Abe, Keishi

Feng, Xu-Chang

Fink, Edwin

Richter, Elmar

English

Open Access LMU ( Ludwig-Maximilians-Univ. München)

STUDIES ON THE INTESTINAL ABSORPTION OF TISSUE K A L L I K R E I N E d w i n F i n k * , Xu-Chang F e n g * * and E l m a r R i c h t e r * * A b t e i l u n g f ü r K l i n i s c h e Chemie und K l i n i s c h e B i o c h e m i e i n d e r C h i r u r g i s c h e n K l i n i k I n n e n s t a d t * and W a l t h e r - S t r a u b - I n s t i t u t f ü r P h a r m a k o l o g i e and T o x i k o l o g i e * * d e r U n i v e r s i t ä t M ü n c h e n INTRODUCTION I t h a s r e c e n t l y b e e n r e p o r t e d t h a t e n t e r a l a d m i n i s t r a t i o n o f t i s s u e k a l l i k r e i n (pig p a n c r e a t i c k a l l i k r e i n ) c a n have a n i m p r o v i n g e f f e c t i n s e v e r a l p a t h o l o g i c d i s o r d e r s s u c h a s an i n c r e a s e i n sperm o u t p u t and sperm m o t i l i t y i n a s t h e n o z o o s p e r m i a and Oligozoospermie ( 1 ) and a d e c r e a s e o f b l o o d p r e s s u r e i n e s s e n t i a l h y p e r t e n s i o n ( 2 ) . T h e s e findings s u g g e s t t h a t t i s s u e k a l l i k r e i n c a n be a b s o r b e d i n t e s t i n a l l y i n a b i o l o g i c a l l y a c t i v e f o r m . T h a t t h i s i s i n d e e d t h e c a s e was r e c e n t l y shown by o t h e r g r o u p s and by us ( 3 , A , 5 ) , H e r e we p r e s e n t r e s u l t s on t h e i n t e s t i n a l a b s o r p t i o n o f enzymatically a c t i v e t i s s u e k a l l i k r e i n o b t a i n e d w i t h a new e x p e r i m e n t a l m o d e l . METHODS P e r f u s i o n o f I n t e s t i n a l Segments Female S p r a g u e - D a w l e y r a t s ( 270-310 g body w e i g h t ) f a s t e d f o r 18 h were o p e r a t e d a s d e s c r i b e d by Windmuel1er and S p a e t h ( 6 ) . B r i e f l y , t h e r a t s were a n e s t h e t i z e d w i t h u r e t h a n e ( 0 . 8 g / k g , i . p . ) and t h e m e s e n t e r i a l v e i n d r a i n i n g a 8-10 cm segment o f i l e u m a s w e l l a s t h e J u g u l a r v e i n and t h e c a -r o t i d a r t e r y w e r e c a n n u l a t e d . L i g a t u r e s were s e c u r e d a r o u n d e a c h end o f t h e s e l e c t e d s e g m e n t . 0 . 2 , 1.0 o r 5.0 mg p i g p a n c r e a t i c k a l l i k r e i n i n 0.5 ml s a l i n e was i n j e c t e d i n t o t h e c l o s e d l o o p . I n c o n t r o l e x p e r i m e n t s C-14 l a -b e l e d p o l y e t h y l e n e g l y c o l 4000 ( 5 0 n C i ) was a d m i n i s t e r e d t o g e t h e r w i t h 0.5 ml s a l i n e o r 5 mg k a l l i k r e i n . B l o o d was c o l l e c t e d f r o m t h e i n t e s t i n a l v e i n f o r 60 min i n 5 rain f r a c t i o n s . The b l o o d l o s t was r e p l a c e d by i n f u s i o n o f h e p a r i n i z e d b l o o d o f d o n o r r a t s i n t o t h e j u g u l a r v e i n . A n a l y t i c a l P r o c e d u r e s I m r o u n o r e a c t l v e p o r c i n e p a n c r e a t i c k a l l i k r e i n was d e t e r m i n e d by a r a -d i o i m m u n o a s s a y ( 7 ) . K i n i n o g e n a s e a c t i v i t i e s were measured by a s s a y i n g t h e k l n i n r e l e a s e d upon i n c u b a t i o n o f an a l i q u o t o f t h e r e s p e c t i v e s a m p l e w i t h p a r t i a l l y p u r i f i e d k i n i n o g e n ( 8 ) . G