1166
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EXPERIENTIA 31/10
T h e effect of t h e p o l y c a t i o n i c m o d i f i e d d e r i v a t i v e s of p o l y g l u t a m i c acid w a s i n v e s t i g a t e d on t h e v i r a l r e s i s t a n c e i n d u c e d b y p o l y I : C in p r i m a r y h u m a n f o e t a l cell c u l t u r e s t o o ; 106 cells were s e e d e d in 1 m l of t h e a b o v e - m e n t i o n e d
m e d i u m a n d g r o w n i n s t a t i o n a r y t u b e s a t 37 ~ for 4 t o 5 d a y s . T h e m i n i m a l p r o t e c t i v e d o s e s of p o l y I : C , b o t h in p r e s e n c e a n d a b s e n c e of p o l y c a t i o n s , w e r e d e t e r m i n e d as d e s c r i b e d a b o v e (Table).
Minimal protective doses of poly I:C against VSV infection in L-929 and human foetal cells in the presence and absence of polyealions
Summary. P o l y c a t i o n i c m o d i f i e d d e r i v a t i v e s of p o l y g l u t a m i c acid are a t l e a s t a s g o o d e n h a n c e r s of p o l y I : C i n d u c e d v i r a l r e s i s t a n c e in v a r i o u s cell c u l t u r e s as a r e D E A E - d e x t r a n or p o l y - L - l y s i n e 7. A. I~0TAI, T. G~NTI a n d I. Ms
Minimal protection doses 9 of poly I : C in [xg/ml of media Type of polycation b
Poly-DMAE-glutamineo Poly-DEAE-glutamine o Poly-L-lysine (Sigma) DEAE-dextran (Pharmaeia) Without polycation
L-cells
Human foetal cells
I.I0 4 1.10 .4
5.10-8 5.10-3 1.10-3 1.10-3 -
1.10-~ 5.10 -4 1.10~ '
Institute o[ Organic Chemistry, University L. Edtv6s, Muzeum krt. d/b, 1088 Budapest (Hungary); Chemical Works o/Gedeon Richter Ltd,, Budapest and Institute o/Microbiology, Medical School, Szeged (Hungary), 5 June 1975.
~Determined as described in the text. bPolycations were used in concentrations of 20 [xg/ml of media, oPolycationic modified derivatives of polyglutamic acid, with characteristic structural units as indicated in the formula.
7 This work was supported by the Chenfical Works of Gedeon Richter Ltd., Budapest.
Norepinephrine in Fetal and Neonatal Rabbit Brain In newborn m a m m a l s , such as the rat, m o u s e a n d rabbit, t h e a m o u n t of n o r e p i n e p h r i n e is r e l a t i v e l y s m a l l , s l o w l y i n c r e a s i n g t o a d u l t v a l u e s o v e r a p e r i o d of 4 t o 6 w e e k s 1, 3. All t h e s e s p e c i e s are c o n s i d e r e d d e v e l o p m e n t a l l y i m m a t u r e a t b i r t h . T h e n e w b o r n g u i n e a - p i g is c o m p a r a t i v e l y well d e v e l o p e d : its b r a i n n o r e p i n e p h r i n e is a t a p p r o x i m a t e l y t h e a d u l t level. I n t h e n e w b o r n r a b b i t (1-day-old) o n l y h a l f of t h i s v a l u e is f o u n d 1. Since we are i n t e r e s t e d in p h y s i o l o g i c a l m o d i f i c a t i o n s o c c u r r i n g in m a m m a l s d u r i n g p a r t u r i t i o n a n d a few h o u r s a f t e r w a r d s , we h a v e u n d e r t a k e n t h i s s t u d y to d e t e r m i n e w h e t h e r or n o t t h e b r a i n n o r e p i n e p h r i n e s t o r e c h a n g e s in f e t u s a n d n e w b o r n r a b b i t s d u r i n g t h i s period. Materials and methods. I n o u r e x p e r i m e n t s we h a v e u s e d t e t u s e s a n d n e w b o r n r a b b i t s of t h e w h i t e N e w Z e a l a n d s t r a i n , in w h i c h t h e t e r m of g e s t a t i o n is 31 d a y s . A t t h e s t a g e of t h e 3 0 t h or 31st d a y , t h e f e m a l e r a b b i t s w e r e killed b y a i r e m b o l i s m , l a p a r o t o m y w a s p e r f o r m e d a n d all t h e f e t u s e s w e r e t a k e n o u t a n d d e c a p i t a t e d a t
601' 501
30
I
30thday 31thday O-lh
|
|
|
2-4h
|
8-12h age
|
Norepinephrine level in fetal and neonatal rabbit brain (ng/g). Mean values and standard error for each group are given, n = number of samples.
