- Email: [email protected]
Puromycin inhibition of in vitro cortical hormone production by the rat adrenal gland
539
P U R O M Y C I N INHIBITION O F IN V I T R O C O R T I C A L H O R M O N E P R O D U C T I O N BY T H E RAT A D R E N A L GLAND G e o r g e W. K i t t i n g e r 1 May Institute for Medical R e s e a r c h , Jewish Hospital of C i n c i n n a t i and D e p a r t m e n t of B i o l o g i c a l C h e m i s t r y , U n i v e r s i t y of C i n c i n n a t i C o l l e g e of M e d i c i n e , C i n c i n n a t i , Ohio
Received August 10, 1964 ABSTRACT S t u d i e s w e r e m a d e of the i n h i b i t o r y e f f e c t s of p u r o m y c i n on in v i t r o r a t a d r e n a l c o r t i c a l h o r m o n e p r o d u c t i o n . P u r o m y c i n ( 7 . 5 x 10-4M) ~-gnific a n t l y r e d u c e d the p r o d u c t i o n of a l d o s t e r o n e , c o r t i c o s t e r o n e and d e o x y c o r t i c o s t e r o n e b y glands t h a t w e r e not s t i m u l a t e d b y A C T H . The p r o d u c t i o n of 1 8 - h y d r o x y d e o x y c o r t i c o s t e r o n e w a s not a f f e c t e d u n d e r t h e s e s a m e c o n d i t i o n s In i n c u b a t i o n s containing a d d e d A C T H , p u r o m y c i n p r e v e n t e d the A C T H i n d u c e d i n c r e a s e s in c o r t i c o s t e r o n e and 1 8 - h y d r o x y d e o x y c o r t i c o s t e r one o b s e r v e d in the c o n t r o l s . In c o n t r a s t , p u r o m y c i n inhibition of a l d o s t e r one p r o d u c t i o n a p p e a r e d to be i n d e p e n d e n t of the e f f e c t s of A C T H . It is c o n c l u d e d that p u r o m y c i n inhibits b i o s y n t h e t i c e v e n t s in the a d r e n a l gland o t h e r than t h o s e s e n s i t i v e to A C T H s t i m u l a t i o n and that t h e s e o t h e r p r o c e s s e s m a y be i m p o r t a n t in the r e g u l a t i o n of the r a t e of a l d o s t e r o n e and c o r t i costerone biosynthesis. INT RODUC TION F e r g u s o n h a s shown that p u r o m y c i n , 9 - ( 3 - D e o x y - 3 - ( ~ - m e t h o x y L-phenylalanylamino) ~-D- ribofuranosyl))-6-dimethylamino/-
p u r i n e , will
inhibit s t i m u l a t i o n of a d r e n o c o r t i c a l h o r m o n e p r o d u c t i o n b y a d r e n o c o r t i c o t r o p h i n (ACTH).
I n c u b a t i o n of r a t o r b o v i n e a d r e n a l s l i c e s with A C T H
c a u s e d an i n c r e a s e in the p r o d u c t i o n of b l u e t e t r a z o l i u m p o s i t i v e s t e r o i d s , but no i n c r e a s e in the i n c o r p o r a t i o n of l e u c i n e - 1 4 C into a d r e n a l p r o t e i n . B o t h of t h e s e p r o c e s s e s ,
ACTH
i . e . the i n c o r p o r a t i o n of l e u c i n e - 1 4 C and the
stimulation of blue tetrazolium positive steroid production, were
540
S T E R 0 I D S
s t r o n g l y inhibited b y p u r o m y c i n 2' 3.
Ferguson
4:4
suggested that these
r e s u l t s s u p p o r t e d t h e c o n c e p t t h a t c o n c o m i t a n t d e n o v o s y n t h e s i s of p r o t e i n m a y b e an e s s e n t i a l p a r t of t h e m e c h a n i s m
by which ACTH stimulates corti-
costeroidogenesis. T h e a n a l y t i c a l m e t h o d s 2' 3 e m p l o y e d b y F e r g u s o n a r e n o t s u f f i c i e n t l y s p e c i f i c t o b e u s e d to s t u d y t h e e f f e c t s of p u r o m y c i n on t h e p r o d u c t i o n r a t e s of i n d i v i d u a l c o r t i c a l s t e r o i d s .
The recent development
of a m o r e s p e c i f i c q u a n t i t a t i v e g a s c h r o m a t o g r a p h i c m a k e s it p o s s i b l e t o s t u d y t h e s e e f f e c t s in g r e a t e r The data presented
method by Kittinger d e t a i l 5.
in t h i s p a p e r d e m o n s t r a t e
that puromycin
s t r o n g l y i n h i b i t s t h e p r o d u c t i o n of a l d o s t e r o n e a n d o t h e r s p e c i f i c c o r t i c a l hormones. presence
T h e s e i n h i b i t o r y e f f e c t s h a v e b e e n s h o w n t o o c c u r b o t h in t h e a n d a b s e n c e of A C T H s t i m u l a t i o n .
