflon h o m o g e n i z e r a n d c e n t r i f u g e d a t 600 g for 10 m i n t o r e m o v e n u c l e a r c o m p o n e n t s , u n b r o k e n cells a n d u n w a n t e d d e b r i s s. T h e s u p e r n a t a n t w a s c e n t r i fuged a t 8,720 g for 10 m i n t o s e p a r a t e t h e h e a v y m i t o c h o n d r i a a n d t w i c e a t 26,700 g for 30 rain t o r e m o v e l i g h t m i t o c h o n d r i a . T h e m i c r o s o m e s were i s o l a t e d f r o m t h e
s u p e r n a t a n t b y c e n t r i f u g a t i o n a t 78,500 g for 90 m i n . T h e y were g e n t l y h a n d - h o m o g e n i z e d in 1-2 m l Tris-HC1, 50 r a M , p H 7.45 c o n t a i n i n g 3 m M MgC12 a n d t h e p r o t e i n concentration determined by the biuret method. Determ i n a t i o n of t h e c y t o c h r o m e P 4 5 0 c o n t e n t of t h e m i c r o s o m a l p r o t e i n was p e r f o r m e d f r o m t h e o p t i c a l e x t i n c t i o n a t 450 n m a f t e r t r e a t m e n t w i t h d i t h i o n i t e a n d c a r b o n m o n o x i d e 9. I n c u b a t i o n s were p e r f o r m e d for 5 m i n w i t h a g i t a t i o n in a D u b n o f f i n c u b a t o r m a i n t a i n e d a t 37~ The reaction m i x t u r e s c o n t a i n e d m i c r o s o m a l e n z y m e (1.7 m g p r o t e i n ) ; t r i s o d i u m isocitrate, 11 v m o l e s ( S i g m a ) ; p i g h e a r t iso-
1 I{. S. HOLT and D. N. EAItCE, Basic Concepts o] Inborn Errors and Dejects o] Steroid Biosynthesis (E. and S. Livingstone, Edinburgh 1966). 2 D. B. VILLEE, Human Endocrinology, a Developmental Approach (Saunders, Philadelphia 1975). 8 F. C. BARTTER, Arch. Dis. Childhood 44, 138 (1969). G. T. BRYAN, B. KLIMAN and 12. C. BARTTER, J. elin. Invest. 44, 957 (1965). R, H. WlCKRA~tASlNGHE,Enzyme 19, 348 (1975). s K. J. RYAN and L. L. ENOEL, J. Am. chem. Soe. 78, 2654 (1956). E. B. NELSON, R. H. WICKRAMASINGHEand G. T. BRYAN, Clin. Res. 22, 79A (1974). s R. H. WICKRAMASINGHE,Int. j. Peptide Protein Res. 6, 187 (1974). 9 T. OMURAand R. SATO, J. biol. Chem. 239, 2379 (1964).
