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Effect of alpha-MSH on the corticosteroid production of isolated zona glomerulosa and zona fasciculata cells
Life Sciences, Vol. 30, pp. 2101-2108 Printed in the U.S.A.
Pergamon Press
E F F E C T OF A L P H A - M S H ON THE C O R T I C O S T E R O I D P R O D U C T I O N OF I S O L A T E D ZONA G L O M E R U L O S A A N D ZONA F A S C I C U L A T A CELLS Katalin Institute
Sz.
Szalay
and E. Stark
of E x p e r i m e n t a l M e d i c i n e , H u n g a r i a n A c a d e m y 1450 B u d a p e s t , P.O.B. 67., H u n g a r y
of S c i e n c e s
(Received in final form April 5, 1982) Summary O~-MSH (10 -9 - 6xlO-7M) p o t e n t i a t e s the e f f e c t of ACTH (i0 -II - 5xlO-9M( on a d r e n o c o r t i c a l s t e r o i d o g e n e s i s d e c r e a s i n g E D 5 0 of ACTH from 220 to 183 p g / m l on zona f a s c i c u l a t a c o r t i c o s t e r o n e - , and from 738 to 437 p g / m l on zona g l o m e r u l o s a a l d o s t e r o n e p r o d u c t i o n . O(-MSH alone i n c r e a s e s a l d o s t e r o n e p r o d u c t i o n of zona g l o m e r u l o s a cells in doses (10 -9 - 6xlO-7M) that do n o t s t i m u l a t e zona f a s c i c u l a t a c o r t i c o s t e r o n e p r o d u c tion. The r e s p o n s e of zona g l o m e r u l o s a a l d o s t e r o n e p r o d u c t i o n to O t - M S H can be c h a r a c t e r i z e d by a b i - p h a s e d o s e - r e s p o n s e curve. R e c e n t l y we have n o t i c e d that in the i n t e r m e d i a t e lobe of the rat there is a h o r m o n e or some o t h e r s u b s t a n c e w h i c h alters the s e n s i t i v i t y of zona g l o m e r u l o s a cells to ACTH (i, 2). F r o m among the p o s s i b l e factors r e s p o n s i b l e for this e f f e c t we have tried first ~ - e n d o r p h i n (3) : in p h y s i o l o g i c a l c o n c e n t r a t i o n s it was found to be i n h i b i t o r y i n s t e a d of b e i n g s t i m u l a t o r y . Since some authors s u g g e s t e d that O~-MSH h a d a role in the r e g u l a t i o n of fetal a d r e n o c o r t i c a l s t e r o i d o g e n e s i s (4, 5) n e x t we s t u d i e d the e f f e c t of C ~ - M S H on a l d o s t e r o n e and c o r t i c o s t e r o n e p r o d u c t i o n of i s o l a t e d zona g l o m e r u l o s a - and on c o r t i c o s t e r o n e p r o d u c t i o n of i s o l a t e d zona f a s c i c u l a t a cells. Materials
and M e t h o d s
Materials S y n t h e t i c h u m a n O t h l - 3 9 A C T H and a n t i - a l d o s t e r o n e a n t i s e r u m (Sheep 088) w e r e s u p p l i e d by the N a t i o n a l I n s t i t u t e of A r t h r i t i s and M e t a b o l i c Diseases, NIH, Bethesda, Maryland, U.S.A. The f o l l o w i n g w e r e p u r c h a s e d : O~-MSH: Reanal, Hungary; b o v i n e s e r u m albumin: Phylaxia, Hungary; c o l l a g e n a s e (type I) : W o r t h i n g t o n C h e m i c a l C o r p o r a t i o n , U.S.A.; d e o x y r i b o n u c l e a s e (DNase I) : S i g m a C h e m i c a l Co. U.S.A.; (I,2-H 3) A l d o s t e r o n e : A m e r s h a m , R a d i o c h e m i c a l Centre, England. Animals Male
CFE rats w e i g h i n g
200-250
g were used
in each e x p e r i m e n t .
0024-3205/82/242 I01-08503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
2102
Effect of ~-MSH on Adrenal Cortex
Preparation
of c__ell s u s p e n s i o n s
Vol. 30, No. 24, 1982
We h a v e p r e v i o u s l y d e s c r i b e d the r a t a d r e n a l c e l l p r e p a r a t i o n u s e d in o u r l a b o r a t o r y (2). B r i e f l y : c e l l s u s p e n s i o n s w e r e p r e p a r e d by c o l l a g e n a s e d i g e s t i o n s of a d r e n a l c a p s u l a r s t r i p p i n g s to y i e l d a p r e p a r a t i o n of z o n a g l o m e r u l o s a c e l l s and of d e c a p s u l a t e d a d r e n a l g l a n d s to y i e l d z o n a f a s c i c u l a t a c e l l s (6). F a s c i c u l a t a c e l l c o n t a m i n a t i o n in the z o n a g l o m e r u l o s a c e l l s u s p e n s i o n w a s less t h a n 5%. In g e n e r a l for e a c h i s o l a t e d a d r e n a l c e l l p r e p a r a t i o n a d r e n a l s f r o m 40 r a t s w e r e u s e d a n d the g l o m e r u l o s a c e l l s f r o m e a c h a d r e n a l g l a n d , f a s c i c u l a t a c e l l s f r o m 40 a d r e n a l g l a n d s w e r e d i g e s t e d . The c e l l s w e r e p r e p a r e d in K r e b s - R i n g e r b i c a r b o n a t e p u f f e r c o n t a i n i n g 2 g / l g l u c o s e a n d 40 g/l b o v i n e s e r u m a l b u m i n (BSA) w i t h p o t a s s i u m c o n c e n t r a t i o n 3.7 m E q / l a n d f i n a l l y s u s p e n d e d in the s a m e s o l u t i o n b u t w i t h 5 g/l BSA. 0.9 ml of 40 g l o m e r u l o s a and f a s c i c u l a t a c e l l s u s p e n s i o n a l i q u o t s ( a p p r . 