MATERIALS AND METHODS A d r e n a l g l a n d s w e r e o b t a i n e d f r o m 235 _+ 5 g v i r g i n f e m a l e r a t s of t h e S p r a g u e - D a w l e y s t r a i n . The animals were killed by decapitation a n d t h e i r a d r e n a l s w e r e i m m e d i a t e l y r e m o v e d a n d t r i m m e d f r e e of extraneous tissue. T h e g l a n d s w e r e t h e n h a l v e d and s t o r e d in i c e d n o r m a l s a l i n e p r i o r t o u s e . E a c h i n c u b a t i o n f l a s k c o n t a i n e d t h e a d r e n a l g l a n d s of t w o r a t s ; t h e p o o l e d t i s s u e w e i g h e d a p p r o x i m a t e l y 125 m g . T h e i n c u b a t i o n s w e r e p e r f o r m e d u n d e r an a t m o s p h e r e of 95% 0 2 and 5% C O 2 a c c o r d i n g t o t h e m e t h o d s of S a f f r a n a n d S c h a l l y 4. D u r i n g t h e p r e i n c u b a t i o n and final incubation each c o n t r o l flask contained a d r e n a l t i s s u e and 5.0 m l of K r e b s - R i n g e r - b i c a r b o n a t e - g l u c o s e m e d i u m , pH 7 . 4 . T h e c o n t e n t s of the e x p e r i m e n t a l f l a s k s w e r e identical to the c o n t r o l s e x c e p t that they c o n t a i n e d , in a d d i t i o n , 7 . 5 x 10 4M p u r o m y c i n d u r i n g t h e p r e i n c u b a t i o n and f i n a l i n c u b a t i o n p e r i o d s . T h e p r e i n c u b a t i o n p e r i o d w a s one h o u r . T h e m e d i u m w a s t h e n d i s c a r d e d and r e p l a c e d b y f r e s h m e d i u m of t h e s a m e c o m p o s i t i o n . T h e i n c u b a t i o n of t h e t i s s u e w a s t h e n c o n t i n u e d u n d e r t h e s a m e c o n d i t i o n s f o r t h r e e h o u r s . W h e r e i n d i c a t e d , 400 m U of p o r c i n e A C T H w e r e a d d e d t o t h e c o n t r o l and e x p e r i m e n t a l f l a s k s at t h e b e g i n n i n g of t h e f i r s t , s e c o n d a n d t h i r d h o u r of i n c u b a t i o n .
Oct. 1 9 6 4
S T E R O I D S
541
The m e d i u m f r o m each incubation was decanted into a ground g l a s s s t o p p e r e d c e n t r i f u g e t u b e . T h e t i s s u e s w e r e w a s h e d with a s m a l l v o l u m e of s a l i n e a n d t h e w a s h i n g s c o m b i n e d with t h e m e d i u m . T h e adrenal tissue was discarded. The combined fluids were eXtracted t h r e e t i m e s w i t h 2 m l . of d i c h l o r o m e t h a n e u s i n g c e n t r i f u g a t i o n to s e p a r a t e t h e s o l v e n t l a y e r s . Q u a n t i t a t i v e d e t e r m i n a t i o n s of t h e i n d i v i d u a l s t e r o i d s in t h e e x t r a c t s w e r e c a r r i e d out b y m e t h o d s p r e v i o u s l y d e s c r i b e d 5. R E S U L T S AND DISCUSSION The results obtained from these experiments are summarized in T a b l e 1.
When the glands w e r e s t i m u l a t e d by ACTH, p u r o m y c i n at a
c o n c e n t r a t i o n of 7 . 5 x 1 0 - 4 M s t r o n g l y i n h i b i t e d t h e p r o d u c t i o n of t o t a l s t e r o i d s a s w e l l a s b l u e t e t r a z o l i u m p o s i t i v e s t e r o i d s 6.
It is a p p a r e n t
t h a t t h e o b s e r v e d d e c r e a s e in s t e r o i d p r o d u c t i o n is l a r g e l y a c c o u n t e d f o r b y a m a r k e d d e c r e a s e in c o r t i c o s t e r o n e ( p r e g n - 4 - e n e - 11/~, 2 1 - d i o l - 3, 20-dione). F e r g u s o n 3.
T h e s e r e s u l t s a r e in a g r e e m e n t with t h o s e r e p o r t e d b y H o w e v e r , in c o n t r a s t t o t h e d a t a of F e r g u s o n ,
the r e s u l t s
in T a b l e 1 d e m o n s t r a t e t h a t a s i g n i f i c a n t i n h i b i t i o n of b l u e t e t r a z o l i u m p o s i t i v e s t e r o i d p r o d u c t i o n c a n o c c u r e v e n in t h e a b s e n c e of A C T H stimulation.
