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I. Catecholamines in developing rat lung
Life Sciences, Vol. 28, pp. 331-337 Printed in the U.S.A.
Pergamon Press
CURRENT CONCEPTS: I. C A T E C H O L A M I N E S
IN D E V E L O P I N G
RAT
LUNG
Christiane Gardey-Levassort, Marie-Odile Richard D a n i ~ l e de L a u t u r e , G e n e v i e v e T h i r o u x and G e o r g e s O l i v e
24,
Laboratoire de P h a r m a c o l o g i e Biochimique P~rinatale rue du F a u ~ o u r g S a i n t - J a c q u e s . F - 7 5 0 1 4 P a r i s (France)
Summary The d e v e l o p m e n t of the c a t e c h o l a m i n e r g i c system of lung was i n v e s t i g a t e d in S p r a g u e - D a w l e y rats d u r i n g the p e r i n a t a l p e r i o d and in y o u n g a d u l t s . A radio-enzymatic p r o c e d u r e a l l o w e d to m e a s u r e as little as I pg of n o r a d r e n a l i n e or d o p a m i n e in each sample. The d o p a m i n e l e v e l s w e r e low and i n d e p e n d a n t of age. The n o r a d r e n a l i n e l e v e l s inc r e a s e d s i g n i f i c a n t l y w i t h the age u n t i l the 20th p o s t - p a r t u m day. No s i g n i f i c a n t d i f f e r e n c e s were found b e t w e e n n o r a d r e n a l i n e l e v e l s for m a l e s vs f e m a l e s at any age. Small d i f f e r e n c e s b e t w e e n m a l e s and f e m a l e s for d o p a m i n e levels e x i s t e d o n l y near birth. The d e v e l o p m e n t of the c a t e c h o l a m i n e r g i c s y s t e m in rat lung not b e e n i n v e s t i g a t e d w i t h b i o c h e m i c a l m e t h o d s . The s e n s i t i v i t i e s of s p e c t r o f l u o r o m e t r i c p r o c e d u r e s and also radioenzymatic ones, such as t h o s e of C o y l e and H e n r y (5), G a u c b y et al. (9) w e r e not s u f f i c i e n t for this r e s e a r c h since the m i n i m u m a m o u n t s they were able to d e t e c t were 50 pg and I0 pg for nor adrenaline (NA) and d o p a m i n e (DA) r e s p e c t i v e l y . To o v e r c o m e this p r o b l e m , we have used the m e t h o d d e s c r i b e d by Da P r a d a and Z~rchef (6) w h i c h has t e n - f o l d i n c r e a s e d s e n s i t i v i t y , compared with the o t h e r m e t h o d s . This has a l l o w e d us to d e t e r m i n e DA and NA levels in the lung t i s s u e d u r i n g p e r i n a t a l d e v e l o p m e n t and in the a d u l t rat, and to c o m p a r e m a l e s w i t h f e m a l e s . has
Materials
and
methods
Lung s a m p l i n g . Sprague-Dawley f e m a l e rats, w e i g h i n g 250 to 300 g, kept at 26 ~ ± ;°C w i t h n a t u r a l light and fed on s t a n d a r d diet w e r e used in this study. T h e y w e r e p l a c e d o v e r n i g h t w i t h m a les and the n e x t m o r n i n g was d e s i g n a t e d as day zero of g e s t a t i o n . U n t i l day 21 of g e s t a t i o n the f e m a l e s w e r e c a g e d in g r o u p s of 6 and then i s o l a t e d . All the l i t t e r s w e r e r e d u c e d to 8 pups. P r e g n a n t rats w e r e k i l l e d by d e c a p i t a t i o n (Decapitator Harw a r d A p p a r a t u s Cy, M a s s , USA) on days 17, 19, 20, 21 of g e s t a t i o n b e t w e e n 10 a.m. and ] p.m. to e l i m i n a t e d i u r n a l v a r i a t i o n s . Uteri
and
f e t u s e s w e r e i m m e d i a t e l y e x c i s e d and 0024-3205/81/040331-07502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
placed
in
332
Catecholamines in Developing Rat Lung
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crushed ice. T h e s e x e of f e t u s e s w a s d e t e r m i n e d and each animal was weighed a n d b l e d by d e c a p i t a t i o n . The lungs were removed, f r o z e n in l i q u i d n i t r o g e n , weighed a n d k e p t at - 8 0 ° C u n t i l a s s a y . Newborn rats were killed immediately after birth before they s t a r t e d to s u c k . T h e o t h e r s , in p o s t n a t a l p e r i o d , w e r e k i l l e d on d a y s 1, 2, 5, 10, 20, 60, u s i n g the s a m e p r o c e s s as b e f o r e . The young rats were separated from mothers on d a y 20 a n d the m a l e s f r o m the f e m a l e s on d a y 30 p o s t - p a r t u m . At d a y 60, t h e r a t s w e r e considered to be an a d u l t . In e a c h c a s e , the l u n