3 - 3 . 5 x l O 5 c e l l s / m l ) w e r e i n c u b a t e d in o n e s e s s i o n in a s h a k i n g w a t e r b a t h at 3 7 ° C u n d e r an a t m o s p h e r e of 95% 02 and 5% CO2 for two h o u r s . In c e r t a i n e x p e r i m e n t s 80 a l i q u o t s of g l o m e r u l o s a c e l l (from 80 rats) or t h a t of f a s c i c u l a t a c e l l (from 40 rats) s u s p e n s i o n s w e r e i n c u b a t e d s e p a r a tely. A C T H a n d O ~ - M S H w e r e d i s s o l v e d in p h y s i o l o g i c a l s a l i n e cont a i n i n g 5 g/l B S A and a d j u s t e d to pH 3.5. T h e e f f e c t of O t - M S H on A C T H a c t i o n w a s e x a m i n e d in 5 e x p e r i m e n t s as f o l l o w s : we h a v e t a k e n i n t o a c c o u n t t h a t the i n t e r m e d i a t e l o b e c o n t a i n s 50 t i m e s as m u c h O ~ - M S H as A C T H (7), t h e r e fore the p r o p o r t i o n of O ~ - M S H a n d A C T H a d d e d to the t u b e s w a s 5 0 : 1 ( e x p r e s s e d in pg). The r a n g e of the A C T H d o s e - r e s p o n s e c u r v e w a s 45 p g - 23 n g / m l (iO -II - 5 . 1 x l O - 9 M ) l a n d the r a n g e of O ~ - M S H a d d e d t o g e t h e r w i t h A C T H was 2.3 - 1 1 5 0 n g / m l ( 1 . 2 x l O - 9 - 6 i x l O -7 M/l). In 3 e x p e r i m e n t s s o l e l y O t - M S H ( l . 2 x l O -9 - 8 x l O -5 M>I) was a d d e d to t e s t its e f f e c t on c o r t i c o s t e r o i d o g e n e s i s . T h e m a t e r i a l s w e r e a d d e d a l t o g e t h e r in 0.i ml v o l u m e to d u p l i c a t e or t r i p l i c a t e b e a k e r s . A c o r r e s p o n d i n g v o l u m e of the s o l v e n t w a s a d d e d to the c o n t r o l i n c u b a t e s ~. E x p e r i m e n t s w e r e p e r f o r m e d in a r a n d o m i s e d b l o c k f o r m a t to e l i m i n a t e b i a s due to systematic error. Steroid
estimation
The c o r t i c o s t e r o n e c o n t e n t s of the i n c u b a t i o n m e d i a (both f r o m f a s c i c u l a t a and g l o m e r u l o s a ) w e r e d e t e r m i n e d by f l u o r i m e t r y (8) a f t e r c h l o r o f o r m e x t r a c t i o n . A l i q u o t s of the c h l o r o f o r m e x t r a c t of the g l o m e r u l o s a i n c u b a t e w e r e a s s a y e d for a l d o s t e r o n e c o n t e n t s by r a d i o i m m u n o a s s a y w i t h o u t c h r o m a t o g r a p h i c s e p a r a t i o n as d e s c r i b e d (2). The a l d o s t e r o n e v a l u e m e a s u r e d d i r e c t l y w a s w e l l c o r r e l a t e d w i t h the v a l u e o b t a i n e d a f t e r p a p e r c h r o m a t o g r a p h i c s e p a r a t i o n (y=l. O l l x + 3.162, r = O . 9 7 7 , p < O . O l , n=24) . Statistical
analysis
S l o p e s of the d o s e - r e s p o n s e c u r v e s of A C T H and A C T H + ~ - M S H and the E D 5 0 v a l u e s w e r e c a l c u l a t e d a f t e r l o g i t - l o g t r a n s f o r m a t i o n
(9).
Vol.
30, No.
24,
1982
Effect of a - M S H . o n A d r e n a l
Cortex
/ /:T
® 20
10
C
e ¢ o
0
<
® 500
~'
20o
U C Q
lOO
O
o
50
20
_- :
....
1,2x10 ~
l
,
•
3.8 xlO"
. :
. . . .
6,1x10 "? - MSH
FIG.
1
Response curves for zona glomerulosa aldosterone (A) a n d z o n a glomerulosa corticosterone (B) to increasing doses of ~-MSH. (Data of 3 experiments, doublelog graph.)
:
• ,
2x10 "~ M/t
2103
2104
Effect of ~-MSH on Adrenal Cortex
Vol. 30, No. 24, 1982
® 40
30
ol ¢:
20
o
0 IA
0
10
® ,ooo
/
II 0 O
500 r,.)
~oo
* ~ . . / . L I
%w"ACTH II
I
i
I
4 ~I0""
i
I
:~x10 "l°
i
!
1,3x10 "l
'
i
5,1M10"s
.
" 3,8~104' 1,5x10-,~ 6,1x10-,W!
a-NSH
FIG.
2
R e s p o n s e c u r v e s for zona glomerulosa aldosterone (A) and zona f a s c i c u l a t a c o r t i c o s t e r o n e (B) to i n c r e a s i n g d o s e s of A C T H (-o-,-/~-) and 0 6 - M S H + A C T H (-o-, - A - ) (Data of a t y p i c a l e x p e r i m e n t , e a c h p o i n t is in t r i p l i c a t e . S e m i - l o g graph.)
geometric
mean
1.194 ~0.iO87
(exp)
of s l o p e
~-MSH.
OF A C T H DOSE
+
~-MSH
OF A C T H :
AND ACTH
os
a
N.S.
N.S.
of S . E . M .
of natural
logarithmic
i.iO1
248.57
~0.0761
1.396
A C T H + 0£-MSH
transformation
1.185
363.0
~O.174
1.218
ACTH
cor t i c o s t e r o n e
r ul
fasciculata
means
1.181
220.46
~O.O528
1.137
ACTH
p
p<0.05
1.209
182.84
~O.O72
1.394
ACTH+Ot-MSH
corticosterone
Zona
C~-MSH: 1.2xlO-9-6.1xlO-7M/1, n=5
OR WITHOUT
logit-log
values
after
1.208
437.04
p< O.01
1.212 c
g 1 ome
DOSE OF
A C T H + 0t-MSH
p< 0.05
737.59 b
c: the r e t r a n s f o r m e d
b:
0.938 a
ACTH
~0.0064
a: m e a n ~ S . E . M .
pg/ml
ED50
SLOPE
a
a l d o s te r o n e
Zon
lO-11-5xlO-9M/1,
i.