It s e e m s l i k e l y t h a t t h i s e f f e c t w a s not n o t e d in F e r g u s o n ' s
s t u d y b e c a u s e of t h e c o n s i d e r a b l y s h o r t e r t i m e s of i n c u b a t i o n , i . e . , 30 a n d 90 m i n u t e s . A s s u g g e s t e d by F e r g u s o n 3 t h e s e d a t a a r e c o n s i s t e n t with t h e h y p o t h e s i s t h a t r a t e s of c o r t i c o s t e r o n e p r o d u c t i o n b y t h e a d r e n a l g l a n d a r e p a r t i a l l y c o n t r o l l e d by p r o c e s s e s w h i c h i n v o l v e de n o v o s y n t h e s i s of one o r m o r e p r o t e i n s .
If t h i s i s c o r r e c t ,
then
p a r t of t h e de n o v o s y n t h e s i s a p p e a r s to be i n d e p e n d e n t of t h e e f f e c t s of A C T H s t i m u l a t i o n .
542
S T E R O I D S
4:4
Table 1 In V i t r o E f f e c t s of P u r o m y c i n ( 7 . 5 x 10-4M) on C o r t i c o s t e r o i d P r o d u c t i o n ~lg/hr/100 m g a d r e n a l t i s s u e No A C T H A d d e d No. Replicates
ALD
18-OH-DOC
B
DO(]
B.T.
Control
7
1.41+. 19"
1.87+. 12
3.60±. 30
0.60+.06
7.51+.28
Experimental
8
0.29±.04
1.70+.23
1.78+.27
0.11+.06
4.29+.26
.001
N.S.
.005
.001
.005
P (t T e s t ) ~f
T.S. 11.70±.50 9.22+1.45 N.S.
ACTH Stimulated Control
5
1.35±.23
3.45±.25
I0.88±.90
1.50±.41
15.424-.64
19.97±.57
Experimental
5
0 . 4 4 ~ . 15
1.66±.30
3.14±.38
0.0 ±.13
5.26±.43
8.24±.7
.005
.001
.001
.001
P (t T e s t ) <
.01
.~
Tetrazolium Positive Steroids Steroids N . S . = Not Significant means + standard errors
= Aldosterone Corticosterone = Deoxyeorticosterone 18-OH-DOC = 18- hydroxydeoxycorticost erone
B.T. T.S.
ALD
B = DOC
= Blue = Total
In a s i m i l a r w a y , the data in T a b l e 1 d e m o n s t r a t e that deoxycorticosterone
( p r e g n - 4- e n e - 21 - o i - 3, 20 - dione) p r o d u c t i o n i s
a l s o i n h i b i t e d by p u r o m y c i n and that o n l y part of t h i s e f f e c t i s due to i n h i b i t i o n of a p r o c e s s w h i c h i s c a p a b l e of b e i n g s t i m u l a t e d by ACTH.
Since deoxycorticosterone
h a s b e e n d e m o n s t r a t e d to be an
e f f e c t i v e p r e c u r s o r of c o r t i c o s t e r o n e 7, the e f f e c t s of p u r o m y c i n on the s y n t h e s i s of t h e s e s t e r o i d s m a y be r e l a t e d and due to i n h i b i t i o n of the f o r m a t i o n of an e a r l i e r i n t e r m e d i a t e on a c o m m o n b i o s y n t h e t i c pathway. C o n t r o l data in T a b l e 1 a r e in a g r e e m e n t w i t h the r e s u l t s of o t h e r s 8 w h o h a v e s h o w n that A C T H s i g n i f i c a n t l y i n c r e a s e s the p r o d u c t i o n of 1 8 - h y d r o x y d e o x y c o r t i c o s t e r o n e 2 0 - d i o n e ) by rat a d r e n a l s .
( p r e g n - 4 - e n e - 18, 2 1 - d i o l - 3,
T h e data o b t a i n e d in t h i s i n v e s t i g a t i o n
Oct. 1 9 6 4
S T E R O I D S
543
d e m o n s t r a t e , in a d d i t i o n , that t h i s e f f e c t of A C T H i s s i g n i f i c a n t l y inhibited by p u r o m y c i n .
It is of i n t e r e s t t h a t inhibition of 1 8 - h y d r o x y -
d e o x y c o r t i c o s t e r o n e p r o d u c t i o n b y p u r o m y c i n w a s not o b s e r v e d in the a b s e n c e of A C T H s t i m u l a t i o n ,
s i n c e p r o d u c t i o n r a t e s of d e o x y c o r t i c o -
sterone were significantly decreased under these conditions.
This
o b s e r v a t i o n d o e s not s u p p o r t t h e h y p o t h e s i s that d e o x y c o r t i c o s t e r o n e is a m a j o r p r e c u r s o r of i t s 1 8 - h y d r o x y l a t e d d e r i v a t i v e .
Independent
e x p e r i m e n t s in o u r l a b o r a t o r y h a v e shown that e x o g e n e o u s 1 8 - h y d r o x y p r o g e s t e r o n e c a n be e f f i c i e n t l y c o n v e r t e d to 1 8 - h y d r o x y d e o x y c o r t i c o s t e r o n e b y a d r e n a l s l i c e s 9.