g s w e r e r e m o v e d as previously. A s s a y of D A and NA. The lung DA and NA levels were determined simultaneously by the r a d i o e n z y m a t i c m e t h o d of Da P r a d a a n d Zurcher (6). T h e rat l i v e r c a t e c h o l - O - m e t h y l transferase (COMT) was purified by the p r o c e d u r e of A x e l r o d and T o m c h i c k (2) m o d i f i e d by Parvez and Gripois (12). T h e e n z y m e a c t i v i t y of e a c h b a t c h w a s c h e c k e d by the A s s i c o t and B o h u o n a s s a y (1). The following modifications were used : --I) as l u n g t i s s u e m a y be c o n t a m i n a t e d with blood, the h o m o g e n a tes w e r e p r e p a r e d with EGTA, a chelating a g e n t for Ca ++ i o n s , to prevent the Ca ++ i n h i b i t o r y e f f e c t on C O M T a c t i v i t y , as the authors suggested for p l a s m a samples. Then, lung tissue was homogeneized in 50 v o l . of i c e - c o l d 0.3 N p e r c h l o r i c acid containing 0 . 1 % E G T A and 0 . 0 1 % M g C I ,6H O. 2
2
-2) as the f e t a l t i s s u e c a t e c h o l a m i n e s were expected to be f o u n d at v e r y l o w l e v e l s , 5 ~ C i / t u b e of S - a d e n o s y l - L - m e t h i o n i n e methyl-3H (SAM) w e r e u s e d i n s t e a d of 2 N C i / t u b e (specific activity 10-12 Ci/mMole, Amersham). In t h e s e c o n d i t i o n s as l i t t l e as I pg of D A or N A in the s a m p l e w a s m e a s u r e d . The last modification changed the s l o p e of the D A s t a n d a r d c u r v e for a r a n g e of v a l u e s f r o m 1 pg to I00 pg, b u t n o t t h a t of NA. T a b l e 1 shows a significantly better labelling of the D A external standard. Moreover, using 5 ~Ci/tube b o t h the D A a n d N A internal standard values were not significantly different f r o m the external standard ones, while they did, when 2 uCi/tube were used as Da P r a d a a n d Z u r c h e r (6), ( t a b l e 2). O n e s a m p l e of e a c h age b e f o r e and a f t e r b i r t h w a s t r e a t e d simultaneously in the s a m e e x p e r i m e n t with both internal and external standards, except the 2 m o n t h s samples treated together in two s e r i e s . The internal standards were distributed at r a n d o m in the s a m p l e s of the d i f f e r e n t a g e s a m o n g the e x p e r i m e n t s . We c h e c k e d w h e ther a degradation of the a u t h e n t i c D A or N A a d d e d as i n t e r n a l standards, occured as a f u n c t i o n of the t i s s u e m a t u r i t y . With this a i m in v i e w , a t w o - w a y a n a l y s i s of v a r i a n c e (anova) was performed taking into account both factors : age and internal or e x t e r n a l standard. A significant difference w a s f o u n d in the D A a n o v a for the a g e f a c t o r ( F = 3 . 5 1 ; df : 1 1 / 7 4 ; ~ < 0 . 0 0 1 ) . N e i t h e r a difference between the i n t e r n a l and e x t e r n a l standards, n o r an i n t e r a c t i o n effect between both anova factors were found significant, t h e n , we can conclude t h a t the two s t a n d a r d s (internal and e x t e r n a l ) varied together. Moreover, the i n t e r n a l standard being independant of a g e , it c a n be d r a w n t h a t the d i f f e r e n c e observed w e r e d u e to i n t r i n -
Vol. 28, No. 4, 1981
Catecholamines
in Developing Rat Lung
333
1
TABLE
External
standard
: 25
pg
3H-SAM/tube NA
DA 772 5 ~Ci
± 33
490
(48)
662
2 ~Ci
± 27
(48)
± 26 ~
434
± 27
(28)
(28)
Efficiency of 3 H - S A M q u a n t i t i e s u s e d to l a b e l the external standards (25 pg) r e s p e c t i v e l y . Comparison of the v a l u e s o b t a i n e d with 5 DCi/tube ( M e a n of c p m ± SEM) ( ) n u m b e r of r e s u l t s significant difference
: 0.02<
2 ~Ci
: 25
±
(13)
662
± 25
(28)
2 NCi/tube
NA
Internal 79
vs
pg
DA
869 5 ~Ci
NA
2
Standard
External
and
P<0.05
TABLE
3H-SAM/tube
DA
706
± 64
External 570
(13)
(13)
470
± 20~
± 53
434
± 27
(28)
(30)
Internal 527
± 54
(13)
343
± 31
(30)
Influence of 3 H - S A M q u a n t i t i e s on the l a b e l l i n g efficiency of the DA and NA i n t e r n a l standards (25 pg) a d d e d to the lung homogenates f r o m the d a y s 19 and 20 of rat g e s t a t i o n . Comparison of the v a l u e s o b t a i n e d with 5 ~Ci/tube and 2 ~Ci/tube vs the e x t e r n a l standard values. ( M e a n of c p m ± SEM) ( ) n u m b e r of r e s u l t s significant difference:
~ 0.02
~
P<0.O0|
334
Catecholamines in Developing Rat Lung
Vol. 28, No. 4, 1981
sic e x p e r i m e n t a l factors (changes in s p e c i f i c activity of 3 H - S A M , COMT activity, etc. a m o n g e x p e r i m e n t s ) . W i t h r e g a r d to NA, the overall analysis of v a r i a n c e was not significant (F=1.34; df : I I / 7 4 ) s h o w i n g t h a t no d i f f e r e n c e between the i n t e r n a l and external standards, a n d no i n f l u e n c e of the age on t h e f o r m e r existed. Accordingly, e a c h r e s u l t w a s the a v e r a g e of the v a l u e s c a l c u lated from the internal and external standards respectively in each experiment. The means ± SEM were expressed in p g / m g p r o t e i n s and computed on 6 or 7 s a m p l e s (in t r i p l i c a t e ) from different litters. In e a c h c a s e , b o t h the m a l e a n d f e m a l e p u p s w e r e f r o m the same litter. A SL-32 liquid scintillation counter (Intertechnique) was used. Proteins were determined by the L o w r y et al. m e t h o d (ll) using bovine serum albumine as s t a n d a r d , in p o r t i o n s of the l u n g homogenates ( 2 0 0 ~i b e f o r e a n d I00 D1 a f t e r b i r t h ) . Statistical significance was assessed by distribution free t e s t s as t h e r e s u l t s w e r e n o t n u m e r o u s e n o u g h to s u p p o s e a n o r m a l distribution. Two different t e s t s w e r e u s e d : the M a n n W h i t n e y t e s t a n d the W i l c o x o n test. The analysis of v a r i a n c e KruskalWallis t e s t w a s u s e d to d e t e r m i n e whether there was a significant difference between t h r e e d a t a and, if t h e r e w a s , the M a n n W h i t n e y t e s t w a s t h e n u s e d to c o m p a r e samples two by two to f i n d w h e r e t h e difference lay. T h e K e n d a l l t e s t of c o r r e l a t i o n w a s u s e d to g i v e a coefficient T w h i c h w a s u s e d to t e s t s i g n i f i c a n c e . A Tecktronic 4051 c o m p u t e r was used with our own non-parametric statistic programs. Results Comparison of p r o t e i n l e v e l s of m a l e s a n d f e m a l e s p e r g r a m of l u n g w e t w e i g h t showed that a large increase occured near birth. This increase began earlier in m a l e t h a n in f e m a l e p u p s : it w a s f o u n d in m a l e s , f r o m d a y 21 of g e s t a t i o n to b i r t h (P<0.01), whereas in f e m a l e s , f r o m b i r t h to p o s t n a t a l day l (P<0.00]) (Fig.l). A statistical analysis was performed to s t u d y the e v o l u t i o n of p r o t e i n s p e r w e t l u n g w e i g h t as a f u n c t i o n of age. A g o o d correlation u n t i l d a y 60 p o s t - p a r t u m was shown (P<0.0|). NA and DA levels during the a n t e n a t a l and postnatal period a r e s h o w e d f i g u r e 2. T h e D A l e v e l s w e r e l o w (not g r e a t e r than 50 p g / m g p r o t e i n s ) and i n d e p e n d a n t of a g e in m a l e s a n d f e m a l e s f r o m d a y 17 of g e s t a t i o n to d a y 20 p o s t - p a r t u m . On the c o n t r a r y , during the s a m e p e r i o d the N A l e v e l s i n c r e a sed s i g n i f i c a n t l y w i t h t h e a g e of a n i m a l s u n t i l d a y 20 p o s t partum (T=0.86) (P<0.001). This relation was not longer signific a n t w h e n the v a l u e s f o r d a y 60 ( a d u l t r a t s ) w e r e i n c l u d e d . S o m e p e r i o d of f e t a l and p o s t n a t a l life ting features : the d e c r e a s e of the N A l e v e l o b s e r v e d from gestation appeared to be s i g n i f i c a n t b o t h in females (P<0.001) (Fig.l) ; -
showed
some
interes-
d a y 17 to d a y ]9 of males (P<0.05) and
Vol. 28, No. 4, 1981
Catecholamines in Developing Rat Lung
pg/mg
wet
335
Lung
160
140
Birth
-'"
1
120
100
80
60 B.rth
40
17
19 21B 1 2
5
Fig.
10 20
60 Days
)
D e v e l o p m e n t of p r o t e i n c o n t e n t in rat lung : r e s u l t s in m a l e s and f e m a l e s (mean ± S . E . M . )
pg/mg
Protein
pg/mg
500
5OO
45(
450
Protein
200
l:i
100 :
Birth
50 25
..., ............ '"-........ J/.
~'--/"~-~--.. B
' ' 'iii 17 21 1
I- ....
I 5
DA
H ....... .
i 10
l 20
/( /~
I 60
Days
Fig.
