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Substrate-dependent angiotensin II formation in the peripheral circulation
Life Sciences, Vol. 46, pp. 335-341 Printed in the U.S.A.
Pergamon Press
S U B S T R A T E - D E P E N D E N T A N G I O T E N S I N II F O R M A T I O N IN THE P E R I P H E R A L C I R C U L A T I O N M u n e h i t o Ideishi, M a n a b u Sasaguri, M a s a h a r u Ikeda and K i k u o A r a k a w a D e p a r t m e n t of Internal M e d i c i n e , F u k u o k a U n i v e r s i t y School of Medicine, Fukuoka, 814-01, J a p a n (Received in final form December 4, 1989) Summary An a l t e r n a t i v e a n g i o t e n s i n I I - f o r m i n g s y s t e m d i s t i n c t f r o m the v a s c u l a r r e n i n - a n g i o t e n s i n s y s t e m was d e m o n s t r a t e d u s i n g a rat h i n d l i m b p e r f u s i o n s y s t e m and synthetic substrates. This p a t h w a y was r e s i s t a n t to c a p t o p r i l and aprotinin, but was h i g h l y s e n s i t i v e to chymostatin. Moreover, a n g i o t e n s i n II f o r m a t i o n w a s s u b s t r a t e - d e p e n d e n t , i.e. a n g i o t e n s i n II f o r m a t i o n f r o m t r i d e c a p e p t i d e human renin s u b s t r a t e in the p r e s e n c e of c a p t o p r i l was m o r e than twice than that from an e q u i m o l a r amount of a n g i o t e n s i n I. Both p a t h w a y s m a y p l a y a role in r e g u l a t i n g the p e r i p h e r a l c i r c u l a t i o n . It is a q e n e r s l ] y held v ~ e w that the v a s o a c t i v e p e p t i d e a n g i o t e n s i n (ANG) II is formed in the p l a s m a f r o m ANG I by the a c t i o n of e n d o t h e l i a l A N G c o n v e r t i n g e n z y m e (ACE), m a i n l y in the p u l m o n a r y c i r c u l a t i o n ( l ) , w h e r e a s ANG I is f o r m e d in the p l a s m a by the r e a c t i o n of k i d n e y - d e r i v e d r e n i n w i t h a n g i o t e n s i n o g e n d e r i v e d f r o m the liver. In a d d i t i o n to this " c l a s s i c a l " r e n i n - a n g i o t e n s i n system, components of the s y s t e m w e r e found in other t i s s u e s ( 2 , 3 ) . The p e r i p h e r a l v a s c u l a r wall is of p a r t i c u l a r i n t e r e s t ( 4 - 6 ) , since its smooth m u s c l e is the m a j o r t a r g e t of the v a s o a c t i v e p e p t i d e ANG IT. On the o t h e r hand, several o t h e r ANG I I - f o r m i n g p a t h w a y s , i n d e D e n d e n t of remin and ACE, have b e e n d i s c o v e r e d and e x a m i n e d recently. Juul et al r e p o r t e d that t e n s i o n d e v e l o p m e n t in rat femoral vessel s e g m e n t s induced by a t e t r a d e c a p e p t i d e renin s u b s t r a t e was not i n h i b i t e d by an ACE i n h i b i t o r ( 7 ) . We also r e o o r t e d on the a l t e r n a t i v e p a t h w a y " k i n i n - t e n s i n system", in w b i c h serine p r o t e a s e s such as t r y p s i n ( 8 ) , k a l l i k r e i n ( 9 , 1 0 ) and t o n i n ( l l ~ form both v a s o d e p r e s s c r k i n i n s and v a s o p r e s s o r A N G II. In addition, the e x i s t e n c e of n e w l y - r e c o g n i z e d enzymes, r e s i s t a n t to ACE i n h i b i t o r s , was r e p o r t e d i n d e p e n d e n t l y by O k u n i s h i et s] (12,]~), L a r z i l l o et el(J4), and J o h n s o n and D r u m m e r ( 1 5 ) . ~ ] t b o u g h these r e p o r t s address the l i k e l i h o o d of local g e n e r a t i o n of ANG If, the a b o v e - m e n t i o n e d p h y s i o l o g i c a l e x p e r i m e n t s did not p r o v i d e c l e a r - c u t e v i d e n c e to s u p p o r t f o r m a t i o n of ANG II in the p e r i p h e r a l c i r c u l a t i o n . A plausible reas~ is tb~t the r e s p o n s e is a f f e c t e d by the p h e n o m e n a of 0024-3205/90 $3.00 +.00 Copyright (c) 1990 Pergamon Press plc