T h e s e c o n s i d e r a t i o n s s u g g e s t the
possibility that 1 8 - h y d r o x y d e o x y c o r t i c o s t e r o n e m a y be f o r m e d f r o m p r o g e s t e r o n e b y s e q u e n t i a l h y d r o x y l a t i o n s at C - 1 8 and C - 2 1 , in that order.
Since A C T H h a s b e e n found to inhibit the c o n v e r s i o n of
e x o g e n o u s 1 8 - h y d r o x y p r o g e s t e r o n e into 1 8 - h y d r o x y d e o x y c o r t i c o s t e r one 10 it a p p e a r s l i k e l y that the A C T H i n d u c e d s t i m u l a t i o n of the p r o d u c t i o n of t h i s s t e r o i d is e f f e c t e d at an e a r l i e r s t a g e in the b i o synthetic pathway. In T a b l e 1 it c a n b e s e e n that s t i m u l a t i o n of a l d o s t e r o n e p r o d u c t i o n w a s not o b s e r v e d when A C T H w a s p r e s e n t in the i n c u b a t i o n s and t h a t p u r o m y c i n i n h i b i t e d a l d o s t e r o n e p r o d u c t i o n to a b o u t the s a m e extent in both the p r e s e n c e and a b s e n c e of A C T H . On the b a s i s of t h e s e data, the inhibition of a l d o s t e r o n e p r o d u c t i o n b y p u r o m y c i n a p p e a r s to b e i n d e p e n d e n t of any A C T H e f f e c t . T h e m e c h a n i s m s r e g u l a t i n g the in v i t r o p r o d u c t i o n of
544
S T E R O I D S
4:4
a l d o s t e r o n e b y t h e r a t a d r e n a l r e m a i n o b s c u r e 11.
T h e a d d i t i o n of
a n g i o t e n s i n to in v i t r o s y s t e m s h a s b e e n r e p o r t e d to b e w i t h o u t e f f e c t on a l d o s t e r o n e p r o d u c t i o n 12.
We h a v e o b t a i n e d s i m i l a r
resultsl0.
The
d a t a p r e s e n t e d h e r e s u g g e s t t h a t t h e r e g u l a t i o n of a l d o s t e r o n e p r o d u c tion b y t h e r a t a d r e n a l m a y d e p e n d in s o m e u n k n o w n w a y u p o n de n o v o s y n t h e s i s of p r o t e i n s i n c e p u r o m y c i n is b e l i e v e d to b e a s p e c i f i c i n h i b i t o r of p r o t e i n s y n t h e s i s 13 It is c l e a r t h a t a g r e a t e r u n d e r s t a n d i n g aldosterone regulatory mechanisms f u r t h e r s t u d i e s on t h e m e c h a n i s m s
is required.
of t h e d e t a i l s of It is h o p e d t h a t
of p u r o m y c i n i n h i b i t i o n of a l d o s t e r -
one p r o d u c t i o n w i l l p r o v i d e e v i d e n c e a l o n g t h e s e l i n e s .
ACKNOWLEDGEMENTS This investigation was supported by a research f r o m the National S c i e n c e F o u n d a t i o n .
grant (G-23686)
Computor facilities were provided by the Medical Computing Center which is supported in part by National Institutes of Health grant FR-O0010. Mrs. Joan Schreiber contributed valuable technical assistance during this investigation.
REFERENCES
.
T h i s w o r k w a s d o n e d u r i n g t h e t e n u r e of an A d v a n c e d R e s e a r c h Fellowship from the American Heart Association.
.
Ferguson, J.J., 618 (1962).
Jr.,
BIOCHIM. ET BIOPHYS. ACTA,
Ferguson,
Jr.,
J . B I O L . C H E M . 238, 2754 (1963}.
3.
J.J.,
57,
Oct. 1964
.
o
6.
o
8.
.
Saffran, (1955). Kittinger,
S T E R 0 I D S
M.,
and Schaily, A.V.,
G.W.,
STEROIDS
545
ENDOCRINOLOGY
56, 523
3, 21 (1964).
The terms "total steroids" and "blue tetrazolium are operationally defined in reference 5.
positive steroids"
Wettstein, A . , EXPERIENTIA 17, 329 (1961). C o r t e s , J . M . , P e r o n , F . G . , and Dorfman, R . I . , ENDOCRINOLOGY 73, 713 (1963). Kittinger, G . W . , PROGRAM OF THE ENDOCRINE SOCIETY, San F r a n c i s c o , 1964.
i0.
Kittinger, G . W . , Unpublished o b s e r v a t i o n s .
ii.
Singer, B . , Losito, C . , and Salmon, S., ENDOCRINOLOGY 74, 325 (1964).
12.
Kaplan, N . M . , and B a r t t e r , F . C . , (1962).
J . CLIN. INVEST. 41, 715
13.
Yarmolinsky, M., and De La Haba, SCI. U.S., 45, 1721 (1959).