CURVES
TABLE DOSE-RESPONSE
OF A C T H W I T H
OF THE
AND ED50
SLOPES
~o
0
>
O
I
O
Oo ~o
to
Z O
~o O
< O
2106
Effect of ~-MSH on Adrenal Cortex
Vol. 30, No. 24, 1982
Results O ~ - M S H i n c r e a s e d a l d o s t e r o n e p r o d u c t i o n of zona g l o m e r u l o s a cells a l r e a d y at d o s e s (l.2xlO -9 - 6 . 1 x l O -7 M/l) w h i c h did n o t i n f l u e n c e c o r t i c o s t e r o n e p r o d u c t i o n of zona f a s c i c u l a t a cells (Fig. i). H i g h e r d o s e s s t i m u l a t e d the c o r t i c o s t e r o i d p r o d u c t i o n of b o t h cell system. The r e s p o n s e of a l d o s t e r o n e p r o d u c t i o n to m 4 - M S H can be c h a r a c t e r i z e d by a b i - p h a s e d o s e - r e s p o n s e curve (Fig. IB). C o r t i c o s t e r o n e p r o d u c t i o n of zona g l o m e r u l o s a cells f o l l o w e d the same p a t t e r n as a l d o s t e r o n e (control: 1 5 . 3 + 2 . 2 5 , 3.9xlO-8M~-M~H: 41.9+4.43, 2xlO-5M~-MSH: 1 1 8 . 3 + 2 . 6 5 ng/ml). The s l o p e s of the d o s e - r e s p o n s e c u r v e s of ACTH + O ~ - M S H w e r e s t e e p e r than those of A C T H w h e n m e a s u r e d on zona f a s c i c u l a t a c o r t i c o s t e r o n e p r o d u c t i o n (p < 0.05), on zona g l o m e r u l o s a aldos t e r o n e (p ~ 0.05) and c o r t i c o s t e r o n e (N.S.) p r o d u c t i o n (Table i.) ~ - M S H d e c r e a s e d E D 5 0 of A C T H in e a c h cell s y s t e m (Table I.) . D a t a of a t y p i c a l e x p e r i m e n t are s h o w n in Fig. 2. A lower dose of ~ - M S H (iO -II - 5 x l O -IO M) did n o t i n f l u e n c e the s l o p e and the E D 5 0 of A C T H (data are n o t p r e s e n t e d ) . Discussion The s t e r o i d o g e n i c e f f e c t of o ~ - 5 ~ H w i t h a p p r o x i m a t e l y 10 -6 of the p o t e n c y of A C T H was f i r s t s h o w n by S a y e r s et al. (i0) and L o w r y et al. (ii). In our e x p e r i m e n t s the t h r e s h o l d dose o f ~ - M S H on zona f a s c i c u l a t a c o r t i c o s t e r o n e p r o d u c t i o n was a b o u t IO-6M, t h a t of ACTH: IO-IIM. ~ - M S H p o t e n t i a t e d the e f f e c t of A C T H b o t h on zona f a s c ~ c u l a t a c o r t i c o s t e r o n e and on zona g l o m e r u l o s a a l d o s t e r o n e p r o d u c t i o n in doses t h a t i t s e l f h a d no effect. The q u e s t i o n r a i s e d at s t a r t i n g our e x p e r i m e n t s was the f o l l o w i n g : is ~ - M S H the f a c t o r that p l a y s a role in the glom e r u l o t r o p i c e f f e c t of the i n t e r m e d i a t e lobe? The a n s w e r is s u p p o s e d to be p o s i t i v e . Our a n s w e r is s u p p o r t e d by the f o l l o w i n g results: i. O{-MSH d e c r e a s e d E D 5 0 of A C T H b o t h on zona g l o m e r u l o s a a l d o s t e r o n e and on zona f a s c i c u l a t a c o r t i c o s t e r o n e p r o d u c t i o n , a l t h o u g h its e f f e c t was m o r e p r o n o u n c e d on zona g l o m e r u l o s a cells. 2. O ~ - M S H i n c r e a s e d zona g l o m e r u l o s a a l d o s t e r o n e p r o d u c t i o n at doses w h i c h did n o t a f f e c t zona f a s c i c u l a t a c o r t i c o s t e r o n e production. W h i l e our e x p e r i m e n t s w e r e a l r e a d y in p r o g r e s s , V i n s o n et al. (12) p u b l i s h e d a p a p e r s h o w i n g e v i d e n c e that ~ - M S H is the c o m p o n e n t of p o s t e r i o r p i t u i t a r y e x t r a c t s t i m u l a t i n g zona glom e r u l o s a . In t h e i r e x p e r i m e n t s 10 -9 - 10 -6 M ~ - M S H i n c r e a s e d glom e r u l o s a - , b u t d i d not a f f e c t zona f a s c i c u l a t a c o r t i c o s t e r o n e p r o d u c t i o n . We h a v e found a b i - p h a s e d o s e - r e s p o n s e curve m e a s u r e d on zona g l o m e r u l o s a a l d o s t e r o n e p r o d u c t i o n ; 10 -9 - 10 -6 M i n d u c i n g the first- and 10 -6 - 8xlO -5 M the s e c o n d p h a s e of the curve. The t h r e s h o l d dose of ~ - M S H (IO-9M) c o r r e s p o n d s to the d a t a of V i n s o n et al. ~12), h o w e v e r , they f o l l o w e d the d o s e - r e s p o n s e curve only up to IO-°M. The b i - p h a s e r e s p o n s e c o u l d be e x p l a i n e d by s u p p o s i n g t h a t O ~ - M S H b i n d s to two d i f f e r e n t r e c e p t o r systems. The first one w i t h h i g h e r a f f i n i t y c o u l d be a s p e c i f i c ~ - M S H r e c e p t o r or is p e r h a p s i d e n t i c a l w i t h the a n g i o t e n s i n r e c e p t o r . To this r e f e r s that S a r a l a s i n , a s p e c i f i c a n g i o t e n s i n a n t a g o n i s t i n h i b i t s p a r t l y the e f f e c t of lower doses of O ~ - M S H ( u n p u b l i s h e d data). The s e c o n d s y s t e m c o u l d be the A C T H r e c e p t o r s y s t e m to w h i c h O C - M S H is b o u n d at I O - 6 M and h i g h e r c o n c e n t r a t i o n s .
