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Dexamethasone increases both catecholamines and methionine-enkephalin in cultured bovine adrenal chromaffin cells and human extramedullary pheochromocytoma cells
Life Sciences, Vol. 35, pp. 1869-1875 Printed in the U.S.A.
Pergamon Press
DEXAMETHASONE INCREASES BOTH CATECHOLAMINES A N D M E T H I O N I N E - E N K E P H A L I N IN C U L T U R E D B O V I N E A D R E N A L C H R O M A F F I N C E L L S A N D HUMAN E X T R A M E D U L L A R Y P H E O C H R O M O C Y T O M A CELLS Toshihiko
Third
Yanase, Ken-ichi
H a j i m e Nawata, Kazumi Higuchi, Kato, Hiroshi Ibayashi
D e p a r t m e n t of Internal Medicine, F a c u l t y of M e d i c i n e , K y u s h u U n i v e r s i t y , F u k u o k a 812, J a p a n (Received in final form August 20, 1984) Summary
The e f f e c t of d e x a m e t h a s o n e on d i s p e r s e d cells in p r i m a r y m o n o l a y e r c u l t u r e from b o v i n e a d r e n a l m e d u l l a and h u m a n e x t r a m e d u l l a r y p h e o c h r o m o c y t o m a was e x a m i n e d by e s t i m a t i n g the level of c a t e c h o l a m i n e s (CAs) and M e t h i o n i n e - e n k e p h a l i n (Met-enk) in the m e d i u m and ceils. In c u l t u r e d b o v i n e adrenal c h r o m a f f i n cells, d e x a m e t h a sone c a u s e d s i g n i f i c a n t increase in M e t - e n k levels 18 hours a f t e r a d m i n i s t r a t i o n . T h e r e was no r e l e a s e of M e t - e n k and CAs in the m e d i u m i0 m i n a f t e r a d m i n i s t r a tion, a l t h o u g h n i c o t i n e did cause a s i g n i f i c a n t r e l e a s e of M e t - e n k and CAs. A dose r e s p o n s e increase in the level of CAs and M e t - e n k in b o v i n e adrenal c h r o m a f f i n cells w a s o b t a i n e d w i t h doses v a r y i n g b e t w e e n 0 and 1 0 - 6 M d e x a m e t h a s o n e 18 hours after a d m i n i s t r a t i o n . In c u l t u r e d h u m a n e x t r a m e d u l l a r y p h e o c h r o m o c y t o m a cells, d e x a m e h t a s o n e s i g n i f i c a n t l y i n c r e a s e d the levels of n o r e p i n e p h r i n e and M e t - e n k in a dose d e p e n d e n t m a n n e r 24 hours after a d m i n i s t r a t i o n . These r e s u l t s suggest that d e x a m e t h a s o n e does not act as a s e c r e t a g o g u e but may be r e l a t e d to the s y n t h e s i s of M e t - e n k and CAs. G l u c o c o r t i c o i d h o r m o n e s s e c r e t e d from the a d r e n a l c o r t e x reach the a d r e n a l m e d u l l a via the adrenal portal veins (i). As for the e f f e c t of g l u c o c o r t i c o i d on c a t e c h o l a m i n e s y n t h e s i s in the a d r e n a l medulla, the m a i n t e n a n c e in a c t i v i t y of phenylethanolam/neN-methyltransferase (PNMT) and d o p a m i n e - B - h y d r o x y l a s e (DBH) by g l u c o c o r t i c o i d s after a d r e n a l d e n e r v a t i o n and h y p o p h y s e c t o m y is well k n o w n (2). Large a m o u n t s of o p i o i d peptides, such as M e t - e n k enk, have been i d e n t i f i e d in the a d r e n a l m e d u l l a (3). logical role has been not e s t a b l i s h e d .