once. T h e b r a i n s w e r e q u i c k l y r e m o v e d , b l o t t e d on filter p a p e r a n d f r o z e n i m m e d i a t e l y . Since e a c h b r a i n c o n t a i n s low a m o u n t s of n o r e p i n e p h r i n e , it w a s n e c e s s a r y t o pool 4 to 5 in e a c h s a m p l e . T h e n e w b o r n r a b b i t s were s e p a r a t e d " f r o m t h e i r m o t h e r j u s t a f t e r b i r t h , d u r i n g t h e 1st h or l a t e r on, w i t h i n 2 to 4 or 8 to 12 h. A s w i t h t h e f e t u s e s , t h e n e w b o r n a n i m a l s f r o m t h e s a m e l i t t e r were d i v i d e d i n t o g r o u p s of 4 to 5 a n d killed b y d e c a p i t a t i o n . T h e i r b r a i n s w e r e r e m o v e d a n d t h e s a m p l e s w e r e p r e p a r e d in t h e s a m e w a y . T h e t i s s u e w a s h o m o g e n i z e d in ice-cold 0.4 M perchloric a c i d by using a Tri-R tissue homogenizer ( G e n e l a b I n t e r n a t i o n a l ) p r o v i d e d w i t h a glass pestle, a n d c e n t r i f u g e d a t 9000 • a t 0~ for 30 rain. T h e e x t r a c t i o n w a s p e r f o r m e d t w i c e m o r e a n d all 3 s u p e r n a t a n t s w e r e pooled ~. T h e p H w a s a d j u s t e d to 8.5 M Tris b u f f e r 4 a n d the samples were adsorbed onto aluminaS. W e used a c t i v e M e r c k a l u m i n i u m oxide, acidic a c t i v i t y I, p r e p a r e d b y t h e m e t h o d of ANTON a n d SAYRE 6. T h e a l u m i n a containing the adsorbed norepinephrine was washed a few t i m e s w i t h bi-distilled w a t e r . T h e e l u t i o n w a s p e r f o r m e d w i t h 3 • 2 m l of 0.3 N acetic acid, t h e a l u m i n a b e i n g m i x e d t h o r o u g h l y w i t h t h e acid b y a m a g n e t i c stirrer. All 3 e l u a t e s w e r e pooled, c e n t r i f u g e d , a d j u s t e d to p H 6.5 a n d u s e d for f l u o r o m e t r i c a s s a y L A n A m i c o B o w m a n S p e c t r o - f l u o r o m e t e r w i t h ellipsoidal m i r r o r z N. KARKI, R. KUNTZMANand t3. B. BRODIE, J. Neurochem. 9, 53 (1962). H. C. AGRAWAL, S. N. GLISSON and W. A. HIMWlCH, Int. J. Neuropharmac. 7, 97 (1968). 8 A. BERTLER, A. CARLSSON and E. ROSENGREN, Acta physiol. scand, d4, 273 (1958). 4 j . p. HANIG, J. ~V[.~V[ORRISONand S. KROP, Analyt. chim. Acta 59, 363 (1972). 5 V. RENZIm, C. A. 13RUNORIand C. VALOR1, Clin. chim. Aeta 30, 587 (1970). 6 A. H. ANTON and D. F. SAYRE, J. Pharmac. exp. Ther. 138, 360 (1962). 7 U. S. voN EULER and F. LISHAJKO, Acta physiol, scand. 57, 348 (1961).
15.10. 1975
1167
Specialia
was used for f l u o r i m e t r i c m e a s u r e m e n t s . R e a d i n g s were m a d e a t a c t i v a t i o n w a v e l e n g t h s of 380 a n d 430 n m a n d a t fluorescence w a v e l e n g t h s of 490 a n d 540 n m . T h e m e a n values 4- S E M are g i v e n in n g / g of tissue. Results. I n t h e fetuses of 30 days, t h e b r a i n n o r e p i n e p h r i n e level is low, o n l y 51 4- 4.9 ng/g. No c h a n g e s occur t h e n e x t day, t h e l a s t one before b i r t h (52 4- 9.7 rig/g), as can be seen in t h e Figure. A i t e r p a r t u r i t i o n , w i t h i n t h e 1st h, t h e n e w b o r n r a b b i t b r a i n c o n t e n t falls to 34 4- 9.9 ng/g n o r e p i n e p h r i n e , a b o u t 37% less t h a n t h a t of t h e 31st day. L a t e r on, w i t h i n 2 to 4 h, t h e a m o u n t of n o r e p h i n e p h r i n e r e t u r n s t o t h e p r e n a t a l level (52 :~ 5.6 ng/g) a n d r e m a i n s p r a c t i c a l l y the s a m e for 8 t o 12 h (51 :j: 2.4 ng/g) (see Figure). Discussion. I n t h e r a t fetus, t h e e n z y m e s i n v o l v e d in t h e b i o s y n t h e s i s of c a t e c h o l a m i n e s are p r e s e n t in t h e b r a i n a t 15 d a y s of g e s t a t i o n s, ~; a t t h i s age, t h e n o r e p i n e p h r i n e is a t o n l y 2% of t h e a d u l t level, w h e r e a s t h e b i o s y n t h e t i c e n z y m e s h a v e specific activities of a b o u t 10% of t h o s e in t h e a d u l t b r a i n ~~ A t b i r t h a n d t h e r e a f t e r , t h e biosynthetic enzymes and their products, dopamine and n o r e p i n e p h r i n e , increase in a