Vol. 30, No. 24, 1982
Effect of e-MSH on Adrenal Cortex
2107
The p h y s i o l o g i c a l role of o ~ - ~ H has n o t b e e n e l u c i d a t e d so far. E f f e c t of ~ - M S H on s o d i u m balance, s e b u m s e c r e t i o n and l e a r n i n g b e h a v i o r have b e e n d e m o n s t r a t e d (for ref. see Thody, 13). The q u e s t i o n arises, w h e t h e r it is p o s s i b l e that O~-MSH p a r t i c i pates in the r e g u l a t i o n of a d r e n o c o r t i c a l f u n c t i o n ? T h e r e are data in the l i t e r a t u r e r e f e r r i n g to this p o s s i b i l i t y . E v i d e n c e was p r e s e n t e d that c o r t i s o l p r o d u c t i o n by fetal h u m a n and s h e e p adrenal cells r e s p o n d e d to Ot-MSH s t i m u l a t i o n (4, 5), a l t h o u g h c o n t r a d i c t o r y results have b e e n o b t a i n e d w i t h rhesus m o n k e y fetal a d r e n a l cells (14). We s u p p o s e that both the g l o m e r u l o t r o p and the ACTH p o t e n t i a t i n g e f f e c t of ~ - M S H may be of p h y s i o l o g i c a l s i g n i f i c a n c e . A l t h o u g h the p l a s m a level of O~-MSH is u s u a l l y lower (Io-IOM) w i t h one o r d e r than the l o w e s t e f f e c t i v e c o n c e n t r a t i o n in vitro, but the s e n s i t i v i t y b e t w e e n in v i t r o and in vivo systems may be different. Under c e r t a i n c o n d i t i o n s the level of ~ - M S H can be h i g h e r than n o r m a l (for e x a m p l e in the third t r i m e s t e r of pregnancy: 15). D a t a of B i r k h ~ u s e r et al~ (16) s e e m to s u p p o r t o u r a s s u m p t i o n : they o b s e r v e d a w e a k s p e c i f i c s t i m u l a t o r y e f f e c t of O ~ - M S H on a l d o s t e r o n e p r o d u c t i o n in p a n h y p o p i t u i t a r y patients. So it is s u g g e s t e d that under c e r t a i n c o n d i t i o n s ~ - M S H m a y p a r t i cipate in the r e g u l a t i o n of a d r e n o c o r t i c a l - m a i n l y m i n e r a l o c o r t i c o i d - s t e r o i d synthesis. Acknowled@ements We thank G. Folly for the p e r f o r m a n c e of m a t h e m a t i c a l a n a l y s e s and to Mrs. A. Falus for h e r v a l u a b l e t e c h n i c a l assistance. References i. 2. 3. 4. 5. 6. 7. 8. 9. IO. ii. 12. 13.
K.Sz. SZALAY, A c t a endocr. (Kbh.) Suppl. 225, 347 (1979). K.Sz. SZALAY, A c t a Physiol. Acad. Sci. Hung. 57, 225-231 (1981). K.Sz. S Z A L A Y and E. STARK, Life Sci. 29, 1 3 5 5 - 1 3 6 1 (1981). J.A. GLICKMAN, G.D. C A R S O N and J.R.G. CHALLIS, E n d o c r i n o l o g y 104, 34-39 (1979). A.J. LLANOS, J. R A M A C H A N D R A N , R.K. CREASY, A.M. RUDOLPH and M. S E R O N - F E R P ~ , E n d o c r i n o l o g y 105, 613-617 (1979). J.F. TAIT, S.A.S. TAIT, R.P. G O U L D and M.S.R. MEE, Proc. roy. Soc. B. 185, 375-407 (1974). R. USATEGUI, C. OLIVER, H. VAUDRY, G. LOMBARDI, I. R O Z E N B E R G and A.M. MORRE, E n d o c r i n o l o g y , 98, 189-196 (1976). R. G U I L L E M I N , G.W. CLAYTON, H.S. L I P S C O M B and J.D. SMITH, J. Lab. Clin. Med. 53, 830-832 (1959). D. R O D B A R D and G.R. FRAZIER, M e t h o d s in E n z y m o l o g y 37, 1-22, A c a d e m i c Press, N e w York (1975). G. SAYERS, R.L. S W A L L O W and N.D. GIORDANO, E n d o c r i n o l o g y , 88, 1 0 6 3 - 1 0 6 8 (1971). ~.J. LOWRY, C. M C M A R T I N and J. PETERS, J. Endocr. 59, 43-55 (1973) . G.P. VINSON, B. W H I T E H O U S E , A. DELL, T. E T I E N N E and H.R. MORRIS, N a t u r e 284, 464-467 (1980). A.J. THODY, A d v a n c e s in Drug R e s e a r c h ii, 23-74 (1977).
2108
14. 15. 16.
Effect of e-MSH on Adrenal Corte~~
Vol. 30, No. 24, 1982
S.W. W A L S H , R.L. N O R M A N and M.J. N O V Y , E n d o c r i n o l o g y 104, 1 8 0 5 - 1 8 1 3 (1979). D. C L A R K , A.J. T H O D Y , S. S H U S T E R a n d H. B O W E R S , N a t u r e 273, 1 6 3 - 1 6 4 (1978). M. B I R K H A U S E R , R. G A I L L A R D , A.M. R I O N D E L a n d G.R. ZAHND, A c t a e n d o c r . (Kbh.) 79, 1 6 - 2 4 (1975).