renal
and LeuThe p h y s i o -
To e l u c i d a t e the p h y s i o l o g i c a l r e l a t i o n s h i p b e t w e e n the adc o r t e x and m e d u l l a , p a r t i c u l a r l y w i t h regard to the r e g u l a -
0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
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tion of c a t e c h o l a m i n e s and o p i o i d p e p t i d e s , we e x a m i n e d the d i r e c t e f f e c t of d e x a m e t h a s o n e on the s t i m u l a t i o n of CAs and M e t - e n k in cultur~ b o v i n e a d r e n a l c h r o m a f f i n cells and h u m a n e x t a m e d u l l a r y p h e o c h r o m o c y t o m a cells. Methods Cell p r e p a r a t i o n . Five a d r e n a l g l a n d s w e r e r a p i d l y e x c i s e d from the a n i m a l s a f t e r the d e a t h and b r o u g h t to the l a b o l a t o r y on ice. A h u m a n e x t r a m e d u l l a r y p h e o c h r o m o c y t o m a w a s o b t a i n e d at surgery. I s o l a t i o n of b o v i n e a d r e n a l c h r o m a f f i n cells and tumor cells was p e r f o r m e d f o l l o w i n 9 a few m o d i f i c a t i o n s of the m e t h o d of K i l p a t r i c k et al. (4). B r i e f l y the m e d u l l a s d i s s e c t e d from the b o v i n e c o r t e x or tumor tissue w e r e minced, i n c u b a t e d w i t h Ca 2~free L o c k e ' s s o l u t i o n c o n t a i n i n 9 10% c o l l a g e n a s e (Sigma, type I) and g e n t l y s t i r r e d at 37°C for 30 min. The cells w e r e r e m o v e d by d e c a n t a t i o n and the r e s i d u e was r e - i n c u b a t e d for an a d d i t i o n a l 20 m i n w i t h 10% c o l l a g e n a s e solution. After filtration through gauze mesh, the cells w e r e p u r i f i e d t h r o u g h a g r a d i e n t of P e r c o l l (Pharmacia) and p l a t e d in p l a s t i c d i s h e s (Corning 35 nun). E a c h d i s h c o n t a i n e d 3 × l0 s cells in 1 ml of I s c o v e ' s M o d i f i e d E a g l e s M e d i u m (IMDM) (GIBCO) s u p p l e m e n t e d w i t h 10% fetal calf serum (GIBCO). The cells w e r e i n c u b a t e d at 37°C in 5% CO2 - 95% air. The f o l l o w ing day, the m e d i u m was c h a n g e d to I M D M c o n t a i n i n g 10% c h a r c o a l t r e a t e d calf s e r u m w h i c h did not c o n t a i n steroids. The cells w e r e u s e d for e x p e r i m e n t s 3 days a f t e r plating. B e f o r e the a d d i t i o n of d e x a m e t h a s o n e or nicotine, e a c h p l a t e was w a s h e d at 37°C for 15 m i n in 1.0 ml of H E P E S - b u f f e r e d K r e b s - R i n g e r saline (KRH) c o n t a i n i n g 0.2% b o v i n e s e r u m albumin, and then e x c h a n g e d by 1.0 ml of KRH cont a i n i n g 2 m M CaCI2 and 5 × 10-SM B a c i t r a c i n (Sigma). N i c o t i n e or d e x a m e t h a s o n e was a d d e d to e a c h plate. A f t e r i n c u b a t i o n , a i00 ~i of the m e d i u m was a c i d i f i e d w i t h 25 ~i of 2 M p e r c h l o l i c acid for s u b s e q u e n t e s t i m a t i o n of c a t e c h o l a m i n e and the r a m a i n i n g 900 ~i w a s a c i d i d i e d w i t h 900 ~i of 2 M acetic a c i d for s u b s e q u e n t e x t r a c t i o n of Met-enk. The r e m a i n i n g cells, w h i c h were s c r a p e d in 1.0 ml of KRH w i t h r u b b e r p o l i c e m a n , w e r e a c i d i f i e d for the assays of CAs and Met-enk respectively. The p r o t e i n c o n t e n t s of the c o n t r o l and exp e r i m e n t a l cells w e r e m e a s u r e d by the m e t h o d of L o w r y (5). Catecholamines determination. N o r e p i n e p h r i n e and e p i n e p h r i n e in the m e d i u m and c u l t u r e d cells w e r e d e t e r m i n e d by HPLC (Hitachi, 635 A) w i t h o u t initial p u r i f i c a t i o n on alumina. HPLC was c a r r i e d out on a N u c l e o s o l sC18 c o l u m n (0.4 × 15 cm). The samples w e r e a p p l i e d to the c o l u m n and e l u t e d w i t h 0.i M p h o s p h a t e b u f f e r (pH 2.9, flow rate 0.6 ml/min) and m e a s u r e d by a u t o a n a l y z e r f l u o r e s cence m e t h o d s and the t r i h y d r o x y i n d o l r e a c t i o n (6). The s e n s i t i v ity of the a s s a y was i0 pg/ml. A s s a y s of M e t - e n k e p h a l i n . The samples for M e t - e n k a s s a y w e r e l y o p h i l i z e d , r e c o n s t i t u t e d in i0 m M s o d i u m p h o s p h a t e b u f f e r (pH 7.5) s u p p l e m e n t e d w i t h 0.01% b o v i n e s e r u m alubumin, e x t r a c t e d u s i n 9 O D S - s i l l i c a c o l l u m n and a s s a y e d by RIA u s i n 9 M e t - k i t s of the Immuno-Nuclear-Corporation (U.S.A.). The s e n s i t i v i t y of the a s s a y was 8 p i c o g r a m s . The % cross r e a c t i v i t y of the used a n t i b o d y was 2.8% to l e u c i n e - e n k e p h a l i n , 0.1% to B-LPH, and b e l o w 0.002% to Bendorphine.