e l f i l t r a t i o n e x p e r i m e n t s were p e r f o r m e d u s i n g an U l t r o g e l AcA 44 c o l u m n , 0.9 x 115 cm, e l u t e d a t a f l o w r a t e o f 4.0 ml/h w i t h 15 « M N a H 2P°4 ' 663 0.15 M N a C l , 0.01 M EDTA, 2 g N a N3/ l , pH 7.4. F r a c t i o n s o f 0.9 ml were c o l l e c t e d and a n a l y z e d f o r k i n i n o g e n a s e a c t i v i t y and i m r a u n o r e a c t i v e p i g p a n c r e a t i c k a l l i k r e i n . RESULTS Complex F o r m a t i o n o f P i g P a n c r e a t i c K a l l i k r e i n ( P P K ) w i t h Rat P l a s m a Component s S i n g l e p l a s m a s a m p l e s o b t a i n e d d u r i n g t h e a b s o r p t i o n e x p e r i m e n t s were s u b j e c t e d t o g e l f i l t r a t i o n . I mraunoreact!ve PPK was e l u t e d i n t h r e e p e a k s ( F i g . 1 ) . The peak maxima a t f r a c t i o n s 3 2 , 40 and 54 c o r r e s p o n d t o m o l e c u -l a r masses o f >130, 82 and 32 k D a . S i n c e PPK h a s a m o l e c u l a r mass a r o u n d 32 kDa t h e two peaks c o r r e s p o n d i n g t o h i g h e r m o l e c u l a r masses o b v i o u s l y r e p r e -s e n t c o m p l e x e s o f PPK w i t h components o f r a t p l a s m a . The i m m u n o r e a c t i v i t i e s o f t h e t h r e e s p e c i e s o f PPK w e r e n o t i d e n t i c a l : when s e r i a l d i l u t i o n s o f s i n g l e f r a c t i o n s o f t h e t h r e e p e a k s were measured by r a d i o i m m u n o a s s a y t h e s l o p e s o f d o s e - r e s p o n s e c u r v e s o f t h e two c o m p l e x e s were n o t i d e n t i c a l and t h e y were a l s o d i f f e r e n t f r o m t h e s l o p e o f t h e s t a n -d a r d c u r v e . The r e l a t i v e amounts o f i m r a u n o r e a c t i v e PPK e l u t e d i n t h e t h r e e p e a k s v a r i e d when d i f f e r e n t p l a s m a s a m p l e s were s u b j e c t e d t o g e l f i l t r a t i o n . I t seems t h a t t h e complex f o r m a t i o n d e pends on d i f f e r e n t c o n c e n t r a t i o n s o f t h e c o m p l e x f o r m i n g components o f t h e i n d i v i d u a l r a t p l a s m a s o r on n o t c o n -t r o l l a b l e c o n d i t i o n s d u r i n g s a m p l e p r e p a r a t i o n . f>Ç/rr^ pmolf /m.n /ml F i g . 1 G e l f i l t r a t i o n o f a s a m p l e o f m e s e n t e r i c v e n o u s p l a s m a c o n t a i n i n g a b s o r b e d p i g p a n c r e a t i c k a l l i k r e i n . The f r a c t i o n s were a s s a y e d f o r immunoreact1 ve k a l l i k r e i n ( f i l l e d c i r c l e s ) and f o r k i n i n o g e n a s e a c t i v i t y ( o p e n c i r c l e s ) . 664 K i n i n o g e n a s e a c t i v i t y was a l s o e l u t e d i n t h r e e p e a k s ( F i g . 1 ) , t h e h i g h e s t a c t i v i t y was c o n t a i n e d i n t h e l m m u n o r e a c t i v i t y p eaks c o r r e s p o n d i n g t o t h e c o m p l e x e s , a l m o s t no a c t i v i t y was f o u n d I n t h e peak o f p r e s u m a b l y f r e e PPK ( a r o u n d f r a c t i o n 5 4 ) . The " f r e e " PPK p r e s e n t i n t h e s a m p l e o b v i -o u s l y r e p r e s e n t s t o a h i g h d e g r e e an e n z y m a t l c a l l y i n a c t i v e f o r m , t h e a c -t i v e PPK i s a l m o s t c o m p l e t e l y bound t o p l a s m a c o m p o n e n t s . No k i n i n o g e n a s e p e a k s w e