Pergamon Press
E F F E C T OF A L P H A - M S H ON THE C O R T I C O S T E R O I D P R O D U C T I O N OF I S O L A T E D ZONA G L O M E R U L O S A A N D ZONA F A S C I C U L A T A CELLS Katalin Institute
Sz.
Szalay
and E. Stark
of E x p e r i m e n t a l M e d i c i n e , H u n g a r i a n A c a d e m y 1450 B u d a p e s t , P.O.B. 67., H u n g a r y
of S c i e n c e s
(Received in final form April 5, 1982) Summary O~-MSH (10 -9 - 6xlO-7M) p o t e n t i a t e s the e f f e c t of ACTH (i0 -II - 5xlO-9M( on a d r e n o c o r t i c a l s t e r o i d o g e n e s i s d e c r e a s i n g E D 5 0 of ACTH from 220 to 183 p g / m l on zona f a s c i c u l a t a c o r t i c o s t e r o n e - , and from 738 to 437 p g / m l on zona g l o m e r u l o s a a l d o s t e r o n e p r o d u c t i o n . O(-MSH alone i n c r e a s e s a l d o s t e r o n e p r o d u c t i o n of zona g l o m e r u l o s a cells in doses (10 -9 - 6xlO-7M) that do n o t s t i m u l a t e zona f a s c i c u l a t a c o r t i c o s t e r o n e p r o d u c tion. The r e s p o n s e of zona g l o m e r u l o s a a l d o s t e r o n e p r o d u c t i o n to O t - M S H can be c h a r a c t e r i z e d by a b i - p h a s e d o s e - r e s p o n s e curve. R e c e n t l y we have n o t i c e d that in the i n t e r m e d i a t e lobe of the rat there is a h o r m o n e or some o t h e r s u b s t a n c e w h i c h alters the s e n s i t i v i t y of zona g l o m e r u l o s a cells to ACTH (i, 2). F r o m among the p o s s i b l e factors r e s p o n s i b l e for this e f f e c t we have tried first ~ - e n d o r p h i n (3) : in p h y s i o l o g i c a l c o n c e n t r a t i o n s it was found to be i n h i b i t o r y i n s t e a d of b e i n g s t i m u l a t o r y . Since some authors s u g g e s t e d that O~-MSH h a d a role in the r e g u l a t i o n of fetal a d r e n o c o r t i c a l s t e r o i d o g e n e s i s (4, 5) n e x t we s t u d i e d the e f f e c t of C ~ - M S H on a l d o s t e r o n e and c o r t i c o s t e r o n e p r o d u c t i o n of i s o l a t e d zona g l o m e r u l o s a - and on c o r t i c o s t e r o n e p r o d u c t i o n of i s o l a t e d zona f a s c i c u l a t a cells. Materials
and M e t h o d s
Materials S y n t h e t i c h u m a n O t h l - 3 9 A C T H and a n t i - a l d o s t e r o n e a n t i s e r u m (Sheep 088) w e r e s u p p l i e d by the N a t i o n a l I n s t i t u t e of A r t h r i t i s and M e t a b o l i c Diseases, NIH, Bethesda, Maryland, U.S.A. The f o l l o w i n g w e r e p u r c h a s e d : O~-MSH: Reanal, Hungary; b o v i n e s e r u m albumin: Phylaxia, Hungary; c o l l a g e n a s e (type I) : W o r t h i n g t o n C h e m i c a l C o r p o r a t i o n , U.S.A.; d e o x y r i b o n u c l e a s e (DNase I) : S i g m a C h e m i c a l Co. U.S.A.; (I,2-H 3) A l d o s t e r o n e : A m e r s h a m , R a d i o c h e m i c a l Centre, England. Animals Male
CFE rats w e i g h i n g
200-250
g were used
in each e x p e r i m e n t .
0024-3205/82/242 I01-08503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
2102
Effect of ~-MSH on Adrenal Cortex
Preparation
of c__ell s u s p e n s i o n s
Vol. 30, No. 24, 1982
We h a v e p r e v i o u s l y d e s c r i b e d the r a t a d r e n a l c e l l p r e p a r a t i o n u s e d in o u r l a b o r a t o r y (2). B r i e f l y : c e l l s u s p e n s i o n s w e r e p r e p a r e d by c o l l a g e n a s e d i g e s t i o n s of a d r e n a l c a p s u l a r s t r i p p i n g s to y i e l d a p r e p a r a t i o n of z o n a g l o m e r u l o s a c e l l s and of d e c a p s u l a t e d a d r e n a l g l a n d s to y i e l d z o n a f a s c i c u l a t a c e l l s (6). F a s c i c u l a t a c e l l c o n t a m i n a t i o n in the z o n a g l o m e r u l o s a c e l l s u s p e n s i o n w a s less t h a n 5%. In g e n e r a l for e a c h i s o l a t e d a d r e n a l c e l l p r e p a r a t i o n a d r e n a l s f r o m 40 r a t s w e r e u s e d a n d the g l o m e r u l o s a c e l l s f r o m e a c h a d r e n a l g l a n d , f a s c i c u l a t a c e l l s f r o m 40 a d r e n a l g l a n d s w e r e d i g e s t e d . The c e l l s w e r e p r e p a r e d in K r e b s - R i n g e r b i c a r b o n a t e p u f f e r c o n t a i n i n g 2 g / l g l u c o s e a n d 40 g/l b o v i n e s e r u m a l b u m i n (BSA) w i t h p o t a s s i u m c o n c e n t r a t i o n 3.7 m E q / l a n d f i n a l l y s u s p e n d e d in the s a m e s o l u t i o n b u t w i t h 5 g/l BSA. 0.9 ml of 40 g l o m e r u l o s a and f a s c i c u l a t a c e l l s u s p e n s i o n a l i q u o t s ( a p p r . 