G.,
PROC.
NATL.
ACAD.
P U R O M Y C I N INHIBITION O F IN V I T R O C O R T I C A L H O R M O N E P R O D U C T I O N BY T H E RAT A D R E N A L GLAND G e o r g e W. K i t t i n g e r 1 May Institute for Medical R e s e a r c h , Jewish Hospital of C i n c i n n a t i and D e p a r t m e n t of B i o l o g i c a l C h e m i s t r y , U n i v e r s i t y of C i n c i n n a t i C o l l e g e of M e d i c i n e , C i n c i n n a t i , Ohio
Received August 10, 1964 ABSTRACT S t u d i e s w e r e m a d e of the i n h i b i t o r y e f f e c t s of p u r o m y c i n on in v i t r o r a t a d r e n a l c o r t i c a l h o r m o n e p r o d u c t i o n . P u r o m y c i n ( 7 . 5 x 10-4M) ~-gnific a n t l y r e d u c e d the p r o d u c t i o n of a l d o s t e r o n e , c o r t i c o s t e r o n e and d e o x y c o r t i c o s t e r o n e b y glands t h a t w e r e not s t i m u l a t e d b y A C T H . The p r o d u c t i o n of 1 8 - h y d r o x y d e o x y c o r t i c o s t e r o n e w a s not a f f e c t e d u n d e r t h e s e s a m e c o n d i t i o n s In i n c u b a t i o n s containing a d d e d A C T H , p u r o m y c i n p r e v e n t e d the A C T H i n d u c e d i n c r e a s e s in c o r t i c o s t e r o n e and 1 8 - h y d r o x y d e o x y c o r t i c o s t e r one o b s e r v e d in the c o n t r o l s . In c o n t r a s t , p u r o m y c i n inhibition of a l d o s t e r one p r o d u c t i o n a p p e a r e d to be i n d e p e n d e n t of the e f f e c t s of A C T H . It is c o n c l u d e d that p u r o m y c i n inhibits b i o s y n t h e t i c e v e n t s in the a d r e n a l gland o t h e r than t h o s e s e n s i t i v e to A C T H s t i m u l a t i o n and that t h e s e o t h e r p r o c e s s e s m a y be i m p o r t a n t in the r e g u l a t i o n of the r a t e of a l d o s t e r o n e and c o r t i costerone biosynthesis. INT RODUC TION F e r g u s o n h a s shown that p u r o m y c i n , 9 - ( 3 - D e o x y - 3 - ( ~ - m e t h o x y L-phenylalanylamino) ~-D- ribofuranosyl))-6-dimethylamino/-
p u r i n e , will
inhibit s t i m u l a t i o n of a d r e n o c o r t i c a l h o r m o n e p r o d u c t i o n b y a d r e n o c o r t i c o t r o p h i n (ACTH).
I n c u b a t i o n of r a t o r b o v i n e a d r e n a l s l i c e s with A C T H
c a u s e d an i n c r e a s e in the p r o d u c t i o n of b l u e t e t r a z o l i u m p o s i t i v e s t e r o i d s , but no i n c r e a s e in the i n c o r p o r a t i o n of l e u c i n e - 1 4 C into a d r e n a l p r o t e i n . B o t h of t h e s e p r o c e s s e s ,
ACTH
i . e . the i n c o r p o r a t i o n of l e u c i n e - 1 4 C and the
stimulation of blue tetrazolium positive steroid production, were
540
S T E R 0 I D S
s t r o n g l y inhibited b y p u r o m y c i n 2' 3.
Ferguson
4:4
suggested that these
r e s u l t s s u p p o r t e d t h e c o n c e p t t h a t c o n c o m i t a n t d e n o v o s y n t h e s i s of p r o t e i n m a y b e an e s s e n t i a l p a r t of t h e m e c h a n i s m
by which ACTH stimulates corti-
costeroidogenesis. T h e a n a l y t i c a l m e t h o d s 2' 3 e m p l o y e d b y F e r g u s o n a r e n o t s u f f i c i e n t l y s p e c i f i c t o b e u s e d to s t u d y t h e e f f e c t s of p u r o m y c i n on t h e p r o d u c t i o n r a t e s of i n d i v i d u a l c o r t i c a l s t e r o i d s .
The recent development
of a m o r e s p e c i f i c q u a n t i t a t i v e g a s c h r o m a t o g r a p h i c m a k e s it p o s s i b l e t o s t u d y t h e s e e f f e c t s in g r e a t e r The data presented
method by Kittinger d e t a i l 5.
in t h i s p a p e r d e m o n s t r a t e
that puromycin
s t r o n g l y i n h i b i t s t h e p r o d u c t i o n of a l d o s t e r o n e a n d o t h e r s p e c i f i c c o r t i c a l hormones. presence
T h e s e i n h i b i t o r y e f f e c t s h a v e b e e n s h o w n t o o c c u r b o t h in t h e a n d a b s e n c e of A C T H s t i m u l a t i o n .