parallel fashion 9,n. Our results show t h a t in t h e r a b b i t f e t u s on t h e 30th a n d 31st d a y of gestation, n o r e p i n e p h r i n e is p r e s e n t a n d t h e level r e m a i n s u n c h a n g e d , as is t h e case w i t h i n 2 to 4 a n d 8 t o 12 h a f t e r p a r t u r i t i o n . On t h e o t h e r h a n d , d u r i n g t h e 1st h following p a r t u r i t i o n , a decrease of n o r e p i n e p h r i n e can be seen a n d t h e v a r i a b i l i t y of t h e i n d i v i d u a l values is larger t h a n in t h e o t h e r stages. W i t h i n 2 to 4 h, t h e a m o u n t of n o r e p i n e p h r i n e rises t o t h e level m e a s u r e d in t h e fetuses, a n d r e m a i n s t h e s a m e for 8 to 12 h. Similar m o d i f i c a t i o n s of t h e a m o u n t of c a t e c h o l a m i n e s h a v e b e e n o b s e r v e d in t h e n e w b o r n r a b b i t , in varigus~.t~ssues. F o r instance, d u r i n g t h e first few h o u r s folloWirig@a~turition, one can observe a decrease of t h e a m o u n t 'of c a t e c h o l a r n i n e ~ i n t h e a d r e n a l s 12, is a n d increase in t h e plasm~ la, 14 a n d in ,tfi e h e a r t aS. These r a p i d a n d t r a n s i t o r y v a r i a t i o n s of t h e a m o u n t s of c a t e c h o l a m i n e s f o u n d in t h e n e w b o r n r a b b i t t h u s seem to be linked to birth. The fall of t h e n o r e p i n e p h r i n e level in t h e b r a i n can be r e l a t e d to t h e fetal suffering special c o n d i t i o n s d u r i n g p a r t u r i t i o n , especially t h e h y p o x i a w h i c h occurs a". I t has been e s t a b l i s h e d t h a t in t h e a d u l t cat, t h e a s p h y x i a p r o d u c e d b y r e b r e a t h i n g can decrease t h e n o r e p i n e p h r i n e level in t h e h y p o t h a l a m u s in less t h a n 2 h~7; in t h e a d u l t rat, t h e h y p o x i a decreases in 1 h t h e n o r e p i n e p h r i n e in t h e b r a i n 18. I n t h e fetus, a s p h y x i a also i n t r o d u c e s a decrease of t h e level of c a t e c h o l a m i n e s in h u m a n ~9, l a m b 2~ calf 2t a n d foal 22 a d r e n a l s a n d in h u m a n ~9 a n d r a b b i t 23 p a r a g a n g l i a as well as in h u m a n 2~ e x t r a - a d r e n a l tissue. I n t h e a d u l t rat, o t h e r severe stresses, such as p r o l o n g e d m u s c u l a r effort 25 a n d e x p o s u r e to cold 26-2s, are also k n o w n t o reduce t h e e n d o g e n o u s level of c a t e c h o l a m i n e s in tissues. C o n s e q u e n t ly, t h e s p o n t a n e o u s decrease of n o r e p i n e p h r i n e in t h e b r a i n w h i c h we h a v e f o u n d in t h e n e w b o r n r a b b i t can
also b e e x p l a i n e d b y stress c o n d i t i o n s to w h i c h t h e a n i m a l s are e x p o s e d d u r i n g p a r t u r i t i o n . As has b e e n s u g g e s t e d 29, n o r e p i n e p h r i n e , b y its v a s o m o t o r action, could c o n t r i b u t e to a b e t t e r d i s t r i b u t i o n of b l o o d t o t h e brain, t h u s increasing t h e b r a i n ' s o x y g e n s t o c k a n d facili t a t i n g t h e defense of t h e n e w b o r n a n i m a l a g a i n s t a s p h y x i a . Therefore, t h i s b r a i n n o r e p i n e p h r i n e l i b e r a t i o n d u r i n g p a r t u r i t i o n could h a v e a n i m p o r t a n t role in a m o s t critical p e r i o d of t h e m a m m a l ' s life 3~
S u m m a r y . I n t h e first h o u r a f t e r p a r t u r i t i o n , t h e n e w b o r n r a b b i t b r a i n n o r e p i n e p h r i n e c o n t e n t is a b o u t 37% less t h a n t h a t of t h e fetus of 30th or 31st day. L a t e r on, w i t h i n 2 to 4 h, t h e n o r e p i n e p h r i n e level r e t u r n s to t h e p r e n a t a l v a l u e a n d r e m a i n s u n c h a n g e d b e t w e e n 8 t o 12 h. This t r a n s i t o r y fall of t h e b r a i n n o r e p i n e p h r i n e seems to be linked t o t h e stress c o n d i t i o n s w h i c h occur d u r i n g p a r t u r i tion. I. MOTELICA-HEINO a n d J. ROFFI
Laboratoire d'Endocrinologie, Bdtiment d97, Universitd de Paris X I , F-91d05 Orsay (France), 28 A p r i l 7975.