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" t a c h y p h y l a x i s " ( 1 6 ) or a u t o p o t e n t i a t i o n ( 1 7 ) to ANG II. Therefore, we c o n d u c t e d e x p e r i m e n t s to q u a n t i t a t e the amount of ANG II in the D e r f u s a t e by HPLC and r a d i o i m m u n o a s s a y in order to e l u c i d a t e h o w the a l t e r n a t i v e and " c l a s s i c a l " p a t h w a y s c o n t r i b u t e to ANG II f o r m a t i o n in the p e r i p h e r a l c i r c u l a t i o n . Materials
and m e t h o d s
Chemicals. S y n t h e t i c A N G I, A N G II and h u m a n t r i d e c a p e p t i d e r e n i n s u b s t r a t e (13RS) and c h y m o s t a t i n w e r e p u r c h a s e d f r o m the P e p t i d e I n s t i t u t e (Osaka, Japan). A p r o t i n i n and c a p t o p r i l w e r e g i f t s k i n d l y p r o v i d e d b y B a y e r (Leverkusen, W e s t Germany) and S a n k y o P h a r m a c e u t i c a l Co. (Tokyo, Japan), r e s p e c t i v e l y . All o t h e r c h e m i c a l s w e r e of r e a g e n t grade. Hind]imb perfusion. E x p e r i m e n t s w e r e c a r r i e d out on m a l e W i s t a r rats (10-14 weeks of age) w h i c h w e r e a n e s t h e t i z e d and n e p h r e c t o m i z e d . A perfusion c a n n u l a was i n t r o d u c e d into the a b d o m i n a l aorta just b e l o w the left renal artery. A n o t h e r c a n n u l a was i n t r o d u c e d into the v e n a c a v a for c o l l e c t i o n of the r e t u r n e d p e r f u s a t e . Both hindquarters w e r e i s o l a t e d by t i g h t l i g a t i o n of the b o d y at the level at w h i c h the p e r f u s i o n c a n n u l a s w e r e i n t r o d u c e d . The p r e p a r a t i o n was p e r f u s e d w i t h m o d i f i e d ~ r e b s - H e n s e l e i t s o l u t i o n at 37°C, c o n t a i n i n g 1 g/L glucose, 5 g/L D e x t r a n T-70, and a m i n o acids(18) and g a s s e d w i t h O 9 / C O 2 ( 9 5 % / 5 % ) . The f l o w rate was kept at 4 ml/min, w h i c h r e s d l t e a in a p e r f u s i o n p r e s s u r e of 30 to 50 mmHg. P r i o r to each e x p e r i m e n t , the p r e p a r a t i o n was w a s h e d for 40-60 m i n to r e m o v e blood. The e f f l u e n t p e r f u s a t e was c o l l e c t e d at e a c h 7 m!n interval in i c e - c h i l l e d tubes. A N G I or 13RS was i n f u s e d for the first 2.5 min of each 7 min interval. D e t e r m i n a t i o n of A N G II. The c o l l e c t e d p e r f u s a t e was c e n t r i f u g e d at 1,000 r p m for 15 min to r e m o v e any t r a c e a m o u n t s of c o n t a m i n a t i n g b l o o d cells. The s u p e r n a t e was a p p l i e d to m e t h a n o l - a c t i v a t e d S e p - P a k C l R c a r t r i d g e s and e l u t e d w i t h 100% m e t h a n o l . The e l u a t e was then e g ~ p o r a t e d to dryness under reduced pressure. R e s i d u e s w e r e r e d i s s o l v e d in 20% a c e t o n i t r i l e in 0.05 M s o d i u m p h o s p h a t e b u f f e r (pH 5.5), and a p p l i e d to an HPLC c o l u m n (Nucleosil C , 0.4 x 20 cm) p r e e q u i l i b r a t e d with the same solution. ~e c o l u m n was e l u t e d i s o c r a t i c a l l y for i0 min, then e l u t e d w i t h a linear g r a d i e n t of a c e t o n i t r i ! e (20% to 35% in 0 . 