3 - 3 . 5 x l O 5 c e l l s / m l ) w e r e i n c u b a t e d in o n e s e s s i o n in a s h a k i n g w a t e r b a t h at 3 7 ° C u n d e r an a t m o s p h e r e of 95% 02 and 5% CO2 for two h o u r s . In c e r t a i n e x p e r i m e n t s 80 a l i q u o t s of g l o m e r u l o s a c e l l (from 80 rats) or t h a t of f a s c i c u l a t a c e l l (from 40 rats) s u s p e n s i o n s w e r e i n c u b a t e d s e p a r a tely. A C T H a n d O ~ - M S H w e r e d i s s o l v e d in p h y s i o l o g i c a l s a l i n e cont a i n i n g 5 g/l B S A and a d j u s t e d to pH 3.5. T h e e f f e c t of O t - M S H on A C T H a c t i o n w a s e x a m i n e d in 5 e x p e r i m e n t s as f o l l o w s : we h a v e t a k e n i n t o a c c o u n t t h a t the i n t e r m e d i a t e l o b e c o n t a i n s 50 t i m e s as m u c h O ~ - M S H as A C T H (7), t h e r e fore the p r o p o r t i o n of O ~ - M S H a n d A C T H a d d e d to the t u b e s w a s 5 0 : 1 ( e x p r e s s e d in pg). The r a n g e of the A C T H d o s e - r e s p o n s e c u r v e w a s 45 p g - 23 n g / m l (iO -II - 5 . 1 x l O - 9 M ) l a n d the r a n g e of O ~ - M S H a d d e d t o g e t h e r w i t h A C T H was 2.3 - 1 1 5 0 n g / m l ( 1 . 2 x l O - 9 - 6 i x l O -7 M/l). In 3 e x p e r i m e n t s s o l e l y O t - M S H ( l . 2 x l O -9 - 8 x l O -5 M>I) was a d d e d to t e s t its e f f e c t on c o r t i c o s t e r o i d o g e n e s i s . T h e m a t e r i a l s w e r e a d d e d a l t o g e t h e r in 0.i ml v o l u m e to d u p l i c a t e or t r i p l i c a t e b e a k e r s . A c o r r e s p o n d i n g v o l u m e of the s o l v e n t w a s a d d e d to the c o n t r o l i n c u b a t e s ~. E x p e r i m e n t s w e r e p e r f o r m e d in a r a n d o m i s e d b l o c k f o r m a t to e l i m i n a t e b i a s due to systematic error. Steroid
estimation
The c o r t i c o s t e r o n e c o n t e n t s of the i n c u b a t i o n m e d i a (both f r o m f a s c i c u l a t a and g l o m e r u l o s a ) w e r e d e t e r m i n e d by f l u o r i m e t r y (8) a f t e r c h l o r o f o r m e x t r a c t i o n . A l i q u o t s of the c h l o r o f o r m e x t r a c t of the g l o m e r u l o s a i n c u b a t e w e r e a s s a y e d for a l d o s t e r o n e c o n t e n t s by r a d i o i m m u n o a s s a y w i t h o u t c h r o m a t o g r a p h i c s e p a r a t i o n as d e s c r i b e d (2). The a l d o s t e r o n e v a l u e m e a s u r e d d i r e c t l y w a s w e l l c o r r e l a t e d w i t h the v a l u e o b t a i n e d a f t e r p a p e r c h r o m a t o g r a p h i c s e p a r a t i o n (y=l. O l l x + 3.162, r = O . 9 7 7 , p < O . O l , n=24) . Statistical
analysis
S l o p e s of the d o s e - r e s p o n s e c u r v e s of A C T H and A C T H + ~ - M S H and the E D 5 0 v a l u e s w e r e c a l c u l a t e d a f t e r l o g i t - l o g t r a n s f o r m a t i o n
(9).
Vol.
30, No.
24,
1982
Effect of a - M S H . o n A d r e n a l
Cortex
/ /:T
® 20
10
C
e ¢ o
0
<
® 500
~'
20o
U C Q
lOO
O
o
50
20
_- :
....
1,2x10 ~
l
,
•
3.8 xlO"
. :
. . . .
6,1x10 "? - MSH
FIG.
1
Response curves for zona glomerulosa aldosterone (A) a n d z o n a glomerulosa corticosterone (B) to increasing doses of ~-MSH. (Data of 3 experiments, doublelog graph.)
:
• ,
2x10 "~ M/t
2103
2104
Effect of ~-MSH on Adrenal Cortex
Vol. 30, No. 24, 1982
® 40
30
ol ¢:
20
o
0 IA
0
10
® ,ooo
/
II 0 O
500 r,.)
~oo
* ~ . . / . L I
%w"ACTH II
I
i
I
4 ~I0""
i
I
:~x10 "l°
i
!
1,3x10 "l
'
i
5,1M10"s
.
" 3,8~104' 1,5x10-,~ 6,1x10-,W!
a-NSH
FIG.
2
R e s p o n s e c u r v e s for zona glomerulosa aldosterone (A) and zona f a s c i c u l a t a c o r t i c o s t e r o n e (B) to i n c r e a s i n g d o s e s of A C T H (-o-,-/~-) and 0 6 - M S H + A C T H (-o-, - A - ) (Data of a t y p i c a l e x p e r i m e n t , e a c h p o i n t is in t r i p l i c a t e . S e m i - l o g graph.)
geometric
mean
1.194 ~0.iO87
(exp)
of s l o p e
~-MSH.
OF A C T H DOSE
+
~-MSH
OF A C T H :
AND ACTH
os
a
N.S.
N.S.
of S . E . M .
of natural
logarithmic
i.iO1
248.57
~0.0761
1.396
A C T H + 0£-MSH
transformation
1.185
363.0
~O.174
1.218
ACTH
cor t i c o s t e r o n e
r ul
fasciculata
means
1.181
220.46
~O.O528
1.137
ACTH
p
p<0.05
1.209
182.84
~O.O72
1.394
ACTH+Ot-MSH
corticosterone
Zona
C~-MSH: 1.2xlO-9-6.1xlO-7M/1, n=5
OR WITHOUT
logit-log
values
after
1.208
437.04
p< O.01
1.212 c
g 1 ome
DOSE OF
A C T H + 0t-MSH
p< 0.05
737.59 b
c: the r e t r a n s f o r m e d
b:
0.938 a
ACTH
~0.0064
a: m e a n ~ S . E . M .
pg/ml
ED50
SLOPE
a
a l d o s te r o n e
Zon
lO-11-5xlO-9M/1,
i.