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Dexamethasone and Adrenal Medulla
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Results The t i m e c o u r s e of the l e v e l of M e t - e n k (medium p l u s cell) s t i m u l a t e d by 1 0 - 6 M d e x a m e t h a s o n e in c u l t u r e d b o v i n e c h r o m a f f i n c e l l s is s h o w n in Fi 9. i. M e t - e n k b e g a n to i n c r e a s e 3 h o u r s a f t e r 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 a n d r e a c h e d the m a x i m u n level 18 h o u r s a f t e r t r e a t m e n t , w h e n it was 1.3 fold g r e a t e r t h a n t h a t of c o n t r o l (p ~ 0.05). The l e v e l of b o t h CAs and M e t - e n k in c u l t u r e d b o v i n e a d r e n a l c h r o m a f f i n c e l l s i n c r e a s e d in a d o s e d e p e n d e n t m a n n e r 18 hours after dexamethasone administration (Fi 9. 2). D u r i n g the a d d i t i o n of d e x a m e t h a s o n e , the p r o t e i n c o n t e n t s of the c o n t r o l and e x p e r i m e n t a l c e l l s w e r e not d i f f e r e n t . 10-~M nicotine stimulated the r e l e a s e of b o t h M e t - e n k and CAs in the m e d i u m of c u l t u r e d b o v i n e a d r e n a l c h r o m a f f i n c e l l s i0 m i n a f t e r a d m i n i s t r a t i o n (Fi 9. 3). In c u l t u r e d h u m a n e x t r a m e d u l l a r y p h e o c h r o m o c y t o m a c e l l s , d e x a m e t h a s o n e s i g n i f i c a n t l y i n c r e a s e d the level of b o t h M e t - e n k and n o r e p i n e p h r i n e in a d o s e d e p e n d e n t m a n n e r (Fig. 4).
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Discussion Not only t r a n s - s y n a p t i c i n d u c t i o n via the s p l a c h n i c n e r v e s (7), but also g l u c o c o r t i c o i d s s e c r e t e d from the a d r e n a l c o r t e x into the m e d u l l a via the a d r e n a l p o r t a l veins play an i m p o r t a n t role in the r e g u l a t i o n of c a t e c h o l a m i n e b i o s y n t h e s i s . It has b e e n r e p o r t e d that g l u c o c o r t i c o i d s cause an i n d u c t i o n of CA e n z y m e s PNMT and DBH in h y p o p h y s e c t o m i z e d rats but not in intact rats (2). F r o m det a i l e d i n v e s t i g a t i o n on DBH (8) and P N M T (9) in CA b i o s y n t h e s i s r e s p e c t i v e l y , it is t h o u g h t that g l u c o c o r t i c o i d m a i n t a i n s the level of DBH and PNMT, s y n t h e s i z e d t r a n s - s y n a p t i c a l l y , by i n h i b i t i n g the enzyme degradation. H e r s e y & D i s t e f a n o (i0) n o t e d the m a i n t e n a n c e of PNMT a c t i v i t y by g l u c o c o r t i c o i d s in c u l t u r e d b o v i n e a d r e n a l c h r o m a f f i n cells. I n d u c t i o n of t y r o s i n e h y d r o x h l a s e (TH) by g l u c o c o r t i c o i d s in the rat a d r e n a l m e d u l l a was r e p o r t e d to be n e g a t i v e (2) or p o s i t i v e by G u i d o t t i et al. (ii). In the a d r e n a l tumor cell
Vol. 35, No. 18, 1984
Dexamethasone and Adrenal Medulla