0 5 M s o d i u m p h o s p h a t e buffer, pH 5.5) for 20 min at a f l o w rate of 1 m l / m i n and m o n i t o r e d at 214 nm. The c o l u m n was s t a n d a r d i z e d w i t h s y n t h e t i c ~ N G I, ANG II and 13RS. A n g i o t e n s i n I I - c o n t a i n i n g f r a c t i o n s w e r e c o l l e c t e d and p r o c e s s e d for r a d i o i m m u n o a s s a y of ANG II. The a n t i b o d y against a n g i o t e n s i n II r a i s e d in male w h i t e rabbit was u s e d at 1 , 0 0 0 , 0 0 0 dilution(ref.10). The cross r e a c t i v i t y of the a n t i s e r u m w i t h ANGI apd 13 RS w e r e 0.3% a~d 0.1%, r e s p e c t i v e l y . The r e c o v e r y of ANG d u r i n g the e x p e r i m e n t a l p r o c e d u r e s was d e t e r m i n e d by a d d i t i o n of s y n t h e t i c ANG II into the p e r f u s a t e b e f o r e S e p - P a k C18 e x t r a c t i o n . S t a t i s t i c a l analyses. T h e e x p e r i m e n t a l r e s u l t s are p r e s e n t e d as m e a n ± S.D. E f f e c t s of i m h i b i t o r s w e r e a n a l y s e d by t w o - t a i l e d S t u d e n t ' s t - t e s t for p a i r e d or u n p a i r e d ~ata, w i t h p < 0 . 0 5 b e i n g taken as ~ i g n i f i c a n t . T u k e y ' s m e t h o d was u s e d to control the e r r o r a s s o c i a t e d w i t h
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comparisons
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for the s i g n i f i c a n c e test in e x p e r i m e n t
337
4.
Experimental ?rotecols. E x p e r i m e n t I: R a t s w e r e i n f u s e d w i t h ANG I (i or 2 ~g, N=8) or ]3RS fl0 or 20 IJg, N=5) at 14 min i n t e r v a l s . A f t e r this c o n t r o l experiment, similar infusions were repeated during continuous i n f u s i o ~ of a p r o t i n i n . E x p e r i m e n t 2: Rats w e r e i n f u s e d w i t h A N G I (I or 2 ug, N=6) or 13RS (10 or 20 ug, N=6) by the same p r o c e d u r e s as in E x p . l d u r i n g c o n t i n u o u s i n f u s i o n of c a p t o p r i l a l o n e or c a p t o p r i l p l u s aprotinin. E x p e r i m e n t 3: E f f e c t s of c o n t i n u o u s i n f u s i o n of c h y m o s t a t i n on the f o r m a t i o n of A N G II f r o m 20 Hg of 13RS or 2 ~g of ANG I w e r e e x a m i n e d (N=5). E x p e r i m e n t 4: ANG II f o r m a t i o n f r o m e q u i m o l a r a m o u n t s (about 3.8 nmol) of the s u b s t r a t e s (5 pg of A N G I, 6.2 ~g 13RS) in the p r e s e n c e of c a p t o p r i l was d e t e r m i n e d b e f o r e and d u r i n g c o n t i n u o u s i n f u s i o n of c h y m o s t a t i n (N=5). W e u s e d t h r e e e n z y m e i n h i b i t o r s - c a p t o p r i l , a p r o t i n i n and c h y m o s t a t J n - w h i c h are k n o w n to be p o t e n t i n h i b J t o r s of ACE, k a ] l i k r e i n and c h y m o t r y p s i n , r e s p e c t i v e l y . These inhibitors were u s e d to d e t e r m i n e w h i c h e n z y m e was r e s p o n s i b l e for the f o r m a t i o n cf ANG II in the system, b e c a u s e k a l l i k r e i n - l i k e and c h y m o t r y p s i n - s e n s i t i v e e n z y m e s have b e e n s u g g e s t e d to f o r m A N G II by us ( 1 0 , 1 1 ) a n d o t h e r s (12,13). The final c o n c e n t r a t i o n s of the e n z y m e i ~ h i b J t ~ r s , c a p t o p r i l , a p r o t i n i n and c h y m o s t a t i n w e r e i0 M, 2x10 M and 4 . 