r e d e t e c t a b l e when an i d e n t i c a l g e l f i l t r a t i o n e x p e r i m e n t was p e r -f o r m e d w i t h a p l a s m a s a m p l e w h i c h d i d n o t c o n t a i n p i g p a n c r e a t i c k a l l i -k r e i n . R a d i o i m m u n o a s s a y o f PPK i n R a t P l a s m a As a c o n s e q u e n c e o f t h e c o m p l e x f o r m a t i o n t h e c o n c e n t r a t i o n s o f PPK measured by r a d i o i m m u n o a s s a y a r e t o o l o w . T h i s was shown by r e c o v e r y e x p e r -i m e n t s . PPK was i n c u b a t e d w i t h r a t p l a s m a i n c o n c e n t r a t i o n s between 2 and 50 ng/ml and t h e PPK c o n c e n t r a t i o n d e t e r m i n e d by r a d i o i m m u n o a s s a y . The r e c o v -e r i e s r a n g e d between 25 and 8 p e r c e n t . T h u s , s i n c e t h e r e c o v e r i e s a r e d o s e d e p e n d e n t , a c o r r e c t i o n o f t h e v a l u e s i s n o t p o s s i b l e . T a k e n t o g e t h e r , PPK and r a t p l a s m a components r e a c t u nder f o r m a t i o n o f c o m p l e x e s w h i c h have i m m u n o r e a c t l v i t l e s d i f f e r e n t f r o m and l o w e r t h a n f r e e PPK. As a r e s u l t , t h e v a l u e o b t a i n e d a s PPK c o n c e n t r a t i o n by r a d i o i m m u n o a s -s a y v a r i e s w i t h t h e d i l u t i o n a t w h i c h t h e s a m p l e i s a s s a y e d and i s s i g n i f i -c a n t l y l o w e r t h a n t h e t r u e PPK c o n c e n t r a t i o n i n t h e s a m p l e . I n a d d i t i o n , t h e r a t i o o f t h e amounts o f t h e t h r e e i m m u n o r e a c t i v e PPK s p e c i e s may v a r y f r o m s a m p l e t o s a m p l e , t h u s , e v e n i f t h e t r u e c o n c e n t r a t i o n s o f PPK i n d i f f e r e n t p l a s m a s a m p l e s a r e i d e n t i c a l t h e c o n c e n t r a t i o n s d e t e r m i n e d by r a d i o i m m u n o a s s a y may s t i l l be d i f f e r e n t . 25-F i g . 2 C u m u l a t i v e amount o f p i g p a n c r e a t i c k a l l i k r e i n i n mes-e n t e r i a l v e nous b l o o d o f an i n t e s t i n a l segment a f t e r a d m i n i s t r a t i o n o f 1.0 mg ( f o u r i d e n t i c a l l y p e r f o r m e d e x p e r i m e n t s , t h e amount o f a b s o r b e d k a l l i k r e i n i s c a l -c u l a t e d p e r cm o f t h e i n t e s t i n a l s e g m e n t ) . 665 A t t h e s e d i f I i ^ y i t c a n n o t be o v e r c o m e and no o ..:, more r e l i a b l e method t o d e t e r m i n e l o w c o n c e n t r a t i o n s o f PPK i n r a t p l a s m a i s a t hand. T h e r e f o r e , i n s p i t e o f t h e p r o b l e m s j u s t p r e s e n t e d we used t h e r a -d i o i m m u n o a s s a y p r o c e d u r e i n o u r s t u d i e s . H o w e v e r , one has t o k e e p i n mind t h a t t h e c o n c e n t r a t i o n s d e t e r m i n e d r e p r e s e n t " i m r a u n o r e a c t i v i t y e q u i v a l e n t s " and n o t t h e t r u e PPK c o n c e n t r a t i o n s . The t r u e PPK c o n c e n t r a t i o n s w i l l be s i g n i f i c a n t l y h i g h e r ( c e r t a i n l y by a f a c t o r o f t e n ) t h a n t h e v a l u e s p r e -s e n t e d h e r e . I n t e s t i n a l A b s o r p t i o n o f P o r c i n e P a n c r e a t i c K a l l i k r e i n P o r c i n e p a n c r e a t i c k a l l i k r e i n was a