MATERIALS AND METHODS A d r e n a l g l a n d s w e r e o b t a i n e d f r o m 235 _+ 5 g v i r g i n f e m a l e r a t s of t h e S p r a g u e - D a w l e y s t r a i n . The animals were killed by decapitation a n d t h e i r a d r e n a l s w e r e i m m e d i a t e l y r e m o v e d a n d t r i m m e d f r e e of extraneous tissue. T h e g l a n d s w e r e t h e n h a l v e d and s t o r e d in i c e d n o r m a l s a l i n e p r i o r t o u s e . E a c h i n c u b a t i o n f l a s k c o n t a i n e d t h e a d r e n a l g l a n d s of t w o r a t s ; t h e p o o l e d t i s s u e w e i g h e d a p p r o x i m a t e l y 125 m g . T h e i n c u b a t i o n s w e r e p e r f o r m e d u n d e r an a t m o s p h e r e of 95% 0 2 and 5% C O 2 a c c o r d i n g t o t h e m e t h o d s of S a f f r a n a n d S c h a l l y 4. D u r i n g t h e p r e i n c u b a t i o n and final incubation each c o n t r o l flask contained a d r e n a l t i s s u e and 5.0 m l of K r e b s - R i n g e r - b i c a r b o n a t e - g l u c o s e m e d i u m , pH 7 . 4 . T h e c o n t e n t s of the e x p e r i m e n t a l f l a s k s w e r e identical to the c o n t r o l s e x c e p t that they c o n t a i n e d , in a d d i t i o n , 7 . 5 x 10 4M p u r o m y c i n d u r i n g t h e p r e i n c u b a t i o n and f i n a l i n c u b a t i o n p e r i o d s . T h e p r e i n c u b a t i o n p e r i o d w a s one h o u r . T h e m e d i u m w a s t h e n d i s c a r d e d and r e p l a c e d b y f r e s h m e d i u m of t h e s a m e c o m p o s i t i o n . T h e i n c u b a t i o n of t h e t i s s u e w a s t h e n c o n t i n u e d u n d e r t h e s a m e c o n d i t i o n s f o r t h r e e h o u r s . W h e r e i n d i c a t e d , 400 m U of p o r c i n e A C T H w e r e a d d e d t o t h e c o n t r o l and e x p e r i m e n t a l f l a s k s at t h e b e g i n n i n g of t h e f i r s t , s e c o n d a n d t h i r d h o u r of i n c u b a t i o n .
Oct. 1 9 6 4
S T E R O I D S
541
The m e d i u m f r o m each incubation was decanted into a ground g l a s s s t o p p e r e d c e n t r i f u g e t u b e . T h e t i s s u e s w e r e w a s h e d with a s m a l l v o l u m e of s a l i n e a n d t h e w a s h i n g s c o m b i n e d with t h e m e d i u m . T h e adrenal tissue was discarded. The combined fluids were eXtracted t h r e e t i m e s w i t h 2 m l . of d i c h l o r o m e t h a n e u s i n g c e n t r i f u g a t i o n to s e p a r a t e t h e s o l v e n t l a y e r s . Q u a n t i t a t i v e d e t e r m i n a t i o n s of t h e i n d i v i d u a l s t e r o i d s in t h e e x t r a c t s w e r e c a r r i e d out b y m e t h o d s p r e v i o u s l y d e s c r i b e d 5. R E S U L T S AND DISCUSSION The results obtained from these experiments are summarized in T a b l e 1.
When the glands w e r e s t i m u l a t e d by ACTH, p u r o m y c i n at a
c o n c e n t r a t i o n of 7 . 5 x 1 0 - 4 M s t r o n g l y i n h i b i t e d t h e p r o d u c t i o n of t o t a l s t e r o i d s a s w e l l a s b l u e t e t r a z o l i u m p o s i t i v e s t e r o i d s 6.
It is a p p a r e n t
t h a t t h e o b s e r v e d d e c r e a s e in s t e r o i d p r o d u c t i o n is l a r g e l y a c c o u n t e d f o r b y a m a r k e d d e c r e a s e in c o r t i c o s t e r o n e ( p r e g n - 4 - e n e - 11/~, 2 1 - d i o l - 3, 20-dione). F e r g u s o n 3.
T h e s e r e s u l t s a r e in a g r e e m e n t with t h o s e r e p o r t e d b y H o w e v e r , in c o n t r a s t t o t h e d a t a of F e r g u s o n ,
the r e s u l t s
in T a b l e 1 d e m o n s t r a t e t h a t a s i g n i f i c a n t i n h i b i t i o n of b l u e t e t r a z o l i u m p o s i t i v e s t e r o i d p r o d u c t i o n c a n o c c u r e v e n in t h e a b s e n c e of A C T H stimulation.