CURVES
TABLE DOSE-RESPONSE
OF A C T H W I T H
OF THE
AND ED50
SLOPES
~o
0
>
O
I
O
Oo ~o
to
Z O
~o O
< O
2106
Effect of ~-MSH on Adrenal Cortex
Vol. 30, No. 24, 1982
Results O ~ - M S H i n c r e a s e d a l d o s t e r o n e p r o d u c t i o n of zona g l o m e r u l o s a cells a l r e a d y at d o s e s (l.2xlO -9 - 6 . 1 x l O -7 M/l) w h i c h did n o t i n f l u e n c e c o r t i c o s t e r o n e p r o d u c t i o n of zona f a s c i c u l a t a cells (Fig. i). H i g h e r d o s e s s t i m u l a t e d the c o r t i c o s t e r o i d p r o d u c t i o n of b o t h cell system. The r e s p o n s e of a l d o s t e r o n e p r o d u c t i o n to m 4 - M S H can be c h a r a c t e r i z e d by a b i - p h a s e d o s e - r e s p o n s e curve (Fig. IB). C o r t i c o s t e r o n e p r o d u c t i o n of zona g l o m e r u l o s a cells f o l l o w e d the same p a t t e r n as a l d o s t e r o n e (control: 1 5 . 3 + 2 . 2 5 , 3.9xlO-8M~-M~H: 41.9+4.43, 2xlO-5M~-MSH: 1 1 8 . 3 + 2 . 6 5 ng/ml). The s l o p e s of the d o s e - r e s p o n s e c u r v e s of ACTH + O ~ - M S H w e r e s t e e p e r than those of A C T H w h e n m e a s u r e d on zona f a s c i c u l a t a c o r t i c o s t e r o n e p r o d u c t i o n (p < 0.05), on zona g l o m e r u l o s a aldos t e r o n e (p ~ 0.05) and c o r t i c o s t e r o n e (N.S.) p r o d u c t i o n (Table i.) ~ - M S H d e c r e a s e d E D 5 0 of A C T H in e a c h cell s y s t e m (Table I.) . D a t a of a t y p i c a l e x p e r i m e n t are s h o w n in Fig. 2. A lower dose of ~ - M S H (iO -II - 5 x l O -IO M) did n o t i n f l u e n c e the s l o p e and the E D 5 0 of A C T H (data are n o t p r e s e n t e d ) . Discussion The s t e r o i d o g e n i c e f f e c t of o ~ - 5 ~ H w i t h a p p r o x i m a t e l y 10 -6 of the p o t e n c y of A C T H was f i r s t s h o w n by S a y e r s et al. (i0) and L o w r y et al. (ii). In our e x p e r i m e n t s the t h r e s h o l d dose o f ~ - M S H on zona f a s c i c u l a t a c o r t i c o s t e r o n e p r o d u c t i o n was a b o u t IO-6M, t h a t of ACTH: IO-IIM. ~ - M S H p o t e n t i a t e d the e f f e c t of A C T H b o t h on zona f a s c ~ c u l a t a c o r t i c o s t e r o n e and on zona g l o m e r u l o s a a l d o s t e r o n e p r o d u c t i o n in doses t h a t i t s e l f h a d no effect. The q u e s t i o n r a i s e d at s t a r t i n g our e x p e r i m e n t s was the f o l l o w i n g : is ~ - M S H the f a c t o r that p l a y s a role in the glom e r u l o t r o p i c e f f e c t of the i n t e r m e d i a t e lobe? The a n s w e r is s u p p o s e d to be p o s i t i v e . Our a n s w e r is s u p p o r t e d by the f o l l o w i n g results: i. O{-MSH d e c r e a s e d E D 5 0 of A C T H b o t h on zona g l o m e r u l o s a a l d o s t e r o n e and on zona f a s c i c u l a t a c o r t i c o s t e r o n e p r o d u c t i o n , a l t h o u g h its e f f e c t was m o r e p r o n o u n c e d on zona g l o m e r u l o s a cells. 2. O ~ - M S H i n c r e a s e d zona g l o m e r u l o s a a l d o s t e r o n e p r o d u c t i o n at doses w h i c h did n o t a f f e c t zona f a s c i c u l a t a c o r t i c o s t e r o n e production. W h i l e our e x p e r i m e n t s w e r e a l r e a d y in p r o g r e s s , V i n s o n et al. (12) p u b l i s h e d a p a p e r s h o w i n g e v i d e n c e that ~ - M S H is the c o m p o n e n t of p o s t e r i o r p i t u i t a r y e x t r a c t s t i m u l a t i n g zona glom e r u l o s a . In t h e i r e x p e r i m e n t s 10 -9 - 10 -6 M ~ - M S H i n c r e a s e d glom e r u l o s a - , b u t d i d not a f f e c t zona f a s c i c u l a t a c o r t i c o s t e r o n e p r o d u c t i o n . We h a v e found a b i - p h a s e d o s e - r e s p o n s e curve m e a s u r e d on zona g l o m e r u l o s a a l d o s t e r o n e p r o d u c t i o n ; 10 -9 - 10 -6 M i n d u c i n g the first- and 10 -6 - 8xlO -5 M the s e c o n d p h a s e of the curve. The t h r e s h o l d dose of ~ - M S H (IO-9M) c o r r e s p o n d s to the d a t a of V i n s o n et al. ~12), h o w e v e r , they f o l l o w e d the d o s e - r e s p o n s e curve only up to IO-°M. The b i - p h a s e r e s p o n s e c o u l d be e x p l a i n e d by s u p p o s i n g t h a t O ~ - M S H b i n d s to two d i f f e r e n t r e c e p t o r systems. The first one w i t h h i g h e r a f f i n i t y c o u l d be a s p e c i f i c ~ - M S H r e c e p t o r or is p e r h a p s i d e n t i c a l w i t h the a n g i o t e n s i n r e c e p t o r . To this r e f e r s that S a r a l a s i n , a s p e c i f i c a n g i o t e n s i n a n t a g o n i s t i n h i b i t s p a r t l y the e f f e c t of lower doses of O ~ - M S H ( u n p u b l i s h e d data). The s e c o n d s y s t e m c o u l d be the A C T H r e c e p t o r s y s t e m to w h i c h O C - M S H is b o u n d at I O - 6 M and h i g h e r c o n c e n t r a t i o n s .