d m i n i s t e r e d a t d o s e l e v e l s o f 0.2, 1.0 and 5.0 mg. When 0.2 mg were a d m i n i s t e r e d , o n l y i n one out o f s i x e x p e r -i m e n t s a b s o r p t i o n o f PPK was d e t e c t a b l e w h e r e a s a t d o s e s 1.0 mg and 5.0 mg o n l y one e x p e r i m e n t o u t o f e i g h t and n i n e , r e s p e c t i v e l y , was n e g a t i v e . The amount a b s o r b e d d u r i n g one h o u r per l cm o f i l e u m was 2.6 ng f o r dose 0.26 mg, up t o 22 ng f o r d o s e 1.0 mg and up t o 2 4 8 ng f o r d o s e 5.0 rag. T h u s , i n s p i t e o f t h e h i g h v a r i a b i l i t y o f a b s o r p t i o n a d o s e dependency i s c l e a r l y r e c o g n i z a b l e . The t i m e c o u r s e o f i n t e s t i n a l a b s o r p t i o n f o r dose l e v e l s 1.0 and 5.0 mg i s s h o w n i n F i g s . 2 a n d 3. The h i g h e s t amount a b s o r b e d i n one o f t h e e x p e r i m e n t s was 0.05 Z o f t h e a d m i n i s t e r e d d o s e . H o w e v e r , one has t o c o n s i d e r t h a t t h i s amount was ab-s o r b e d by o n l y 10 cm o f i l e u m w h e r e a s t h e t o t a l l e n g t h o f r a t s m a l l i n t e s -t i n e i s a b o u t 100 cm and t h a t t h e shape o f t h e c u r v e s c l e a r l y d e m o n s t r a t e s t h a t t h e a b s o r p t i o n was s t i l l i n p r o g r e s s a t t h e end o f t h e e x p e r i m e n t . I n a d d i t i o n , a s d i s c u s s e d a b o v e , t h e PPK c o n c e n t r a t i o n s i n t h e p l a s m a s a m p l e s a r e h i g h l y u n d e r e s t i m a t e d by t h e r a d i o i m m u n o a s s a y . 240-220-200-180-160-140-120-100-80-60-40-?0-04 T * 1 , , , , , , , r , -0 S K) 15 20 25 30 35 40 4 5 50 S5 60 m.n F i g . 3 C u m u l a t i v e amount o f p i g p a n c r e a t i c k a l l i k r e i n i n mes-e n t e r i a l v enous b l o o d o f an i n t e s t i n a l segment a f t e r a d m i n i s t r a t i o n o f 5.0 mg ( f o u r i d e n t i c a l l y p e r f o r m e d e x p e r i m e n t s , t h e amount o f a b s o r b e d k a l l i k r e i n i s c a l -c u l a t e d p e r cm o f t h e i n t e s t i n a l s e g m e n t ) . 666 I n a c o n t r o l e x p e r i m e n t C-14 l a b e l l e d PEG 4000 was a d m i n i s t e r e d t o -g e t h e r w i t h 5 mg k a l l i k r e i n . O n l y a b o u t 0.8 Z o f t h e r a d i o a c t i v i t y were found i n t h e b l o o d w h i c h i s i n t h e same r a n g e a s i n t h e a b s e n c e o f k a l l i -k r e i n . Thus, n e i t h e r t h e e x p e r i m e n t a l p r o c e d u r e n o r t h e p r e s e n c e o f t h e t i s s u e k a l l i k r e i n c a u s e d any s i g n i f i c a n t u n s p e c i f i c p e r m e a b i l i t y i n c r e a s e o f t h e i n t e s t i n a l w a l l f o r h i g h m o l e c u l a r s u b s t a n c e s . DISCUSSION E a r l i e r i n v e s t i g a t i o n s ( 3 - 5 ) h a ve shown t h a t i n t e s t i n a l a b s o r p t i o n o f t i s s u e k a l l i k r e i n i s p o s s i b l e and some e v i d e n c e h a s been p r e s e n t e d s u g g e s t -i n g t h a t t h e a b s o r b e d k a l l i k r e i n i s t o some d e g r e e s t i l l e n z y m a t i c a l i y a c t i v e . The e x p e r i m e n t a l m o d e l e m p l o y e d h e r e w i l l a l l o w t o s t u d y t h e i n t e s t i -n a l a b s o r p t i o n o f t i s s u e k a l l i k