It s e e m s l i k e l y t h a t t h i s e f f e c t w a s not n o t e d in F e r g u s o n ' s
s t u d y b e c a u s e of t h e c o n s i d e r a b l y s h o r t e r t i m e s of i n c u b a t i o n , i . e . , 30 a n d 90 m i n u t e s . A s s u g g e s t e d by F e r g u s o n 3 t h e s e d a t a a r e c o n s i s t e n t with t h e h y p o t h e s i s t h a t r a t e s of c o r t i c o s t e r o n e p r o d u c t i o n b y t h e a d r e n a l g l a n d a r e p a r t i a l l y c o n t r o l l e d by p r o c e s s e s w h i c h i n v o l v e de n o v o s y n t h e s i s of one o r m o r e p r o t e i n s .
If t h i s i s c o r r e c t ,
then
p a r t of t h e de n o v o s y n t h e s i s a p p e a r s to be i n d e p e n d e n t of t h e e f f e c t s of A C T H s t i m u l a t i o n .
542
S T E R O I D S
4:4
Table 1 In V i t r o E f f e c t s of P u r o m y c i n ( 7 . 5 x 10-4M) on C o r t i c o s t e r o i d P r o d u c t i o n ~lg/hr/100 m g a d r e n a l t i s s u e No A C T H A d d e d No. Replicates
ALD
18-OH-DOC
B
DO(]
B.T.
Control
7
1.41+. 19"
1.87+. 12
3.60±. 30
0.60+.06
7.51+.28
Experimental
8
0.29±.04
1.70+.23
1.78+.27
0.11+.06
4.29+.26
.001
N.S.
.005
.001
.005
P (t T e s t ) ~f
T.S. 11.70±.50 9.22+1.45 N.S.
ACTH Stimulated Control
5
1.35±.23
3.45±.25
I0.88±.90
1.50±.41
15.424-.64
19.97±.57
Experimental
5
0 . 4 4 ~ . 15
1.66±.30
3.14±.38
0.0 ±.13
5.26±.43
8.24±.7
.005
.001
.001
.001
P (t T e s t ) <
.01
.~
Tetrazolium Positive Steroids Steroids N . S . = Not Significant means + standard errors
= Aldosterone Corticosterone = Deoxyeorticosterone 18-OH-DOC = 18- hydroxydeoxycorticost erone
B.T. T.S.
ALD
B = DOC
= Blue = Total
In a s i m i l a r w a y , the data in T a b l e 1 d e m o n s t r a t e that deoxycorticosterone
( p r e g n - 4- e n e - 21 - o i - 3, 20 - dione) p r o d u c t i o n i s
a l s o i n h i b i t e d by p u r o m y c i n and that o n l y part of t h i s e f f e c t i s due to i n h i b i t i o n of a p r o c e s s w h i c h i s c a p a b l e of b e i n g s t i m u l a t e d by ACTH.
Since deoxycorticosterone
h a s b e e n d e m o n s t r a t e d to be an
e f f e c t i v e p r e c u r s o r of c o r t i c o s t e r o n e 7, the e f f e c t s of p u r o m y c i n on the s y n t h e s i s of t h e s e s t e r o i d s m a y be r e l a t e d and due to i n h i b i t i o n of the f o r m a t i o n of an e a r l i e r i n t e r m e d i a t e on a c o m m o n b i o s y n t h e t i c pathway. C o n t r o l data in T a b l e 1 a r e in a g r e e m e n t w i t h the r e s u l t s of o t h e r s 8 w h o h a v e s h o w n that A C T H s i g n i f i c a n t l y i n c r e a s e s the p r o d u c t i o n of 1 8 - h y d r o x y d e o x y c o r t i c o s t e r o n e 2 0 - d i o n e ) by rat a d r e n a l s .
( p r e g n - 4 - e n e - 18, 2 1 - d i o l - 3,
T h e data o b t a i n e d in t h i s i n v e s t i g a t i o n
Oct. 1 9 6 4
S T E R O I D S
543
d e m o n s t r a t e , in a d d i t i o n , that t h i s e f f e c t of A C T H i s s i g n i f i c a n t l y inhibited by p u r o m y c i n .
It is of i n t e r e s t t h a t inhibition of 1 8 - h y d r o x y -
d e o x y c o r t i c o s t e r o n e p r o d u c t i o n b y p u r o m y c i n w a s not o b s e r v e d in the a b s e n c e of A C T H s t i m u l a t i o n ,
s i n c e p r o d u c t i o n r a t e s of d e o x y c o r t i c o -
sterone were significantly decreased under these conditions.
This
o b s e r v a t i o n d o e s not s u p p o r t t h e h y p o t h e s i s that d e o x y c o r t i c o s t e r o n e is a m a j o r p r e c u r s o r of i t s 1 8 - h y d r o x y l a t e d d e r i v a t i v e .
Independent
e x p e r i m e n t s in o u r l a b o r a t o r y h a v e shown that e x o g e n e o u s 1 8 - h y d r o x y p r o g e s t e r o n e c a n be e f f i c i e n t l y c o n v e r t e d to 1 8 - h y d r o x y d e o x y c o r t i c o s t e r o n e b y a d r e n a l s l i c e s 9.