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E f f e c t s of n i c o t i n e and d e x a m e t h a s o n e on c a t e c h o l a m i n e and M e t - e n k levels s e c r e t e d in the m e d i u m for 10 min in c u l t u r e d b o v i n e adrenal c h r o m a f f i n cells. 0: Control, N: 1 0 - S M nicotine, D: 1 0 - 6 M d e x a m e t h a s o n e The v a l u e r e p r e s e n t s m e a n ± SE. n=3. **p ~ 0.01 line, little is k n o w n of CA s y n t h e s i s o t h e r than the findings that g l u c o c o r t i c o i d c a u s e d an increase of CA s y n t h e s i s and TH a c t i v i t y in the PC 12 cell line (rat p h e o c h r o m o c y t o m a ) (12, 13). We i n v e s t i g a t e d the d i r e c t effect of d e x a m e t h a s o n e by e s t i m a ting CA levels in c u l t u r e d b o v i n e adrenal c h r o m a f f i n cells and h u m a n e x t r a m e d u l l a r y p h e o c h r o m o c y t o m a cells. We found an increase in CA c o n c e n t r a t i o n by d e x a m e t h a s o n e . A l t h o u g h the exzyme a c t i v i t y r e g u l a t i n g CA s y n t h e s i s was not m e a s u r e d , the i n c r e a s e in norepinephrine levels s u g g e s t e d that d e x a m e t h a s o n e a f f e c t e d b o t h PNMT and DBH or TH. Since the c u l t u r e was m a i n t a i n e d in 10% c h a r c o a l t r e a t e d calf serum, the results we o b t a i n e d suggest that the dec l i n e of CA e n z y m e a c t i v i t y for lack of g l u c o c o r t i c o i d s was restored w i t h the a d d i t i o n of d e x a m e t h a s o n e . At the same time, we found a slight but s i g n i f i c a n t increase in M e t - e n k by d e x a m e t h a s o n e after long t r e a t m e n t in both c u l t u r e d
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Dexamethasone and Adrenal Medulla
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E f f e c t of d e x a m e t h a s o n e on n o r e p i n e p h r i n e a n d M e t - e n k l e v e l s in c u l t u r e d h u m a n e x t r a m e d u l l a r y p h e o c h r o m o c y t o m a c e l l s 24 h o u r s a f t e r the a d d i t i o n of d e x a methasone. The v a l u e r e p r e s e n t s m e a n ± SE. n=3. *p < 0.05, • *p < 0 . 0 1 b o v i n e a d r e n a l c h r o m a f f i n c e l l s and h u m a n e x t r a m e d u l l a r y p h e o chromocytoma cells. T h e s e f i n d i n g s are in a c c o r d a n c e w i t h a r e p o r t t h a t d e x a m e t h a s o n e c a u s e d a b o u t a 2 f o l d i n c r e a s e in i n t r a c e l l u l a r c o n c e n t r a t i o n s of o p i o i d p e p t i d e s in n e u r o b l a s t o m a x g l i o m a h y b r i d c e l l s in c u l t u r e for 24 h o u r s i n c u b a t i o n (14). In c o n t r a s t , B o h n et al. (15) r e p o r t e d t h a t c h r o n i c a d m i n i s t r a t i o n of d e x a m e t h a s o n e d i d not l e a d to an e n k e p h a l i n i m m u n o r e a c t i v i t y in the rat a d r e n a l medulla. T h e i r m e t h o d of i m m u n o f l u o r e s c e n c e d e t e c t i o n m a y be t o o g l o b a l to d e t e c t s l i g h t i n c r e a s e s in l e v e l s of o p i o i d p e p t i d e s . T h e i n c r e a s e in the l e v e l of M e t - e n k by d e x a m e t h a s o n e m a y be exp l a i n e d by v a r i o u s p o s s i b i l i t i e s ; e n h a n c e d s y n t h e s i s , d e c r e a s e d r a t e of d e g r a d a t i o n or i n c r e a s e d p r o c e s s i n g of a p r e v i o u s l y p e r formed precursor. Dexamethasone
did not
have
the
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of
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n i c o t i n e w h i c h is well known to increase the s e c r e t i o n of CA and o p i o i d p e p t i d e s s i m u l t a n e o u s l y from bovine adrenal c h r o m a f f i n cells (16). G l u c o c o r t i c o i d s from the adrenal cortex m a y p r o m o t e the s y n t h e s i s of both CAs and M e t - e n k in the b o v i n e adrenal medulla. Acknowledgement We thank T. S h i m a b u k u r o levels of c a t e c h o l o m i n e s .
for t e c h n i c a l
assistance
in m e a s u r i n g
References i. 2. 3. 4. 5. 6.