17
21 1
5
10
20
//
2
D e v e l o p m e n t of DA and NA l e v e l s in rat lung : r e s u l t s in m a l e s and f e m a l e s (mean -+ S . E . M . )
6 0 Days
336
Catecholamines in Developing Rat Lung
Vol. 28, No. 4, 1981
- the p e a k of N A o b s e r v e d at b i r t h w a s n o t s i g n i f i c a n t and likely in r e l a t i o n to t h e s t r e s s d u e to b i r t h ; - the a n a l y s i s of v a r i a n c e between D A or N A d a t a on d a y s 10, 20, a n d 60 p o s t - p a r t u m showed a significant difference (P<0.05) (Fig.l). This difference is d u e , in f a c t , to the d e c r e a s e of the a m i n e l e v e l s f r o m d a y 20 to d a y 60. T h e v a l u e s of d a y 60 a r e a l m o s t the s a m e as t h o s e of d a y lO. Discussion The evolution of c a t e c h o l a m i n e levels reflected the m a t u r a t i o n of t h e d o p a m i n e r g i c and n o r a d r e n e r g i c systems in r a t l u n g . This evolution is t h e s a m e in m a l e s a n d f e m a l e s w i t h the s o l e exception of the D A l e v e l s (P<0.05). An analysis of v a r i a n c e w i t h the D A r e s u l t s carried out separ a t e l y for m a l e s a n d f e m a l e s on the d a t a o b t a i n e d at b i r t h ± o n e day showed a significant difference only for males (P
Vol. 28, No. 4, 1981
Catecholamines in Developing Rat Lung
337
E I - B e r m a n i and B l o o m q u i s t (8) u s i n g a f l u o r e s c e n c e h i s t o l o g i c a l m e t h o d , s h o w e d the p r e s e n c e of n o r a d r e n e r g i c n e r v e s in the rat lung. T h e s e n e r v e s e n t e r e d in the o r g a n at the h i l u m in a s s o c i a tion w i t h the b r o n c h i a l a r t e r i e s . In a d d i t i o n , the t e r m i n a l v a r i c o s i t i e s w e r e found in all d i v i s i o n s of the b r o n c h i a l a r t e r i e s . F i n a l l y , it is l i k e l y that the q u a n t i t y of b l o o d r e m a i n i n g in the lung t i s s u e a f t e r b l e e d i n g the adult rats r e p r e s e n t a more i m p o r t a n t f r a c t i o n of the s a m p l e w e i g h t than in the l u n g s r e m o v e d d u r i n g the p e r i n a t a l or fetal p e r i o d s . T h e r e f o r e , the r e l a t i v e v a l u e s for the NA l e v e l s c o u l d be p a r t i a l l y a l t e r e d , as d i s c u s s e d p r e v i o u s l y by H o d s o n et al. (I0), w h e n they s t u d i e d the lung development in the fetal p r i m a t e M a c a c a N e m e s t r i n a . References I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
14.
M. A S S I C O T and C. B O H U O N , Life Sci. 8 9 3 - ] 0 0 (1969) J. A X E L R O D and R. T O M C H I C K , J. Biol. Chem. 233 7 0 2 - 7 0 5 (1958) W,A. B E H R E N S , S. L A C E L L E and F. D E P O C A S , Can. J. P h y s i o l . P h a r m a c o l . 57 3 8 0 - 3 8 4 (1979) N° B E N - J O N A T H A N and R.E. M A X S O N , E n d o c r i n o l o g y 102 6 4 9 - 6 5 2 (1978) J.T. COYLE a n d D. HENRY, J . N e u r o c h e m . 21 6 ] - 6 3 (1973) M. DA P R A D A and G. Z ~ R C H E R , Life Sci. 19 1 1 6 ] - ] 1 7 4 (1976) A . W . I . E L - B E R M A N I , T h o r a x 33 1 6 7 - ] 7 4 (1978) A . W . I . E L - B E R M A N I and E.I. B L O O M Q U I S T , J. E m b r y o l . Exp. M o r p h . 48 1 7 7 - 1 8 3 (1978) C ~ G A U C H Y , J.P. T A S S I N , J. G L O W I N S K I and A. C H E R A M Y , J. N e u r o chem. 26 4 7 1 - 4 8 0 (;976) W.A° H O D S O N , S. P A L M E R , G.A. B R A K E L Y , J.H. M U R P H Y , D.E. W O O D RUM and T.E. M O R G A N , P e d i a t . Res. II 1 0 0 9 - 1 0 1 4 (1977) O.H. L O W R Y , N.J. R O S E B R O U G H , A.L. F A R R and R.J. R A N D A L L , J. Biol. Chem. ]93 2 6 5 - 2 7 5 (1951) H. P A R V E Z and D. G R I P O I S , Clin. Chim. A c t a 39 2 5 3 - 2 5 4 (1972) S. S I E G E L in N o n P a r a m e t r i c S t a t i s t i c s for the B e h a v i o r a l S c i e n c e s , p. 312, I n t e r n a t i o n a l s t u d e n t Ed., Mc G r a w - H i l l K o g a k u s h a Ltd. (;956) R.M. W E I N S H I L B O U M , P h a r m a c o l . Rev. 30 1 3 3 - ; 6 6 (1979)
Pergamon Press
CURRENT CONCEPTS: I. C A T E C H O L A M I N E S
IN D E V E L O P I N G
RAT
LUNG