1 x 1 0 M, r e s p e c t i v e l y . C a p t o p r i l in this c o n c e n t r a t i o n has b e e n r e p o r t e d to i n h i b i t lung A C E ( 1 9 ) . The a m o u m t of a p r o t J n i n was the same as that u t i l i z e d in a p r e v i o u s e x p e r i m e n t ( 1 0 ) in w h i c h it showed ~ull i n h i b i t i o n of t i s s u e ka]]ikrein. The c o n c e n t r a t i o n of c h y m o s t a t i n in this e x p e r i m e n t was o n e - t e n t h of t h a t u s e d in an e x p e r i m e n t r e p o r t e d b y O k u n i s h i et al(!3), b e c a u s e of its i n s o l u b i l i t y in a q u e o u s s o l u t i o n . Results The p e p t i d e r e c o v e r y from the e x t r a c t i o n p r o c e d u r e s in the p r e s e n t e x p e r i m e n t was e x a m i n e d u s i n g s y n t h e t i c A N G II. W h e n the p e p t i d e was p e r f u s e d ms w i t h ANG I and 13RS, a p p r o x i m a t e l y 25% of the i n f u s e d a m o u n t was d e t e c t e d by r a d i o i m m u n o a s s a y . This r e c o v e r y was not a f f e c t e d s i g n i f i c a n t l y by any of the t h r e e ]nhibitors utilized. W h e n s y m t h e t i c A N G II was a d d e d to the p e r f u s a t e just b e f o r e S e o - P a k e x t r a c t i o n , the r e c o v e r y was 88.7 ± 5.~%. R e t e n t i o ~ times of s y t h e t i c ANG II, A N G I and ]3RS w e r e 14.7 min, 26.4 mJ~ and 30.6 min, r e s p e c t i v e l y , w i t h the H P L C c o n d i t i o n s shown in " M a t e r i a l s and M e t h o d s " . The e l u a t e was c o l l e c t e d for ANG II r a d J o i m m u n o a s s a y from ]4 t h r o u g h ]6 min. F r a g m e n t s of A N G p e p t i d e s , H i s - L e u , A s p - A r g - V a l - T y r and I l e - H i s - P r o - P h e w e r e not e l u t e d ~n th~s p e r i o d . The e f f e c t s of a p r o t i n i n on the a m o u n t of ANG II r e c o v e r e d in the a b s e n c e or p r e s e n c e of c a p t o p r i l w h e n A N G I or 13RS was ~ n f u s e d are s n m m a r i z e d Jn T a b l e I. D a t a are shown as the a m o u n t of ANG TT d e r i v e d f r o m 1 pg of e a c h s u b s t r a t e . E f f e c t s of
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a p r o t i n i n d i d not r e a c h a s t a t i s t i c a l l y s i g n i f i c a n t l e v e l , b u t t h e r e was a t e n d e n c y for r e d u c t i o n in A N G II f o r m a t i o n f r o m 1 3 R S in t h e p r e s e n c e of c a p t o p r i l . Conversely, captopril suppressed s i g n i f i c a n t l y the f o r m a t i o n of A N G II f r o m A N G I, b u t not t h a t f r o m 13RS. TABLE
I
E f f e c t of A p r o t ~ n i n on the A m o u n t of A n g i o t e n s i n II R e c o v e r e d in the A b s e n c e a n d P r e s e n c e of C a p t o p r i l Inhibitor Captopril
-
Aprotinin
+
-
+
-
+
4.5±4.6* 7.3±2.4
3.1±2.2" 5.7±1.5
Substrate ANG I 13RS
95.1±41.8 5.5± 2.8
113.4±55.6 5 . 4 ± 2.1
ANG II/ug substrate, mean ± SD * p < 0 . 0 5 c o m p a r e d w i t h v a l u e s in the
ng
absence
of c a p t o p r i l
C h y m o s t a t i n i n h i b i t e d A N G II f o r m a t i o n f r o m 1 3 R S ( 2 . 9 ± 1 . 9 to 0 . 6 ± 0 . 3 ng A N G I I / ~ g 13RS, p < 0 . 0 2 5 ) , w h e r e a s no s u p p r e s s i o n w a s o b s e r v e d w h e n A N G I s e r v e d as the s u b s t r a t e ( i i i . 2 ± 3 3 . 6 to 1 0 4 . 8 ± 6 1 . 5 ng A N G I I / ~ g A N G I, NS).