7 8 9 i0 ii 12 13 14 15 16
R.G. HRRISON, M.J. HOEY; Blaekwell, Oxford, pp 1-77 (1960). J. AXELROD; A n n . N . Y . A c a d . Sci. 297, 275-283 (1977). A.M. DI GIULIO, H.Y. YANG, W. F R A T T A and E. COSTA; Nature 278, 646-647 (1979). D.L. K I L P A T R I C K , F.H. LEDBETTER, K.A. CARSON, A.G. KIRSHNER, R. S L E P E R T I S and N. KIRSHNER; J . N e u r o c h e m 35, 679-692 (1980). O.H. Lowry, N.J. Rosebrouqh, A.L. F A R R and R.J. RUDALL; J . B i o l . C h e m . 193, 265- 275 (1951). E. UEDA, N. YOSHIDA, K. NISHIMURA, T. JOH, S. ANTOKU, K. TSUKADA, S. G A N N O and T. KOKUBO; C l i n . C l i n . A c t a . 80, 447453 (1977). D.R. C I A R A N E L L O , F.G. W O O T E N and J. AXELROD; J . B i o l . C h e m . 250, 3204-4311 (1975). D.R. C I A R A N E L L O , F.G. W O O T E N and J. AXELROD; Brain. Res. 113, 349-362 (1976). D.R. C I A R A N E L L O , F.G. W O O T E N and J. AXELROD; M o l . P h a r m a c o l 14, 478-489 (1978). M.R. H E R S E Y and V. DISTEFANO; J . P h a r m a c o l . E x p . T h e r a p . 209, 147-152 (1979). A. GUIDOTTI, I. H A B A U E R and E. COSTA; A d v a n c e s in C y c l i c N u c l e o t i d e R e s e a r c h 15, 619-639 (1975). D. SCHBERT, M. LACORBIERE, F.G. K L I E R and J.H. STEINBACH; Brain. Res. 190, 67-79 (1980). A.S. TISHLER, R.L. PERLMAN, G.M. MORSE and B.E. SHEARD; J . N e u r o c h e m . 40, 364-370 (1983). T. GLASER, K . H U B N E R and B. HAMPRECHT; FEBS LETTERS 131, 63-67 (1981). M.C. BOHN, J.A. KESSLER, L. G O L I G H T L Y and I.B. BLACK; Cell T i s s u e Res 231, 469-479 (1983). B.G. LIVETT, D.M. DEAN, L.G. WHELAN, S. U D E N F R I E N D and J. ROSSIER; N a t u r e 289, 317-318 (1981).
Pergamon Press
DEXAMETHASONE INCREASES BOTH CATECHOLAMINES A N D M E T H I O N I N E - E N K E P H A L I N IN C U L T U R E D B O V I N E A D R E N A L C H R O M A F F I N C E L L S A N D HUMAN E X T R A M E D U L L A R Y P H E O C H R O M O C Y T O M A CELLS Toshihiko
Third
Yanase, Ken-ichi
H a j i m e Nawata, Kazumi Higuchi, Kato, Hiroshi Ibayashi
D e p a r t m e n t of Internal Medicine, F a c u l t y of M e d i c i n e , K y u s h u U n i v e r s i t y , F u k u o k a 812, J a p a n (Received in final form August 20, 1984) Summary
The e f f e c t of d e x a m e t h a s o n e on d i s p e r s e d cells in p r i m a r y m o n o l a y e r c u l t u r e from b o v i n e a d r e n a l m e d u l l a and h u m a n e x t r a m e d u l l a r y p h e o c h r o m o c y t o m a was e x a m i n e d by e s t i m a t i n g the level of c a t e c h o l a m i n e s (CAs) and M e t h i o n i n e - e n k e p h a l i n (Met-enk) in the m e d i u m and ceils. In c u l t u r e d b o v i n e adrenal c h r o m a f f i n cells, d e x a m e t h a sone c a u s e d s i g n i f i c a n t increase in M e t - e n k levels 18 hours a f t e r a d m i n i s t r a t i o n . T h e r e was no r e l e a s e of M e t - e n k and CAs in the m e d i u m i0 m i n a f t e r a d m i n i s t r a tion, a l t h o u g h n i c o t i n e did cause a s i g n i f i c a n t r e l e a s e of M e t - e n k and CAs. A dose r e s p o n s e increase in the level of CAs and M e t - e n k in b o v i n e adrenal c h r o m a f f i n cells w a s o b t a i n e d w i t h doses v a r y i n g b e t w e e n 0 and 1 0 - 6 M d e x a m e t h a s o n e 18 hours after a d m i n i s t r a t i o n . In c u l t u r e d h u m a n e x t r a m e d u l l a r y p h e o c h r o m o c y t o m a cells, d e x a m e h t a s o n e s i g n i f i c a n t l y i n c r e a s e d the levels of n o r e p i n e p h r i n e and M e t - e n k in a dose d e p e n d e n t m a n n e r 24 hours after a d m i n i s t r a t i o n . These r e s u l t s suggest that d e x a m e t h a s o n e does not act as a s e c r e t a g o g u e but may be r e l a t e d to the s y n t h e s i s of M e t - e n k and CAs. G l u c o c o r t i c o i d h o r m o n e s s e c r e t e d from the a d r e n a l c o r t e x reach the a d r e n a l m e d u l l a via the adrenal portal veins (i). As for the e f f e c t of g l u c o c o r t i c o i d on c a t e c h o l a m i n e s y n t h e s i s in the a d r e n a l medulla, the m a i n t e n a n c e in a c t i v i t y of phenylethanolam/neN-methyltransferase (PNMT) and d o p a m i n e - B - h y d r o x y l a s e (DBH) by g l u c o c o r t i c o i d s after a d r e n a l d e n e r v a t i o n and h y p o p h y s e c t o m y is well k n o w n (2). Large a m o u n t s of o p i o i d peptides, such as M e t - e n k enk, have been i d e n t i f i e d in the a d r e n a l m e d u l l a (3). logical role has been not e s t a b l i s h e d .