r e i n on a f u l l y q u a n t i t a t i v e b a s i s a s so o n as t h e p r o b l e m s o f t h e r a d i o i m m u n o a s s a y can be o v e r c o m e . The model i s w e l l e s t a b l i s h e d f o r s t u d i e s on t h e a b s o r p t i o n o f s m a l l m o l e c u l e s and seems t o be e q u a l l y w e l l s u i t e d f o r i n v e s t i g a t i o n s on t h e t i s s u e k a l l i k r e i n a b s o r p -t i o n though f u r t h e r c o n t r o l e x p e r i m e n t s w i l l be n e c e s s a r y t o c o n f i r m t h a t t h e o b s e r v e d a b s o r p t i o n i s not an a r t e f a c t o f t h e e x p e r i m e n t a l s e t u p . The p r e s e n t s t u d i e s c o r r o b o r a t e t h e e a r l i e r f i n d i n g s (3-4) and demon-s t r a t e c l e a r l y t h a t t i s s u e k a l l i k r e i n c a n i n d e e d be a b s o r b e d i n an enzyma-t i c a l i y a c t i v e f o r m . V i r t u a l l y a l l o f t h e a c t i v e t i s s u e k a l l i k r e i n i s bound t o p l a s m a c o m p o n e n t s , b u t t h e s e k a l l i k r e i n c o m p l e x e s s t i l l d i s p l a y k i n i n o -g enase a c t i v i t y . I t i s n o t c l e a r w h e t h e r t h i s k i n i n o g e n a s e a c t i v i t y i s i n -t r i n s i c o r due t o a p a r t i a l d i s s o c i a t i o n o f t h e c o m p l e x e s u n d e r t h e c o n d i -t i o n s o f o u r a s s a y . At p r e s e n t , n o t h i n g i s known on t h e n a t u r e and t h e p h y s i o l o g i c a l f u n c -t i o n o f the c o m p l e x f o r m i n g p r o t e i n s . One m i g h t s p e c u l a t e t h a t t h e y a c t b o t h a s i n h i b i t o r s and a s c a r r i e r s f o r t i s s u e k a l l i k r e l n s p r e v e n t i n g unde-s i r a b l e k i n i n r e l e a s e d u r i n g t h e t r a n s p o r t t o t h e t a r g e t s i t e . Compared t o t h e a d m i n i s t e r e d d o s e t h e f r a c t i o n o f a b s o r b e d t i s s u e k a l -l i k r e i n was q u i t e l o w and i t may be r e g a r d e d a s q u e s t i o n a b l e w h e t h e r s u c h s m a l l amounts c a n e x e r t t h e p h a r m a c o l o g i c a l e f f e c t s o b s e r v e d i n human. How-e v e r , f o r t h e t r e a t m e n t o f a s t h e n o z o o s p e r m i a and o l i g o z o o s p e r m i a a s w e l l a s of e s s e n t i a l h y p e r t e n s i o n t h e t i s s u e k a l l i k r e i n i s a d m i n i s t e r e d o r a l l y f o r a t l e a s t s e v e r a l weeks and u n d e r t h e s e c o n d i t i o n s a p h a r m a c o l o g i c a l l y a c -t i v e l e v e l may b u i l d up. ACKNOWLEDGMENTS T h i s work was s u p p o r t e d by t h e D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t , S o n d e r f o r -s c h u n g s b e r e i c h 2 0 7 , B2. P i g p a n c r e a t i c k a l l i k r e i n was k i n d l y p r o v i d e d by Dr. P. H u b e r , B a y e r AG, W u p p e r t a l , FRG. We a r e v e r y g r a t e f u l t o M r s . G. Godec and Mr s . C H i r a c h a u e r f o r t h e i r e x c e l l e n t t e c h n i c a l a s s i s t a n c e . REFERENCES 1. 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Studies on the Intestinal Absorption of Tissue Kallikrein

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Studies on the Intestinal Absorption of Tissue Kallikrein

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