Chymostatir Captopril h\\\\\\\\\\\9
lOOlE •,=
~
B
"
.8
13RS Angl13RS Angl 6.2pg 5/Jg621Jg 5pg Substrate FIG.
1
E f f e c t of c h y m o s t a t i n on a n g i o t e n s i n II f o r m a t i o n in t h e D r e s e n c e of c a p t o p r i l (mean ± SD). 13RS, t r i d e c a p e p t i d e h u m a n r e n i n s u b s t r a t e ; A n g I, a n g i o t e n s i n I; M e a n s w i t h s a m e l e t t e r are n o t s i g n i f i c a n t l y d i f f e r e n t .
the
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In the p r e s e n c e of captopril, s i g n i f i c a n t l y m o r e A N G II w a s g e n e r a t e d from 13RS t h a n f r o m e q u i m o l a r a m o u n t s of A N G I. The a m o u n t s of ANG II f o r m e d f r o m b o t h s u b s t r a t e s in the p r e s e n c e of c a p t o p r i l was s u p p r e s s e d by c h y m o s t a t i n , a l t h o u g h s t a t i s t i c a l s i g n i f i c a n c e was not a c h i e v e d in the case of ANG I (FIG.l). Discussion The p r e s e n t s t u d y c o n f i r m e d the f o r m a t i o n of A N G II in the p e r i p h e r a l c i r c u l a t i o n , by the q u a n t i t a t i o n of A N G II in the p e r f u s a t e , as p r e v i o u s l y s u g g e s t e d by h i n d l i m b p e r f u s i o n e x p e r i m e n t s on rats(5) and dogs(6), and b y h u m a n f o r e a r m experiments(20). In the p r e s e n t e x p e r i m e n t , a r o u n d 1 0 - 1 5 % of i n f u s e d ANG I was c o n v e r t e d to a n g i e t e n s i n II w h e n m e a s u r e d as i m m u n o r e a c t i v e ANG II in the p e r f u s a t e a f t e r e x t r a c t i o n . This v a l u e is c o n s i s t e n t w i t h r e p o r t s in w h i c h the c o n v e r s i o n of A N G I to ANG II was e s t i m a t e d as 40%, and d e g r a d a t i o n of the A N G II thus f o r m e d was 65% i~ the p e r i p h e r a l t i s s u e ( 2 1 ) . In addition, it was p r o v e n that f o r m a t i o n of ANG II in this s y s t e m d e p e n d s on b o t h the v a s c u l a r r e n i n - a n g i o t e n s i n s y s t e m and an a l t e r n a t i v e p a t h w a y . Namely, an ACE inhibitor, c a p t o p r i l d i d not a f f e c t A N G II g e n e r a t i o n f r o m 13RS, w h i l s t the g e n e r a t i o n f r o m A N G I was s u p p r e s s e d b y a p p r o x i m a t e l y 90% or more. The i n c o m p l e t e i n h i b i t i o n of ANG II f c r m a t i o n from ANG I b y c a p t o p r i l does not s i m p l y i n d i c a t e that t h e r e is an a l t e r n a t i v e pathway, r a t h e r it m a y i n d i c a t e d i f f e r e n t i a l s e n s i t i v i t y of t i s s u e ACEs to the ACE J~hibitors. The e f f i c i e n c y of 13RS as a s u b s t r a t e for the f o r m a t i o n of A N G II was o n e - t w e n t i e t h of that of ANG I in the h i n d l i m b p e r f u s i o n system, b a s e d on m o l a r considerations. However, the c o n c e n t r a t i o n of 13RS in the p e r f u s a t e was not e x t r e m e l y high b u t was s i m i l a r t o that of the renin s u b s t r a t e c o n c e n t r a t i o n in n e p h r e c t o m i z e d animals(22). The human p l a s m a c o n c e n t r a t i o n of renin s u b s t r a t e has also b e e n r e p o r t e d as about 100 ng A n g I e q u i v a l e n t / m l , w h i c h is m u c h h i g h e r than the c o n c e n t r a t i o n of A n g I(23). Thus the reqllirement for h i g h e r c o n c