renal
and LeuThe p h y s i o -
To e l u c i d a t e the p h y s i o l o g i c a l r e l a t i o n s h i p b e t w e e n the adc o r t e x and m e d u l l a , p a r t i c u l a r l y w i t h regard to the r e g u l a -
0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
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tion of c a t e c h o l a m i n e s and o p i o i d p e p t i d e s , we e x a m i n e d the d i r e c t e f f e c t of d e x a m e t h a s o n e on the s t i m u l a t i o n of CAs and M e t - e n k in cultur~ b o v i n e a d r e n a l c h r o m a f f i n cells and h u m a n e x t a m e d u l l a r y p h e o c h r o m o c y t o m a cells. Methods Cell p r e p a r a t i o n . Five a d r e n a l g l a n d s w e r e r a p i d l y e x c i s e d from the a n i m a l s a f t e r the d e a t h and b r o u g h t to the l a b o l a t o r y on ice. A h u m a n e x t r a m e d u l l a r y p h e o c h r o m o c y t o m a w a s o b t a i n e d at surgery. I s o l a t i o n of b o v i n e a d r e n a l c h r o m a f f i n cells and tumor cells was p e r f o r m e d f o l l o w i n 9 a few m o d i f i c a t i o n s of the m e t h o d of K i l p a t r i c k et al. (4). B r i e f l y the m e d u l l a s d i s s e c t e d from the b o v i n e c o r t e x or tumor tissue w e r e minced, i n c u b a t e d w i t h Ca 2~free L o c k e ' s s o l u t i o n c o n t a i n i n 9 10% c o l l a g e n a s e (Sigma, type I) and g e n t l y s t i r r e d at 37°C for 30 min. The cells w e r e r e m o v e d by d e c a n t a t i o n and the r e s i d u e was r e - i n c u b a t e d for an a d d i t i o n a l 20 m i n w i t h 10% c o l l a g e n a s e solution. After filtration through gauze mesh, the cells w e r e p u r i f i e d t h r o u g h a g r a d i e n t of P e r c o l l (Pharmacia) and p l a t e d in p l a s t i c d i s h e s (Corning 35 nun). E a c h d i s h c o n t a i n e d 3 × l0 s cells in 1 ml of I s c o v e ' s M o d i f i e d E a g l e s M e d i u m (IMDM) (GIBCO) s u p p l e m e n t e d w i t h 10% fetal calf serum (GIBCO). The cells w e r e i n c u b a t e d at 37°C in 5% CO2 - 95% air. The f o l l o w ing day, the m e d i u m was c h a n g e d to I M D M c o n t a i n i n g 10% c h a r c o a l t r e a t e d calf s e r u m w h i c h did not c o n t a i n steroids. The cells w e r e u s e d for e x p e r i m e n t s 3 days a f t e r plating. B e f o r e the a d d i t i o n of d e x a m e t h a s o n e or nicotine, e a c h p l a t e was w a s h e d at 37°C for 15 m i n in 1.0 ml of H E P E S - b u f f e r e d K r e b s - R i n g e r saline (KRH) c o n t a i n i n g 0.2% b o v i n e s e r u m albumin, and then e x c h a n g e d by 1.0 ml of KRH cont a i n i n g 2 m M CaCI2 and 5 × 10-SM B a c i t r a c i n (Sigma). N i c o t i n e or d e x a m e t h a s o n e was a d d e d to e a c h plate. A f t e r i n c u b a t i o n , a i00 ~i of the m e d i u m was a c i d i f i e d w i t h 25 ~i of 2 M p e r c h l o l i c acid for s u b s e q u e n t e s t i m a t i o n of c a t e c h o l a m i n e and the r a m a i n i n g 900 ~i w a s a c i d i d i e d w i t h 900 ~i of 2 M acetic a c i d for s u b s e q u e n t e x t r a c t i o n of Met-enk. The r e m a i n i n g cells, w h i c h were s c r a p e d in 1.0 ml of KRH w i t h r u b b e r p o l i c e m a n , w e r e a c i d i f i e d for the assays of CAs and Met-enk respectively. The p r o t e i n c o n t e n t s of the c o n t r o l and exp e r i m e n t a l cells w e r e m e a s u r e d by the m e t h o d of L o w r y (5). Catecholamines determination. N o r e p i n e p h r i n e and e p i n e p h r i n e in the m e d i u m and c u l t u r e d cells w e r e d e t e r m i n e d by HPLC (Hitachi, 635 A) w i t h o u t initial p u r i f i c a t i o n on alumina. HPLC was c a r r i e d out on a N u c l e o s o l sC18 c o l u m n (0.4 × 15 cm). The samples w e r e a p p l i e d to the c o l u m n and e l u t e d w i t h 0.i M p h o s p h a t e b u f f e r (pH 2.9, flow rate 0.6 ml/min) and m e a s u r e d by a u t o a n a l y z e r f l u o r e s cence m e t h o d s and the t r i h y d r o x y i n d o l r e a c t i o n (6). The s e n s i t i v ity of the a s s a y was i0 pg/ml. A s s a y s of M e t - e n k e p h a l i n . The samples for M e t - e n k a s s a y w e r e l y o p h i l i z e d , r e c o n s t i t u t e d in i0 m M s o d i u m p h o s p h a t e b u f f e r (pH 7.5) s u p p l e m e n t e d w i t h 0.01% b o v i n e s e r u m alubumin, e x t r a c t e d u s i n 9 O D S - s i l l i c a c o l l u m n and a s s a y e d by RIA u s i n 9 M e t - k i t s of the Immuno-Nuclear-Corporation (U.S.A.). The s e n s i t i v i t y of the a s s a y was 8 p i c o g r a m s . The % cross r e a c t i v i t y of the used a n t i b o d y was 2.8% to l e u c i n e - e n k e p h a l i n , 0.1% to B-LPH, and b e l o w 0.002% to Bendorphine.