s j. T. COYLE and J. AXELROD, J. Neurochem. 70, 449 (1972). 9 F. LAMPRECHTand J. T. COYLE, Brain Res. dl, 503 (1972). 10 j. T. COYLE and D. HENRY, J. Neuroehem. 21, 61 (1973). 11 G. R. BREESEand T. D. TRAYLOR,Br. J. Pharmae. 44, 210 (1972). 12 j. ROFFI, J. L. FROGERand M. F. DEBRAY,C. r. Aead. Sci., Paris 277, 595 (1973). la I. MOTELICA-HEINO, M. F. DEBRAY and J. ROFFI, J. Physiol., Paris, in press. 14 K. ORVAYUZand R. GHAMmR, Experientia 29, 1472 (1973). 15 j. ROFFI and I. MOTELICA-HEINO, Experientia 31, 194 (1975). 10 G. S. DAWES,in Foetal and Neonatal Physiology (Year Book Medical Publ., Chicago 1969), p. 117. 17 M. VOGT, J. Physiol., Loud. 123, 451 (1954). 18 M. STUPFEL and J. ROFFI, C. r. Goc. Bid., Paris 155, 237 (1961). 10 A. HERVONENand O. KORKALA,Acta obstet, gynee, scand. 51, 17 (1972). 20 Z. S. COMLINE and M. SILVER, Nature, Loud. 781, ~.~3"{1953)21 R. S. COMHNE and M. SILVER, J. Physiol., I.ond. 783, 305 (,1966). 2z R. S. COMLINEand M. SILVER, J. Physiol., Loud. 215, 659 (1971). 23 T. BRUNmN, Aeta physiol, scand., suppl. 280, 70 (1966). '~4 A. HERVONEN and O. KORKALA,Seand. J. clin. Lab. Invest. 27, suppl, ll6, 73 (1971). 25 j. FUGAZZA,J. Physiol. Paris, suppl. 5, 55 (1963). 2o j. D. BARCHAS and D. X. FREEDMAN, Biochem. l?harmae. 12, 1232 (1963). 27 j. LEDUC, Aeta physiol, seand., suppl. 183, 52 (1961). 28 I. MOTELICA,Acta physiol, scand. 76, 393 (1969). 29 j. BOETHIUS, ~1". BRUNDIN and N. A. PERSSON, Aeta physiol. seand. 78, 269 (1970). a0 This work was supported by a grant from DGRST (D616gation G6ndrale t~ la Recherche Scientifi~tue et Technique, contrat No. 71 73219).
C o m p a r a t i v e S t u d y of t h e E l e c t r i c a l a n d M e c h a n i c a l B e h a v i o u r of an I n t a c t , S e m i - I n t a c t a n d I s o l a t e d G a s t r o p o d e (Helix pomatia) S m o o t h M u s c l e P r e p a r a t i o n U n d e r a p p r o p r i a t e s t i m u l a t i o n conditions, t h e isolated penis r e t r a c t o r muscle (PRM) of Helix pomatia L., a g a s t r o p o d e s m o o t h muscle, can be m a d e t o p e r f o r m a phasic contraction and a prolonged contraction known as ' c a t c h ' (WABNITZl,~). W h e t h e r t h e t w o d i s t i n c t t y p e s of c o n t r a c t i o n p l a y a p a r t in t h e n o r m a l b e h a v i o u r of t h e penis r e t r a c t o r muscle in t h e i n t a c t a n i m a l is u n k n o w n .
The aim of t h e p r e s e n t e x p e r i m e n t s is to c o m p a r e t h e n o r m a l electrical a n d m e c h a n i c a l b e h a v i o u r of t h e i n t a v f penis retractor muscle;nerve-brain preparation with the p r o p e r t i e s of t h e muscle a t d i f f e r e n t stages of surgical 1 R. W. WABNITZ, Dipl.-Arbeit Univ. G6ttingen (1970). 2 R. W. WABNITZ, Cell Tiss. Res. 756, 253 (1975).