e n t r a t i o n s of s u b s t r a t e for the a l t e r n a t i v e p a t h w a y d o e s not imply that it is p h y s i o l o g i c a l l y iDsignificant. It c o u l d be m o r e s i g n i f i c a n t w h e n ACE i n h i b i t o r s are a d m i n i s t e r e d . C h y m o s t a t i n s u p p r e s s e d ANG II f o r m a t i o n f r o m 13RS b y about 80%, w h i l s t no s u p p r e s s i o n was s h o w n w h e n A N G I s e r v e d as the s u b s t r a t e in the a b s e n c e of c a p t o p r i l . T h i s is t a k e n to suggest that c o n v e r s i o n of ANG I to A N G II d e p e n d s h e a v i l y on ACE. This was also s u p p o r t e d by the fact t h a t A N G II f o r m a t i o n f r o m ANG I was a l m o s t c o m p l e t e l y i n h i b i t e d by c a p t o p r i l . In o r d e r to d e t e r m i n e w h e t h e r or not the a l t e r n a t i v e p a t h w a y shows s u b s t r a t e d e p e n d e n c y , e q u i m o ] a r a m o u n t s of 13RS a n d A N G I w e r e u s e d as ~ u b s t r a t e s in the p r e s e n c e of captopril. Significantly m e r e ANG II was r e c o v e r e d when 13ES was u t i l i z e d . We u t i l i z e d this s y n t h e t i c RS as the model of the n a t u r a l substrate, a n g i o t e n s i n o g e n , as h~ve o t h e r i n v e s t i g a t o r s ( 5 , 7 ) . In summary, a n e w p u t a t i v e A N G I I - f o r m i n g p a t h w a y was d e m o n s t r a t e d in the rat p e r i p h e r a l v a s c u l a r bed. This pathway was s e n s i t i v e to c h y m o s t a t i n , but r e s i s t a n t to c a p t o p r i l . The s e n s i t i v i t y of the p r e s e n t s y s t e m to c h y m o s t a t i n seems to be s i m i l a r to that of the e n z y m e d e s c r i b e d b y O k u n i s h i et al(13).
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However, d i f f e r e n c e s in s u b s t r a t e s p e c i f i c i t y still r e m a i n obscure, since ANG I I - f o r m i n g a c t i v i t y was d e t e c t e d o n l y w i t h ANG I as a substrate. Further, it should be e l u c i d a t e d w h e t h e r the s n b s t r a t e d e p e n d e n c y of the p r e s e n t s y s t e m was due e i t h e r to its b i o c h e m i c a l nature or to its anatomical localization. It is p o s s i b l e that a c c e s s i b i l i t y of the e n z y m e causes s u b s t r a t e d e p e n d e n c y since the n e w enzyme r e c o g n i z e d by O k u n i s h i et al. is u b i q u i t o u s in the adventitia. ~ n o t h e r c a n d i d a t e in the p r e s e n t s y s t e m is the mast cell c h y m o t r y p s i n - l i k e enzyme r e p o r t e d by W i n t r o u b et ai(24). However, d e f i n i t i v e b i o c h e m i c a l c o m p a r i s o n s must await complete i s o l a t i o n and c h a r a c t e r i z a t i o n of these enzymes. It remains to be e l u c i d a t e d w h e t h e r the a l t e r n a t i v e p a t h w a y d e s c r i b e d here cleaves natural a n g i o t e n s i n o g e n and h o w it c o n t r i b u t e s to r e g u l a t i o n of the p e r i p h e r a l circulation. Acknowledgments T h i s w o r k was s u p p o r t e d in part by a G r a n t - i n - A i d f r o m the M i n i s t r y of Education, Science and C u l t u r e of Japan (No.62480221). We are grateful to Miss M i t s u k o O n i k u r a for technical a s s i s t a n c e and to Miss H i s a k o Oka for s e c r e t a r i a l work.