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Results The t i m e c o u r s e of the l e v e l of M e t - e n k (medium p l u s cell) s t i m u l a t e d by 1 0 - 6 M d e x a m e t h a s o n e in c u l t u r e d b o v i n e c h r o m a f f i n c e l l s is s h o w n in Fi 9. i. M e t - e n k b e g a n to i n c r e a s e 3 h o u r s a f t e r 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 a n d r e a c h e d the m a x i m u n level 18 h o u r s a f t e r t r e a t m e n t , w h e n it was 1.3 fold g r e a t e r t h a n t h a t of c o n t r o l (p ~ 0.05). The l e v e l of b o t h CAs and M e t - e n k in c u l t u r e d b o v i n e a d r e n a l c h r o m a f f i n c e l l s i n c r e a s e d in a d o s e d e p e n d e n t m a n n e r 18 hours after dexamethasone administration (Fi 9. 2). D u r i n g the a d d i t i o n of d e x a m e t h a s o n e , the p r o t e i n c o n t e n t s of the c o n t r o l and e x p e r i m e n t a l c e l l s w e r e not d i f f e r e n t . 10-~M nicotine stimulated the r e l e a s e of b o t h M e t - e n k and CAs in the m e d i u m of c u l t u r e d b o v i n e a d r e n a l c h r o m a f f i n c e l l s i0 m i n a f t e r a d m i n i s t r a t i o n (Fi 9. 3). In c u l t u r e d h u m a n e x t r a m e d u l l a r y p h e o c h r o m o c y t o m a c e l l s , d e x a m e t h a s o n e s i g n i f i c a n t l y i n c r e a s e d the level of b o t h M e t - e n k and n o r e p i n e p h r i n e in a d o s e d e p e n d e n t m a n n e r (Fig. 4).
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Discussion Not only t r a n s - s y n a p t i c i n d u c t i o n via the s p l a c h n i c n e r v e s (7), but also g l u c o c o r t i c o i d s s e c r e t e d from the a d r e n a l c o r t e x into the m e d u l l a via the a d r e n a l p o r t a l veins play an i m p o r t a n t role in the r e g u l a t i o n of c a t e c h o l a m i n e b i o s y n t h e s i s . It has b e e n r e p o r t e d that g l u c o c o r t i c o i d s cause an i n d u c t i o n of CA e n z y m e s PNMT and DBH in h y p o p h y s e c t o m i z e d rats but not in intact rats (2). F r o m det a i l e d i n v e s t i g a t i o n on DBH (8) and P N M T (9) in CA b i o s y n t h e s i s r e s p e c t i v e l y , it is t h o u g h t that g l u c o c o r t i c o i d m a i n t a i n s the level of DBH and PNMT, s y n t h e s i z e d t r a n s - s y n a p t i c a l l y , by i n h i b i t i n g the enzyme degradation. H e r s e y & D i s t e f a n o (i0) n o t e d the m a i n t e n a n c e of PNMT a c t i v i t y by g l u c o c o r t i c o i d s in c u l t u r e d b o v i n e a d r e n a l c h r o m a f f i n cells. I n d u c t i o n of t y r o s i n e h y d r o x h l a s e (TH) by g l u c o c o r t i c o i d s in the rat a d r e n a l m e d u l l a was r e p o r t e d to be n e g a t i v e (2) or p o s i t i v e by G u i d o t t i et al. (ii). In the a d r e n a l tumor cell
Vol. 35, No. 18, 1984
Dexamethasone and Adrenal Medulla
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E f f e c t s of n i c o t i n e and d e x a m e t h a s o n e on c a t e c h o l a m i n e and M e t - e n k levels s e c r e t e d in the m e d i u m for 10 min in c u l t u r e d b o v i n e adrenal c h r o m a f f i n cells. 