Christiane Gardey-Levassort, Marie-Odile Richard D a n i ~ l e de L a u t u r e , G e n e v i e v e T h i r o u x and G e o r g e s O l i v e
24,
Laboratoire de P h a r m a c o l o g i e Biochimique P~rinatale rue du F a u ~ o u r g S a i n t - J a c q u e s . F - 7 5 0 1 4 P a r i s (France)
Summary The d e v e l o p m e n t of the c a t e c h o l a m i n e r g i c system of lung was i n v e s t i g a t e d in S p r a g u e - D a w l e y rats d u r i n g the p e r i n a t a l p e r i o d and in y o u n g a d u l t s . A radio-enzymatic p r o c e d u r e a l l o w e d to m e a s u r e as little as I pg of n o r a d r e n a l i n e or d o p a m i n e in each sample. The d o p a m i n e l e v e l s w e r e low and i n d e p e n d a n t of age. The n o r a d r e n a l i n e l e v e l s inc r e a s e d s i g n i f i c a n t l y w i t h the age u n t i l the 20th p o s t - p a r t u m day. No s i g n i f i c a n t d i f f e r e n c e s were found b e t w e e n n o r a d r e n a l i n e l e v e l s for m a l e s vs f e m a l e s at any age. Small d i f f e r e n c e s b e t w e e n m a l e s and f e m a l e s for d o p a m i n e levels e x i s t e d o n l y near birth. The d e v e l o p m e n t of the c a t e c h o l a m i n e r g i c s y s t e m in rat lung not b e e n i n v e s t i g a t e d w i t h b i o c h e m i c a l m e t h o d s . The s e n s i t i v i t i e s of s p e c t r o f l u o r o m e t r i c p r o c e d u r e s and also radioenzymatic ones, such as t h o s e of C o y l e and H e n r y (5), G a u c b y et al. (9) w e r e not s u f f i c i e n t for this r e s e a r c h since the m i n i m u m a m o u n t s they were able to d e t e c t were 50 pg and I0 pg for nor adrenaline (NA) and d o p a m i n e (DA) r e s p e c t i v e l y . To o v e r c o m e this p r o b l e m , we have used the m e t h o d d e s c r i b e d by Da P r a d a and Z~rchef (6) w h i c h has t e n - f o l d i n c r e a s e d s e n s i t i v i t y , compared with the o t h e r m e t h o d s . This has a l l o w e d us to d e t e r m i n e DA and NA levels in the lung t i s s u e d u r i n g p e r i n a t a l d e v e l o p m e n t and in the a d u l t rat, and to c o m p a r e m a l e s w i t h f e m a l e s . has
Materials
and
methods
Lung s a m p l i n g . Sprague-Dawley f e m a l e rats, w e i g h i n g 250 to 300 g, kept at 26 ~ ± ;°C w i t h n a t u r a l light and fed on s t a n d a r d diet w e r e used in this study. T h e y w e r e p l a c e d o v e r n i g h t w i t h m a les and the n e x t m o r n i n g was d e s i g n a t e d as day zero of g e s t a t i o n . U n t i l day 21 of g e s t a t i o n the f e m a l e s w e r e c a g e d in g r o u p s of 6 and then i s o l a t e d . All the l i t t e r s w e r e r e d u c e d to 8 pups. P r e g n a n t rats w e r e k i l l e d by d e c a p i t a t i o n (Decapitator Harw a r d A p p a r a t u s Cy, M a s s , USA) on days 17, 19, 20, 21 of g e s t a t i o n b e t w e e n 10 a.m. and ] p.m. to e l i m i n a t e d i u r n a l v a r i a t i o n s . Uteri
and
f e t u s e s w e r e i m m e d i a t e l y e x c i s e d and 0024-3205/81/040331-07502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
placed
in
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crushed ice. T h e s e x e of f e t u s e s w a s d e t e r m i n e d and each animal was weighed a n d b l e d by d e c a p i t a t i o n . The lungs were removed, f r o z e n in l i q u i d n i t r o g e n , weighed a n d k e p t at - 8 0 ° C u n t i l a s s a y . Newborn rats were killed immediately after birth before they s t a r t e d to s u c k . T h e o t h e r s , in p o s t n a t a l p e r i o d , w e r e k i l l e d on d a y s 1, 2, 5, 10, 20, 60, u s i n g the s a m e p r o c e s s as b e f o r e . The young rats were separated from mothers on d a y 20 a n d the m a l e s f r o m the f e m a l e s on d a y 30 p o s t - p a r t u m . At d a y 60, t h e r a t s w e r e considered to be an a d u l t . In e a c h c a s e , the l u n g s w e r e r e m o v e d as previously. A s s a y of D A and NA. The lung DA and NA levels were determined simultaneously by the r a d i o e n z y m a t i c m e t h o d of Da P r a d a a n d Zurcher (6). T h e rat l i v e r c a t e c h o l - O - m e t h y l transferase (COMT) was purified by the p r o c e d u r e of A x e l r o d and T o m c h i c k (2) m o d i f i e d by Parvez and Gripois (12). T h e e n z y m e a c t i v i t y of e a c h b a t c h w a s c h e c k e d by the A s s i c o t and B o h u o n a s s a y (1). The following modifications were used : --I) as l u n g t i s s u e m a y be c o n t a m i n a t e d with blood, the h o m o g e n a tes w e r e p r e p a r e d with EGTA, a chelating a g e n t for Ca ++ i o n s , to prevent the Ca ++ i n h i b i t o r y e f f e c t on C O M T a c t i v i t y , as the authors suggested for p l a s m a samples. Then, lung tissue was homogeneized in 50 v o l . of i c e - c o l d 0.3 N p e r c h l o r i c acid containing 0 . 1 % E G T A and 0 . 0 1 % M g C I ,6H O. 2
2
-2) as the f e t a l t i s s u e c a t e c h o l a m i n e s were expected to be f o u n d at v e r y l o w l e v e l s , 5 ~ C i / t u b e of S - a d e n o s y l - L - m e t h i o n i n e methyl-3H (SAM) w e r e u s e d i n s t e a d of 2 N C i / t u b e (specific activity 10-12 Ci/mMole, Amersham). In t h e s e c o n d i t i o n s as l i t t l e as I pg of D A or N A in the s a m p l e w a s m e a s u r e d . The last modification changed the s l o p e of the D A s t a n d a r d c u r v e for a r a n g e of v a l u e s f r o m 1 pg to I00 pg, b u t n o t t h a t of NA. T a b l e 1 shows a significantly better labelling of the D A external standard. Moreover, using 5 ~Ci/tube b o t h the D A a n d N A internal standard values were not significantly different f r o m the external standard ones, while they did, when 2 uCi/tube were used as Da P r a d a a n d Z u r c h e r (6), ( t a b l e 2). O n e s a m p l e of e a c h age b e f o r e and a f t e r b i r t h w a s t r e a t e d simultaneously in the s a m e e x p e r i m e n t with both internal and external standards, except the 2 m o n t h s samples treated together in two s e r i e s . The internal standards were distributed at r a n d o m in the s a m p l e s of the d i f f e r e n t a g e s a m o n g the e x p e r i m e n t s . We c h e c k e d w h e ther a degradation of the a u t h e n t i c D A or N A a d d e d as i n t e r n a l standards, occured as a f u n c t i o n of the t i s s u e m a t u r i t y . With this a i m in v i e w , a t w o - w a y a n a l y s i s of v a r i a n c e (anova) was performed taking into account both factors : age and internal or e x t e r n a l standard. A significant difference w a s f o u n d in the D A a n o v a for the a g e f a c t o r ( F = 3 . 5 1 ; df : 1 1 / 7 4 ; ~ < 0 . 0 0 1 ) . N e i t h e r a difference between the i n t e r n a l and e x t e r n a l standards, n o r an i n t e r a c t i o n effect between both anova factors were found significant, t h e n , we can conclude t h a t the two s t a n d a r d s (internal and e x t e r n a l ) varied together. Moreover, the i n t e r n a l standard being independant of a g e , it c a n be d r a w n t h a t the d i f f e r e n c e observed w e r e d u e to i n t r i n -
Vol. 28, No. 4, 1981
Catecholamines
in Developing Rat Lung
333
1
TABLE
External
standard
: 25
pg
3H-SAM/tube NA
DA 772 5 ~Ci
± 33
490
(48)
662
2 ~Ci
± 27
(48)
± 26 ~
434
± 27
(28)
(28)
Efficiency of 3 H - S A M q u a n t i t i e s u s e d to l a b e l the external standards (25 pg) r e s p e c t i v e l y . Comparison of the v a l u e s o b t a i n e d with 5 DCi/tube ( M e a n of c p m ± SEM) ( ) n u m b e r of r e s u l t s significant difference
: 0.02<
2 ~Ci
: 25
±
(13)
662
± 25
(28)
2 NCi/tube
NA
Internal 79
vs
pg
DA
869 5 ~Ci
NA
2
Standard
External
and
P<0.05
TABLE
3H-SAM/tube
DA
706
± 64
External 570
(13)
(13)
470
± 20~
± 53
434
± 27
(28)
(30)
Internal 527
± 54
(13)
343
± 31
(30)
Influence of 3 H - S A M q u a n t i t i e s on the l a b e l l i n g efficiency of the DA and NA i n t e r n a l standards (25 pg) a d d e d to the lung homogenates f r o m the d a y s 19 and 20 of rat g e s t a t i o n . Comparison of the v a l u e s o b t a i n e d with 5 ~Ci/tube and 2 ~Ci/tube vs the e x t e r n a l standard values. ( M e a n of c p m ± SEM) ( ) n u m b e r of r e s u l t s significant difference:
~ 0.02
~
P<0.O0|
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sic e x p e r i m e n t a l factors (changes in s p e c i f i c activity of 3 H - S A M , COMT activity, etc. a m o n g e x p e r i m e n t s ) . W i t h r e g a r d to NA, the overall analysis of v a r i a n c e was not significant (F=1.34; df : I I / 7 4 ) s h o w i n g t h a t no d i f f e r e n c e between the i n t e r n a l and external standards, a n d no i n f l u e n c e of the age on t h e f o r m e r existed. Accordingly, e a c h r e s u l t w a s the a v e r a g e of the v a l u e s c a l c u lated from the internal and external standards respectively in each experiment. The means ± SEM were expressed in p g / m g p r o t e i n s and computed on 6 or 7 s a m p l e s (in t r i p l i c a t e ) from different litters. In e a c h c a s e , b o t h the m a l e a n d f e m a l e p u p s w e r e f r o m the same litter. A SL-32 liquid scintillation counter (Intertechnique) was used. Proteins were determined by the L o w r y et al. m e t h o d (ll) using bovine serum albumine as s t a n d a r d , in p o r t i o n s of the l u n g homogenates ( 2 0 0 ~i b e f o r e a n d I00 D1 a f t e r b i r t h ) . Statistical significance was assessed by distribution free t e s t s as t h e r e s u l t s w e r e n o t n u m e r o u s e n o u g h to s u p p o s e a n o r m a l distribution. Two different t e s t s w e r e u s e d : the M a n n W h i t n e y t e s t a n d the W i l c o x o n test. The analysis of v a r i a n c e KruskalWallis t e s t w a s u s e d to d e t e r m i n e whether there was a significant difference between t h r e e d a t a and, if t h e r e w a s , the M a n n W h i t n e y t e s t w a s t h e n u s e d to c o m p a r e samples two by two to f i n d w h e r e t h e difference lay. T h e K e n d a l l t e s t of c o r r e l a t i o n w a s u s e d to g i v e a coefficient T w h i c h w a s u s e d to t e s t s i g n i f i c a n c e . A Tecktronic 4051 c o m p u t e r was used with our own non-parametric statistic programs. Results Comparison of p r o t e i n l e v e l s of m a l e s a n d f e m a l e s p e r g r a m of l u n g w e t w e i g h t showed that a large increase occured near birth. This increase began earlier in m a l e t h a n in f e m a l e p u p s : it w a s f o u n d in m a l e s , f r o m d a y 21 of g e s t a t i o n to b i r t h (P<0.01), whereas in f e m a l e s , f r o m b i r t h to p o s t n a t a l day l (P<0.00]) (Fig.l). A statistical analysis was performed to s t u d y the e v o l u t i o n of p r o t e i n s p e r w e t l u n g w e i g h t as a f u n c t i o n of age. A g o o d correlation u n t i l d a y 60 p o s t - p a r t u m was shown (P<0.0|). NA and DA levels during the a n t e n a t a l and postnatal period a r e s h o w e d f i g u r e 2. T h e D A l e v e l s w e r e l o w (not g r e a t e r than 50 p g / m g p r o t e i n s ) and i n d e p e n d a n t of a g e in m a l e s a n d f e m a l e s f r o m d a y 17 of g e s t a t i o n to d a y 20 p o s t - p a r t u m . On the c o n t r a r y , during the s a m e p e r i o d the N A l e v e l s i n c r e a sed s i g n i f i c a n t l y w i t h t h e a g e of a n i m a l s u n t i l d a y 20 p o s t partum (T=0.86) (P<0.001). This relation was not longer signific a n t w h e n the v a l u e s f o r d a y 60 ( a d u l t r a t s ) w e r e i n c l u d e d . S o m e p e r i o d of f e t a l and p o s t n a t a l life ting features : the d e c r e a s e of the N A l e v e l o b s e r v e d from gestation appeared to be s i g n i f i c a n t b o t h in females (P<0.001) (Fig.l) ; -
showed
some
interes-
d a y 17 to d a y ]9 of males (P<0.05) and
Vol. 28, No. 4, 1981
Catecholamines in Developing Rat Lung
pg/mg
wet
335
Lung
160
140
Birth
-'"
1
120
100
80
60 B.rth
40
17
19 21B 1 2
5
Fig.
10 20
60 Days
)
D e v e l o p m e n t of p r o t e i n c o n t e n t in rat lung : r e s u l t s in m a l e s and f e m a l e s (mean ± S . E . M . )
pg/mg
Protein
pg/mg
500
5OO
45(
450
Protein
200
l:i
100 :
Birth
50 25
..., ............ '"-........ J/.
~'--/"~-~--.. B
' ' 'iii 17 21 1
I- ....
I 5
DA
H ....... .
i 10
l 20
/( /~
I 60
Days
Fig.