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The p h y s i o l o g i c a l role of o ~ - ~ H has n o t b e e n e l u c i d a t e d so far. E f f e c t of ~ - M S H on s o d i u m balance, s e b u m s e c r e t i o n and l e a r n i n g b e h a v i o r have b e e n d e m o n s t r a t e d (for ref. see Thody, 13). The q u e s t i o n arises, w h e t h e r it is p o s s i b l e that O~-MSH p a r t i c i pates in the r e g u l a t i o n of a d r e n o c o r t i c a l f u n c t i o n ? T h e r e are data in the l i t e r a t u r e r e f e r r i n g to this p o s s i b i l i t y . E v i d e n c e was p r e s e n t e d that c o r t i s o l p r o d u c t i o n by fetal h u m a n and s h e e p adrenal cells r e s p o n d e d to Ot-MSH s t i m u l a t i o n (4, 5), a l t h o u g h c o n t r a d i c t o r y results have b e e n o b t a i n e d w i t h rhesus m o n k e y fetal a d r e n a l cells (14). We s u p p o s e that both the g l o m e r u l o t r o p and the ACTH p o t e n t i a t i n g e f f e c t of ~ - M S H may be of p h y s i o l o g i c a l s i g n i f i c a n c e . A l t h o u g h the p l a s m a level of O~-MSH is u s u a l l y lower (Io-IOM) w i t h one o r d e r than the l o w e s t e f f e c t i v e c o n c e n t r a t i o n in vitro, but the s e n s i t i v i t y b e t w e e n in v i t r o and in vivo systems may be different. Under c e r t a i n c o n d i t i o n s the level of ~ - M S H can be h i g h e r than n o r m a l (for e x a m p l e in the third t r i m e s t e r of pregnancy: 15). D a t a of B i r k h ~ u s e r et al~ (16) s e e m to s u p p o r t o u r a s s u m p t i o n : they o b s e r v e d a w e a k s p e c i f i c s t i m u l a t o r y e f f e c t of O ~ - M S H on a l d o s t e r o n e p r o d u c t i o n in p a n h y p o p i t u i t a r y patients. So it is s u g g e s t e d that under c e r t a i n c o n d i t i o n s ~ - M S H m a y p a r t i cipate in the r e g u l a t i o n of a d r e n o c o r t i c a l - m a i n l y m i n e r a l o c o r t i c o i d - s t e r o i d synthesis. Acknowled@ements We thank G. Folly for the p e r f o r m a n c e of m a t h e m a t i c a l a n a l y s e s and to Mrs. A. Falus for h e r v a l u a b l e t e c h n i c a l assistance. References i. 2. 3. 4. 5. 6. 7. 8. 9. IO. ii. 12. 13.
K.Sz. SZALAY, A c t a endocr. (Kbh.) Suppl. 225, 347 (1979). K.Sz. SZALAY, A c t a Physiol. Acad. Sci. Hung. 57, 225-231 (1981). K.Sz. S Z A L A Y and E. STARK, Life Sci. 29, 1 3 5 5 - 1 3 6 1 (1981). J.A. GLICKMAN, G.D. C A R S O N and J.R.G. CHALLIS, E n d o c r i n o l o g y 104, 34-39 (1979). A.J. LLANOS, J. R A M A C H A N D R A N , R.K. CREASY, A.M. RUDOLPH and M. S E R O N - F E R P ~ , E n d o c r i n o l o g y 105, 613-617 (1979). J.F. TAIT, S.A.S. TAIT, R.P. G O U L D and M.S.R. MEE, Proc. roy. Soc. B. 185, 375-407 (1974). R. USATEGUI, C. OLIVER, H. VAUDRY, G. LOMBARDI, I. R O Z E N B E R G and A.M. MORRE, E n d o c r i n o l o g y , 98, 189-196 (1976). R. G U I L L E M I N , G.W. CLAYTON, H.S. L I P S C O M B and J.D. SMITH, J. Lab. Clin. Med. 53, 830-832 (1959). D. R O D B A R D and G.R. FRAZIER, M e t h o d s in E n z y m o l o g y 37, 1-22, A c a d e m i c Press, N e w York (1975). G. SAYERS, R.L. S W A L L O W and N.D. GIORDANO, E n d o c r i n o l o g y , 88, 1 0 6 3 - 1 0 6 8 (1971). ~.J. LOWRY, C. M C M A R T I N and J. PETERS, J. Endocr. 59, 43-55 (1973) . G.P. VINSON, B. W H I T E H O U S E , A. DELL, T. E T I E N N E and H.R. MORRIS, N a t u r e 284, 464-467 (1980). A.J. THODY, A d v a n c e s in Drug R e s e a r c h ii, 23-74 (1977).
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14. 15. 16.
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S.W. W A L S H , R.L. N O R M A N and M.J. N O V Y , E n d o c r i n o l o g y 104, 1 8 0 5 - 1 8 1 3 (1979). D. C L A R K , A.J. T H O D Y , S. S H U S T E R a n d H. B O W E R S , N a t u r e 273, 1 6 3 - 1 6 4 (1978). M. B I R K H A U S E R , R. G A I L L A R D , A.M. R I O N D E L a n d G.R. ZAHND, A c t a e n d o c r . (Kbh.) 79, 1 6 - 2 4 (1975).