0: Control, N: 1 0 - S M nicotine, D: 1 0 - 6 M d e x a m e t h a s o n e The v a l u e r e p r e s e n t s m e a n ± SE. n=3. **p ~ 0.01 line, little is k n o w n of CA s y n t h e s i s o t h e r than the findings that g l u c o c o r t i c o i d c a u s e d an increase of CA s y n t h e s i s and TH a c t i v i t y in the PC 12 cell line (rat p h e o c h r o m o c y t o m a ) (12, 13). We i n v e s t i g a t e d the d i r e c t effect of d e x a m e t h a s o n e by e s t i m a ting CA levels in c u l t u r e d b o v i n e adrenal c h r o m a f f i n cells and h u m a n e x t r a m e d u l l a r y p h e o c h r o m o c y t o m a cells. We found an increase in CA c o n c e n t r a t i o n by d e x a m e t h a s o n e . A l t h o u g h the exzyme a c t i v i t y r e g u l a t i n g CA s y n t h e s i s was not m e a s u r e d , the i n c r e a s e in norepinephrine levels s u g g e s t e d that d e x a m e t h a s o n e a f f e c t e d b o t h PNMT and DBH or TH. Since the c u l t u r e was m a i n t a i n e d in 10% c h a r c o a l t r e a t e d calf serum, the results we o b t a i n e d suggest that the dec l i n e of CA e n z y m e a c t i v i t y for lack of g l u c o c o r t i c o i d s was restored w i t h the a d d i t i o n of d e x a m e t h a s o n e . At the same time, we found a slight but s i g n i f i c a n t increase in M e t - e n k by d e x a m e t h a s o n e after long t r e a t m e n t in both c u l t u r e d
1874
Dexamethasone and Adrenal Medulla
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E f f e c t of d e x a m e t h a s o n e on n o r e p i n e p h r i n e a n d M e t - e n k l e v e l s in c u l t u r e d h u m a n e x t r a m e d u l l a r y p h e o c h r o m o c y t o m a c e l l s 24 h o u r s a f t e r the a d d i t i o n of d e x a methasone. The v a l u e r e p r e s e n t s m e a n ± SE. n=3. *p < 0.05, • *p < 0 . 0 1 b o v i n e a d r e n a l c h r o m a f f i n c e l l s and h u m a n e x t r a m e d u l l a r y p h e o chromocytoma cells. T h e s e f i n d i n g s are in a c c o r d a n c e w i t h a r e p o r t t h a t d e x a m e t h a s o n e c a u s e d a b o u t a 2 f o l d i n c r e a s e in i n t r a c e l l u l a r c o n c e n t r a t i o n s of o p i o i d p e p t i d e s in n e u r o b l a s t o m a x g l i o m a h y b r i d c e l l s in c u l t u r e for 24 h o u r s i n c u b a t i o n (14). In c o n t r a s t , B o h n et al. (15) r e p o r t e d t h a t c h r o n i c a d m i n i s t r a t i o n of d e x a m e t h a s o n e d i d not l e a d to an e n k e p h a l i n i m m u n o r e a c t i v i t y in the rat a d r e n a l medulla. T h e i r m e t h o d of i m m u n o f l u o r e s c e n c e d e t e c t i o n m a y be t o o g l o b a l to d e t e c t s l i g h t i n c r e a s e s in l e v e l s of o p i o i d p e p t i d e s . T h e i n c r e a s e in the l e v e l of M e t - e n k by d e x a m e t h a s o n e m a y be exp l a i n e d by v a r i o u s p o s s i b i l i t i e s ; e n h a n c e d s y n t h e s i s , d e c r e a s e d r a t e of d e g r a d a t i o n or i n c r e a s e d p r o c e s s i n g of a p r e v i o u s l y p e r formed precursor. Dexamethasone
did not
have
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of
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Dexamethasone and Adrenal Medulla
1875
n i c o t i n e w h i c h is well known to increase the s e c r e t i o n of CA and o p i o i d p e p t i d e s s i m u l t a n e o u s l y from bovine adrenal c h r o m a f f i n cells (16). G l u c o c o r t i c o i d s from the adrenal cortex m a y p r o m o t e the s y n t h e s i s of both CAs and M e t - e n k in the b o v i n e adrenal medulla. Acknowledgement We thank T. S h i m a b u k u r o levels of c a t e c h o l o m i n e s .
for t e c h n i c a l
assistance
in m e a s u r i n g
References i. 2. 3. 4. 5. 6.
7 8 9 i0 ii 12 13 14 15 16
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