17
21 1
5
10
20
//
2
D e v e l o p m e n t of DA and NA l e v e l s in rat lung : r e s u l t s in m a l e s and f e m a l e s (mean -+ S . E . M . )
6 0 Days
336
Catecholamines in Developing Rat Lung
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- the p e a k of N A o b s e r v e d at b i r t h w a s n o t s i g n i f i c a n t and likely in r e l a t i o n to t h e s t r e s s d u e to b i r t h ; - the a n a l y s i s of v a r i a n c e between D A or N A d a t a on d a y s 10, 20, a n d 60 p o s t - p a r t u m showed a significant difference (P<0.05) (Fig.l). This difference is d u e , in f a c t , to the d e c r e a s e of the a m i n e l e v e l s f r o m d a y 20 to d a y 60. T h e v a l u e s of d a y 60 a r e a l m o s t the s a m e as t h o s e of d a y lO. Discussion The evolution of c a t e c h o l a m i n e levels reflected the m a t u r a t i o n of t h e d o p a m i n e r g i c and n o r a d r e n e r g i c systems in r a t l u n g . This evolution is t h e s a m e in m a l e s a n d f e m a l e s w i t h the s o l e exception of the D A l e v e l s (P<0.05). An analysis of v a r i a n c e w i t h the D A r e s u l t s carried out separ a t e l y for m a l e s a n d f e m a l e s on the d a t a o b t a i n e d at b i r t h ± o n e day showed a significant difference only for males (P
Vol. 28, No. 4, 1981
Catecholamines in Developing Rat Lung
337
E I - B e r m a n i and B l o o m q u i s t (8) u s i n g a f l u o r e s c e n c e h i s t o l o g i c a l m e t h o d , s h o w e d the p r e s e n c e of n o r a d r e n e r g i c n e r v e s in the rat lung. T h e s e n e r v e s e n t e r e d in the o r g a n at the h i l u m in a s s o c i a tion w i t h the b r o n c h i a l a r t e r i e s . In a d d i t i o n , the t e r m i n a l v a r i c o s i t i e s w e r e found in all d i v i s i o n s of the b r o n c h i a l a r t e r i e s . F i n a l l y , it is l i k e l y that the q u a n t i t y of b l o o d r e m a i n i n g in the lung t i s s u e a f t e r b l e e d i n g the adult rats r e p r e s e n t a more i m p o r t a n t f r a c t i o n of the s a m p l e w e i g h t than in the l u n g s r e m o v e d d u r i n g the p e r i n a t a l or fetal p e r i o d s . T h e r e f o r e , the r e l a t i v e v a l u e s for the NA l e v e l s c o u l d be p a r t i a l l y a l t e r e d , as d i s c u s s e d p r e v i o u s l y by H o d s o n et al. (I0), w h e n they s t u d i e d the lung development in the fetal p r i m a t e M a c a c a N e m e s t r i n a . References I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
14.
M. A S S I C O T and C. B O H U O N , Life Sci. 8 9 3 - ] 0 0 (1969) J. A X E L R O D and R. T O M C H I C K , J. Biol. Chem. 233 7 0 2 - 7 0 5 (1958) W,A. B E H R E N S , S. L A C E L L E and F. D E P O C A S , Can. J. P h y s i o l . P h a r m a c o l . 57 3 8 0 - 3 8 4 (1979) N° B E N - J O N A T H A N and R.E. M A X S O N , E n d o c r i n o l o g y 102 6 4 9 - 6 5 2 (1978) J.T. COYLE a n d D. HENRY, J . N e u r o c h e m . 21 6 ] - 6 3 (1973) M. DA P R A D A and G. Z ~ R C H E R , Life Sci. 19 1 1 6 ] - ] 1 7 4 (1976) A . W . I . E L - B E R M A N I , T h o r a x 33 1 6 7 - ] 7 4 (1978) A . W . I . E L - B E R M A N I and E.I. B L O O M Q U I S T , J. E m b r y o l . Exp. M o r p h . 48 1 7 7 - 1 8 3 (1978) C ~ G A U C H Y , J.P. T A S S I N , J. G L O W I N S K I and A. C H E R A M Y , J. N e u r o chem. 26 4 7 1 - 4 8 0 (;976) W.A° H O D S O N , S. P A L M E R , G.A. B R A K E L Y , J.H. M U R P H Y , D.E. W O O D RUM and T.E. M O R G A N , P e d i a t . Res. II 1 0 0 9 - 1 0 1 4 (1977) O.H. L O W R Y , N.J. R O S E B R O U G H , A.L. F A R R and R.J. R A N D A L L , J. Biol. Chem. ]93 2 6 5 - 2 7 5 (1951) H. P A R V E Z and D. G R I P O I S , Clin. Chim. A c t a 39 2 5 3 - 2 5 4 (1972) S. S I E G E L in N o n P a r a m e t r i c S t a t i s t i c s for the B e h a v i o r a l S c i e n c e s , p. 312, I n t e r n a t i o n a l s t u d e n t Ed., Mc G r a w - H i l l K o g a k u s h a Ltd. (;956) R.M. W E I N S H I L B O U M , P h a r m a c o l . Rev. 30 1 3 3 - ; 6 6 (1979)