Specialia
EXPERIENTIA 31/10
T h e effect of t h e p o l y c a t i o n i c m o d i f i e d d e r i v a t i v e s of p o l y g l u t a m i c acid w a s i n v e s t i g a t e d on t h e v i r a l r e s i s t a n c e i n d u c e d b y p o l y I : C in p r i m a r y h u m a n f o e t a l cell c u l t u r e s t o o ; 106 cells were s e e d e d in 1 m l of t h e a b o v e - m e n t i o n e d
m e d i u m a n d g r o w n i n s t a t i o n a r y t u b e s a t 37 ~ for 4 t o 5 d a y s . T h e m i n i m a l p r o t e c t i v e d o s e s of p o l y I : C , b o t h in p r e s e n c e a n d a b s e n c e of p o l y c a t i o n s , w e r e d e t e r m i n e d as d e s c r i b e d a b o v e (Table).
Minimal protective doses of poly I:C against VSV infection in L-929 and human foetal cells in the presence and absence of polyealions
Summary. P o l y c a t i o n i c m o d i f i e d d e r i v a t i v e s of p o l y g l u t a m i c acid are a t l e a s t a s g o o d e n h a n c e r s of p o l y I : C i n d u c e d v i r a l r e s i s t a n c e in v a r i o u s cell c u l t u r e s as a r e D E A E - d e x t r a n or p o l y - L - l y s i n e 7. A. I~0TAI, T. G~NTI a n d I. Ms
Minimal protection doses 9 of poly I : C in [xg/ml of media Type of polycation b
Poly-DMAE-glutamineo Poly-DEAE-glutamine o Poly-L-lysine (Sigma) DEAE-dextran (Pharmaeia) Without polycation
L-cells
Human foetal cells
I.I0 4 1.10 .4
5.10-8 5.10-3 1.10-3 1.10-3 -
1.10-~ 5.10 -4 1.10~ '
Institute o[ Organic Chemistry, University L. Edtv6s, Muzeum krt. d/b, 1088 Budapest (Hungary); Chemical Works o/Gedeon Richter Ltd,, Budapest and Institute o/Microbiology, Medical School, Szeged (Hungary), 5 June 1975.
~Determined as described in the text. bPolycations were used in concentrations of 20 [xg/ml of media, oPolycationic modified derivatives of polyglutamic acid, with characteristic structural units as indicated in the formula.
7 This work was supported by the Chenfical Works of Gedeon Richter Ltd., Budapest.
Norepinephrine in Fetal and Neonatal Rabbit Brain In newborn m a m m a l s , such as the rat, m o u s e a n d rabbit, t h e a m o u n t of n o r e p i n e p h r i n e is r e l a t i v e l y s m a l l , s l o w l y i n c r e a s i n g t o a d u l t v a l u e s o v e r a p e r i o d of 4 t o 6 w e e k s 1, 3. All t h e s e s p e c i e s are c o n s i d e r e d d e v e l o p m e n t a l l y i m m a t u r e a t b i r t h . T h e n e w b o r n g u i n e a - p i g is c o m p a r a t i v e l y well d e v e l o p e d : its b r a i n n o r e p i n e p h r i n e is a t a p p r o x i m a t e l y t h e a d u l t level. I n t h e n e w b o r n r a b b i t (1-day-old) o n l y h a l f of t h i s v a l u e is f o u n d 1. Since we are i n t e r e s t e d in p h y s i o l o g i c a l m o d i f i c a t i o n s o c c u r r i n g in m a m m a l s d u r i n g p a r t u r i t i o n a n d a few h o u r s a f t e r w a r d s , we h a v e u n d e r t a k e n t h i s s t u d y to d e t e r m i n e w h e t h e r or n o t t h e b r a i n n o r e p i n e p h r i n e s t o r e c h a n g e s in f e t u s a n d n e w b o r n r a b b i t s d u r i n g t h i s period. Materials and methods. I n o u r e x p e r i m e n t s we h a v e u s e d t e t u s e s a n d n e w b o r n r a b b i t s of t h e w h i t e N e w Z e a l a n d s t r a i n , in w h i c h t h e t e r m of g e s t a t i o n is 31 d a y s . A t t h e s t a g e of t h e 3 0 t h or 31st d a y , t h e f e m a l e r a b b i t s w e r e killed b y a i r e m b o l i s m , l a p a r o t o m y w a s p e r f o r m e d a n d all t h e f e t u s e s w e r e t a k e n o u t a n d d e c a p i t a t e d a t
601' 501
30
I
30thday 31thday O-lh
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2-4h
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8-12h age
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Norepinephrine level in fetal and neonatal rabbit brain (ng/g). Mean values and standard error for each group are given, n = number of samples.