References I. 2. 3. 4. 5. 6. 7. 8. 9. 10. II. 12. 13. 14. 15. 16.
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S.OPARIL, G . W . T R E G E A R , T.J.KOERNER, B . A . B A R N E S and E.HABER, Circ.Res. 29 682-690 (1971). N.J.SAMANI~--K.MORGAN, W . J . B R A M M A R and J.D.SWALES, J.Hypertens. 5(Suppl 2) S19-S21 (1987). H.OHKUBO, K.NAKAYAMA, T . T A N A K A and S.NAKANISHI, J.Biol.Chem. 261 319-323 (1986). I.KIFOR and V.J.DZAU, Circ.Res. 60 422-428 (1987). J . A . O L I V E R and R . R . S C I A C C A , J.Cl~-n. Invest. 74 1247-1251 (1984). P . J . M E T T I N G and S.L.BRITTON, Can. J . P h y s i o l . P h a r m a c o l . 65 544549 (1986). B.JUUL, C . A A L K J A E R and M . J . M U L V A N Y , J.Hypertens. 5 ( S u p p l 2) $7-S10 (1987). K.ARAKAWA, M.YUKI amd M.IKEDA, Biochem.J. 187 647-653 (1980). K . A R A K A W A and H.MARUTA, Nature 288 705-706 (1980). M.IDEISHI, M . I K E D A and K.ARAKAWA, J.Biochem. 102 859-868 (1987). M . I K E D A and K.ARAKAWA, H y p e r t e n s i o n 6 222-228 (1984). H.OKUNISHI, M . M I Y A Z A K I and N.TODA, JTHypertens. 2 277-284 (1984). H.OKUNISHI, M . M I Y A Z A K I , T . O K A M U R A and N.TODA, Biochem. Biophys. Res.Commun. 149 1186-1192 (1987). J.J.LANZILLO, Y . D A S A R A T H Y , J . S T E V E N S J and B.L.FANBURG, Biochem.Biophys.Res.Commun. 134 770-776 (1986). H . J O H N S O N and O . H . D R U M M E R , B i o c h e m . P h a r m a c o l . 37 1131-1136 (1988). J.M.STEWART, T a c h y p h ~ l a x i s to an~iotensin, H a n d b o o k of P h a r m a c o l o g y Voi.37, I.H.PAGE and F . M . B U M P U S (eds), 170-184, S p r i n g e r Verlag, N e w Y o r k (1974). T.GODFRAIND, B r . J . P h a r m a c o l . 40 5 4 2 p - 5 4 3 p (1970). G . D E M E L L O and T.MAACK, A m . J . P ~ s i o l . 231 1699-1707 (1976). D.W.CUSHMAN, H.S.CHEUNG, E . F . S A B O and M.A.ONDETTI, Prog. Cardiovasc.Dis. 21 176-182 (1978).
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D . J . W E B B and J.G.COLLIER, J . C a r d i o v a s c . P h a r m a c o l . 8 ( S u p p l i0) $40-$44 (1986). D . J . C A M P B E L L , J . H y p e r t e n s . 3 199-207 (1985). O . C A R R E T E R O and F.GROSS, A m . J . P h y s i o l . 213 695-700 (1967). P.EGGENA, J.D.BARRETT, H.HIDAKA, C.L.CHU, C . T H A N A N O P A V A R N , M . S . G L O U B and M . P . S A M B H I , Circ.Res. 41 I I - 3 4 - I I - 3 7 (1977). B . U . W I N T R O U B , C . E . K A E M P F E R , N.M.SCHECH---TER and D.PROUD, J.Clin. Invest. 77 196-201 (1986).