T h e s e c o n s i d e r a t i o n s s u g g e s t the
possibility that 1 8 - h y d r o x y d e o x y c o r t i c o s t e r o n e m a y be f o r m e d f r o m p r o g e s t e r o n e b y s e q u e n t i a l h y d r o x y l a t i o n s at C - 1 8 and C - 2 1 , in that order.
Since A C T H h a s b e e n found to inhibit the c o n v e r s i o n of
e x o g e n o u s 1 8 - h y d r o x y p r o g e s t e r o n e into 1 8 - h y d r o x y d e o x y c o r t i c o s t e r one 10 it a p p e a r s l i k e l y that the A C T H i n d u c e d s t i m u l a t i o n of the p r o d u c t i o n of t h i s s t e r o i d is e f f e c t e d at an e a r l i e r s t a g e in the b i o synthetic pathway. In T a b l e 1 it c a n b e s e e n that s t i m u l a t i o n of a l d o s t e r o n e p r o d u c t i o n w a s not o b s e r v e d when A C T H w a s p r e s e n t in the i n c u b a t i o n s and t h a t p u r o m y c i n i n h i b i t e d a l d o s t e r o n e p r o d u c t i o n to a b o u t the s a m e extent in both the p r e s e n c e and a b s e n c e of A C T H . On the b a s i s of t h e s e data, the inhibition of a l d o s t e r o n e p r o d u c t i o n b y p u r o m y c i n a p p e a r s to b e i n d e p e n d e n t of any A C T H e f f e c t . T h e m e c h a n i s m s r e g u l a t i n g the in v i t r o p r o d u c t i o n of
544
S T E R O I D S
4:4
a l d o s t e r o n e b y t h e r a t a d r e n a l r e m a i n o b s c u r e 11.
T h e a d d i t i o n of
a n g i o t e n s i n to in v i t r o s y s t e m s h a s b e e n r e p o r t e d to b e w i t h o u t e f f e c t on a l d o s t e r o n e p r o d u c t i o n 12.
We h a v e o b t a i n e d s i m i l a r
resultsl0.
The
d a t a p r e s e n t e d h e r e s u g g e s t t h a t t h e r e g u l a t i o n of a l d o s t e r o n e p r o d u c tion b y t h e r a t a d r e n a l m a y d e p e n d in s o m e u n k n o w n w a y u p o n de n o v o s y n t h e s i s of p r o t e i n s i n c e p u r o m y c i n is b e l i e v e d to b e a s p e c i f i c i n h i b i t o r of p r o t e i n s y n t h e s i s 13 It is c l e a r t h a t a g r e a t e r u n d e r s t a n d i n g aldosterone regulatory mechanisms f u r t h e r s t u d i e s on t h e m e c h a n i s m s
is required.
of t h e d e t a i l s of It is h o p e d t h a t
of p u r o m y c i n i n h i b i t i o n of a l d o s t e r -
one p r o d u c t i o n w i l l p r o v i d e e v i d e n c e a l o n g t h e s e l i n e s .
ACKNOWLEDGEMENTS This investigation was supported by a research f r o m the National S c i e n c e F o u n d a t i o n .
grant (G-23686)
Computor facilities were provided by the Medical Computing Center which is supported in part by National Institutes of Health grant FR-O0010. Mrs. Joan Schreiber contributed valuable technical assistance during this investigation.
REFERENCES
.
T h i s w o r k w a s d o n e d u r i n g t h e t e n u r e of an A d v a n c e d R e s e a r c h Fellowship from the American Heart Association.
.
Ferguson, J.J., 618 (1962).
Jr.,
BIOCHIM. ET BIOPHYS. ACTA,
Ferguson,
Jr.,
J . B I O L . C H E M . 238, 2754 (1963}.
3.
J.J.,
57,
Oct. 1964
.
o
6.
o
8.
.
Saffran, (1955). Kittinger,
S T E R 0 I D S
M.,
and Schaily, A.V.,
G.W.,
STEROIDS
545
ENDOCRINOLOGY
56, 523
3, 21 (1964).
The terms "total steroids" and "blue tetrazolium are operationally defined in reference 5.
positive steroids"
Wettstein, A . , EXPERIENTIA 17, 329 (1961). C o r t e s , J . M . , P e r o n , F . G . , and Dorfman, R . I . , ENDOCRINOLOGY 73, 713 (1963). Kittinger, G . W . , PROGRAM OF THE ENDOCRINE SOCIETY, San F r a n c i s c o , 1964.
i0.
Kittinger, G . W . , Unpublished o b s e r v a t i o n s .
ii.
Singer, B . , Losito, C . , and Salmon, S., ENDOCRINOLOGY 74, 325 (1964).
12.
Kaplan, N . M . , and B a r t t e r , F . C . , (1962).
J . CLIN. INVEST. 41, 715
13.
Yarmolinsky, M., and De La Haba, SCI. U.S., 45, 1721 (1959).
G.,
PROC.
NATL.
ACAD.