once. T h e b r a i n s w e r e q u i c k l y r e m o v e d , b l o t t e d on filter p a p e r a n d f r o z e n i m m e d i a t e l y . Since e a c h b r a i n c o n t a i n s low a m o u n t s of n o r e p i n e p h r i n e , it w a s n e c e s s a r y t o pool 4 to 5 in e a c h s a m p l e . T h e n e w b o r n r a b b i t s were s e p a r a t e d " f r o m t h e i r m o t h e r j u s t a f t e r b i r t h , d u r i n g t h e 1st h or l a t e r on, w i t h i n 2 to 4 or 8 to 12 h. A s w i t h t h e f e t u s e s , t h e n e w b o r n a n i m a l s f r o m t h e s a m e l i t t e r were d i v i d e d i n t o g r o u p s of 4 to 5 a n d killed b y d e c a p i t a t i o n . T h e i r b r a i n s w e r e r e m o v e d a n d t h e s a m p l e s w e r e p r e p a r e d in t h e s a m e w a y . T h e t i s s u e w a s h o m o g e n i z e d in ice-cold 0.4 M perchloric a c i d by using a Tri-R tissue homogenizer ( G e n e l a b I n t e r n a t i o n a l ) p r o v i d e d w i t h a glass pestle, a n d c e n t r i f u g e d a t 9000 • a t 0~ for 30 rain. T h e e x t r a c t i o n w a s p e r f o r m e d t w i c e m o r e a n d all 3 s u p e r n a t a n t s w e r e pooled ~. T h e p H w a s a d j u s t e d to 8.5 M Tris b u f f e r 4 a n d the samples were adsorbed onto aluminaS. W e used a c t i v e M e r c k a l u m i n i u m oxide, acidic a c t i v i t y I, p r e p a r e d b y t h e m e t h o d of ANTON a n d SAYRE 6. T h e a l u m i n a containing the adsorbed norepinephrine was washed a few t i m e s w i t h bi-distilled w a t e r . T h e e l u t i o n w a s p e r f o r m e d w i t h 3 • 2 m l of 0.3 N acetic acid, t h e a l u m i n a b e i n g m i x e d t h o r o u g h l y w i t h t h e acid b y a m a g n e t i c stirrer. All 3 e l u a t e s w e r e pooled, c e n t r i f u g e d , a d j u s t e d to p H 6.5 a n d u s e d for f l u o r o m e t r i c a s s a y L A n A m i c o B o w m a n S p e c t r o - f l u o r o m e t e r w i t h ellipsoidal m i r r o r z N. KARKI, R. KUNTZMANand t3. B. BRODIE, J. Neurochem. 9, 53 (1962). H. C. AGRAWAL, S. N. GLISSON and W. A. HIMWlCH, Int. J. Neuropharmac. 7, 97 (1968). 8 A. BERTLER, A. CARLSSON and E. ROSENGREN, Acta physiol. scand, d4, 273 (1958). 4 j . p. HANIG, J. ~V[.~V[ORRISONand S. KROP, Analyt. chim. Acta 59, 363 (1972). 5 V. RENZIm, C. A. 13RUNORIand C. VALOR1, Clin. chim. Aeta 30, 587 (1970). 6 A. H. ANTON and D. F. SAYRE, J. Pharmac. exp. Ther. 138, 360 (1962). 7 U. S. voN EULER and F. LISHAJKO, Acta physiol, scand. 57, 348 (1961).
15.10. 1975
1167
Specialia
was used for f l u o r i m e t r i c m e a s u r e m e n t s . R e a d i n g s were m a d e a t a c t i v a t i o n w a v e l e n g t h s of 380 a n d 430 n m a n d a t fluorescence w a v e l e n g t h s of 490 a n d 540 n m . T h e m e a n values 4- S E M are g i v e n in n g / g of tissue. Results. I n t h e fetuses of 30 days, t h e b r a i n n o r e p i n e p h r i n e level is low, o n l y 51 4- 4.9 ng/g. No c h a n g e s occur t h e n e x t day, t h e l a s t one before b i r t h (52 4- 9.7 rig/g), as can be seen in t h e Figure. A i t e r p a r t u r i t i o n , w i t h i n t h e 1st h, t h e n e w b o r n r a b b i t b r a i n c o n t e n t falls to 34 4- 9.9 ng/g n o r e p i n e p h r i n e , a b o u t 37% less t h a n t h a t of t h e 31st day. L a t e r on, w i t h i n 2 to 4 h, t h e a m o u n t of n o r e p h i n e p h r i n e r e t u r n s t o t h e p r e n a t a l level (52 :~ 5.6 ng/g) a n d r e m a i n s p r a c t i c a l l y the s a m e for 8 t o 12 h (51 :j: 2.4 ng/g) (see Figure). Discussion. I n t h e r a t fetus, t h e e n z y m e s i n v o l v e d in t h e b i o s y n t h e s i s of c a t e c h o l a m i n e s are p r e s e n t in t h e b r a i n a t 15 d a y s of g e s t a t i o n s, ~; a t t h i s age, t h e n o r e p i n e p h r i n e is a t o n l y 2% of t h e a d u l t level, w h e r e a s t h e b i o s y n t h e t i c e n z y m e s h a v e specific activities of a b o u t 10% of t h o s e in t h e a d u l t b r a i n ~~ A t b i r t h a n d t h e r e a f t e r , t h e biosynthetic enzymes and their products, dopamine and n o r e p i n e p h r i n e , increase in a parallel fashion 9,n. Our results show t h a t in t h e r a b b i t f e t u s on t h e 30th a n d 31st d a y of gestation, n o r e p i n e p h r i n e is p r e s e n t a n d t h e level r e m a i n s u n c h a n g e d , as is t h e case w i t h i n 2 to 4 a n d 8 t o 12 h a f t e r p a r t u r i t i o n . On t h e o t h e r h a n d , d u r i n g t h e 1st h following p a r t u r i t i o n , a decrease of n o r e p i n e p h r i n e can be seen a n d t h e v a r i a b i l i t y of t h e i n d i v i d u a l values is larger t h a n in t h e o t h e r stages. W i t h i n 2 to 4 h, t h e a m o u n t of n o r e p i n e p h r i n e rises t o t h e level m e a s u r e d in t h e fetuses, a n d r e m a i n s t h e s a m e for 8 to 12 h. Similar m o d i f i c a t i o n s of t h e a m o u n t of c a t e c h o l a m i n e s h a v e b e e n o b s e r v e d in t h e n e w b o r n r a b b i t , in varigus~.t~ssues. F o r instance, d u r i n g t h e first few h o u r s folloWirig@a~turition, one can observe a decrease of t h e a m o u n t 'of c a t e c h o l a r n i n e ~ i n t h e a d r e n a l s 12, is a n d increase in t h e plasm~ la, 14 a n d in ,tfi e h e a r t aS. These r a p i d a n d t r a n s i t o r y v a r i a t i o n s of t h e a m o u n t s of c a t e c h o l a m i n e s f o u n d in t h e n e w b o r n r a b b i t t h u s seem to be linked to birth. The fall of t h e n o r e p i n e p h r i n e level in t h e b r a i n can be r e l a t e d to t h e fetal suffering special c o n d i t i o n s d u r i n g p a r t u r i t i o n , especially t h e h y p o x i a w h i c h occurs a". I t has been e s t a b l i s h e d t h a t in t h e a d u l t cat, t h e a s p h y x i a p r o d u c e d b y r e b r e a t h i n g can decrease t h e n o r e p i n e p h r i n e level in t h e h y p o t h a l a m u s in less t h a n 2 h~7; in t h e a d u l t rat, t h e h y p o x i a decreases in 1 h t h e n o r e p i n e p h r i n e in t h e b r a i n 18. I n t h e fetus, a s p h y x i a also i n t r o d u c e s a decrease of t h e level of c a t e c h o l a m i n e s in h u m a n ~9, l a m b 2~ calf 2t a n d foal 22 a d r e n a l s a n d in h u m a n ~9 a n d r a b b i t 23 p a r a g a n g l i a as well as in h u m a n 2~ e x t r a - a d r e n a l tissue. I n t h e a d u l t rat, o t h e r severe stresses, such as p r o l o n g e d m u s c u l a r effort 25 a n d e x p o s u r e to cold 26-2s, are also k n o w n t o reduce t h e e n d o g e n o u s level of c a t e c h o l a m i n e s in tissues. C o n s e q u e n t ly, t h e s p o n t a n e o u s decrease of n o r e p i n e p h r i n e in t h e b r a i n w h i c h we h a v e f o u n d in t h e n e w b o r n r a b b i t can
also b e e x p l a i n e d b y stress c o n d i t i o n s to w h i c h t h e a n i m a l s are e x p o s e d d u r i n g p a r t u r i t i o n . As has b e e n s u g g e s t e d 29, n o r e p i n e p h r i n e , b y its v a s o m o t o r action, could c o n t r i b u t e to a b e t t e r d i s t r i b u t i o n of b l o o d t o t h e brain, t h u s increasing t h e b r a i n ' s o x y g e n s t o c k a n d facili t a t i n g t h e defense of t h e n e w b o r n a n i m a l a g a i n s t a s p h y x i a . Therefore, t h i s b r a i n n o r e p i n e p h r i n e l i b e r a t i o n d u r i n g p a r t u r i t i o n could h a v e a n i m p o r t a n t role in a m o s t critical p e r i o d of t h e m a m m a l ' s life 3~
S u m m a r y . I n t h e first h o u r a f t e r p a r t u r i t i o n , t h e n e w b o r n r a b b i t b r a i n n o r e p i n e p h r i n e c o n t e n t is a b o u t 37% less t h a n t h a t of t h e fetus of 30th or 31st day. L a t e r on, w i t h i n 2 to 4 h, t h e n o r e p i n e p h r i n e level r e t u r n s to t h e p r e n a t a l v a l u e a n d r e m a i n s u n c h a n g e d b e t w e e n 8 t o 12 h. This t r a n s i t o r y fall of t h e b r a i n n o r e p i n e p h r i n e seems to be linked t o t h e stress c o n d i t i o n s w h i c h occur d u r i n g p a r t u r i tion. I. MOTELICA-HEINO a n d J. ROFFI
Laboratoire d'Endocrinologie, Bdtiment d97, Universitd de Paris X I , F-91d05 Orsay (France), 28 A p r i l 7975.
s j. T. COYLE and J. AXELROD, J. Neurochem. 70, 449 (1972). 9 F. LAMPRECHTand J. T. COYLE, Brain Res. dl, 503 (1972). 10 j. T. COYLE and D. HENRY, J. Neuroehem. 21, 61 (1973). 11 G. R. BREESEand T. D. TRAYLOR,Br. J. Pharmae. 44, 210 (1972). 12 j. ROFFI, J. L. FROGERand M. F. DEBRAY,C. r. Aead. Sci., Paris 277, 595 (1973). la I. MOTELICA-HEINO, M. F. DEBRAY and J. ROFFI, J. Physiol., Paris, in press. 14 K. ORVAYUZand R. GHAMmR, Experientia 29, 1472 (1973). 15 j. ROFFI and I. MOTELICA-HEINO, Experientia 31, 194 (1975). 10 G. S. DAWES,in Foetal and Neonatal Physiology (Year Book Medical Publ., Chicago 1969), p. 117. 17 M. VOGT, J. Physiol., Loud. 123, 451 (1954). 18 M. STUPFEL and J. ROFFI, C. r. Goc. Bid., Paris 155, 237 (1961). 10 A. HERVONENand O. KORKALA,Acta obstet, gynee, scand. 51, 17 (1972). 20 Z. S. COMLINE and M. SILVER, Nature, Loud. 781, ~.~3"{1953)21 R. S. COMHNE and M. SILVER, J. Physiol., I.ond. 783, 305 (,1966). 2z R. S. COMLINEand M. SILVER, J. Physiol., Loud. 215, 659 (1971). 23 T. BRUNmN, Aeta physiol, scand., suppl. 280, 70 (1966). '~4 A. HERVONEN and O. KORKALA,Seand. J. clin. Lab. Invest. 27, suppl, ll6, 73 (1971). 25 j. FUGAZZA,J. Physiol. Paris, suppl. 5, 55 (1963). 2o j. D. BARCHAS and D. X. FREEDMAN, Biochem. l?harmae. 12, 1232 (1963). 27 j. LEDUC, Aeta physiol, seand., suppl. 183, 52 (1961). 28 I. MOTELICA,Acta physiol, scand. 76, 393 (1969). 29 j. BOETHIUS, ~1". BRUNDIN and N. A. PERSSON, Aeta physiol. seand. 78, 269 (1970). a0 This work was supported by a grant from DGRST (D616gation G6ndrale t~ la Recherche Scientifi~tue et Technique, contrat No. 71 73219).
C o m p a r a t i v e S t u d y of t h e E l e c t r i c a l a n d M e c h a n i c a l B e h a v i o u r of an I n t a c t , S e m i - I n t a c t a n d I s o l a t e d G a s t r o p o d e (Helix pomatia) S m o o t h M u s c l e P r e p a r a t i o n U n d e r a p p r o p r i a t e s t i m u l a t i o n conditions, t h e isolated penis r e t r a c t o r muscle (PRM) of Helix pomatia L., a g a s t r o p o d e s m o o t h muscle, can be m a d e t o p e r f o r m a phasic contraction and a prolonged contraction known as ' c a t c h ' (WABNITZl,~). W h e t h e r t h e t w o d i s t i n c t t y p e s of c o n t r a c t i o n p l a y a p a r t in t h e n o r m a l b e h a v i o u r of t h e penis r e t r a c t o r muscle in t h e i n t a c t a n i m a l is u n k n o w n .
The aim of t h e p r e s e n t e x p e r i m e n t s is to c o m p a r e t h e n o r m a l electrical a n d m e c h a n i c a l b e h a v i o u r of t h e i n t a v f penis retractor muscle;nerve-brain preparation with the p r o p e r t i e s of t h e muscle a t d i f f e r e n t stages of surgical 1 R. W. WABNITZ, Dipl.-Arbeit Univ. G6ttingen (1970). 2 R. W. WABNITZ, Cell Tiss. Res. 756, 253 (1975).