Pergamon Press
S U B S T R A T E - D E P E N D E N T A N G I O T E N S I N II F O R M A T I O N IN THE P E R I P H E R A L C I R C U L A T I O N M u n e h i t o Ideishi, M a n a b u Sasaguri, M a s a h a r u Ikeda and K i k u o A r a k a w a D e p a r t m e n t of Internal M e d i c i n e , F u k u o k a U n i v e r s i t y School of Medicine, Fukuoka, 814-01, J a p a n (Received in final form December 4, 1989) Summary An a l t e r n a t i v e a n g i o t e n s i n I I - f o r m i n g s y s t e m d i s t i n c t f r o m the v a s c u l a r r e n i n - a n g i o t e n s i n s y s t e m was d e m o n s t r a t e d u s i n g a rat h i n d l i m b p e r f u s i o n s y s t e m and synthetic substrates. This p a t h w a y was r e s i s t a n t to c a p t o p r i l and aprotinin, but was h i g h l y s e n s i t i v e to chymostatin. Moreover, a n g i o t e n s i n II f o r m a t i o n w a s s u b s t r a t e - d e p e n d e n t , i.e. a n g i o t e n s i n II f o r m a t i o n f r o m t r i d e c a p e p t i d e human renin s u b s t r a t e in the p r e s e n c e of c a p t o p r i l was m o r e than twice than that from an e q u i m o l a r amount of a n g i o t e n s i n I. Both p a t h w a y s m a y p l a y a role in r e g u l a t i n g the p e r i p h e r a l c i r c u l a t i o n . It is a q e n e r s l ] y held v ~ e w that the v a s o a c t i v e p e p t i d e a n g i o t e n s i n (ANG) II is formed in the p l a s m a f r o m ANG I by the a c t i o n of e n d o t h e l i a l A N G c o n v e r t i n g e n z y m e (ACE), m a i n l y in the p u l m o n a r y c i r c u l a t i o n ( l ) , w h e r e a s ANG I is f o r m e d in the p l a s m a by the r e a c t i o n of k i d n e y - d e r i v e d r e n i n w i t h a n g i o t e n s i n o g e n d e r i v e d f r o m the liver. In a d d i t i o n to this " c l a s s i c a l " r e n i n - a n g i o t e n s i n system, components of the s y s t e m w e r e found in other t i s s u e s ( 2 , 3 ) . The p e r i p h e r a l v a s c u l a r wall is of p a r t i c u l a r i n t e r e s t ( 4 - 6 ) , since its smooth m u s c l e is the m a j o r t a r g e t of the v a s o a c t i v e p e p t i d e ANG IT. On the o t h e r hand, several o t h e r ANG I I - f o r m i n g p a t h w a y s , i n d e D e n d e n t of remin and ACE, have b e e n d i s c o v e r e d and e x a m i n e d recently. Juul et al r e p o r t e d that t e n s i o n d e v e l o p m e n t in rat femoral vessel s e g m e n t s induced by a t e t r a d e c a p e p t i d e renin s u b s t r a t e was not i n h i b i t e d by an ACE i n h i b i t o r ( 7 ) . We also r e o o r t e d on the a l t e r n a t i v e p a t h w a y " k i n i n - t e n s i n system", in w b i c h serine p r o t e a s e s such as t r y p s i n ( 8 ) , k a l l i k r e i n ( 9 , 1 0 ) and t o n i n ( l l ~ form both v a s o d e p r e s s c r k i n i n s and v a s o p r e s s o r A N G II. In addition, the e x i s t e n c e of n e w l y - r e c o g n i z e d enzymes, r e s i s t a n t to ACE i n h i b i t o r s , was r e p o r t e d i n d e p e n d e n t l y by O k u n i s h i et s] (12,]~), L a r z i l l o et el(J4), and J o h n s o n and D r u m m e r ( 1 5 ) . ~ ] t b o u g h these r e p o r t s address the l i k e l i h o o d of local g e n e r a t i o n of ANG If, the a b o v e - m e n t i o n e d p h y s i o l o g i c a l e x p e r i m e n t s did not p r o v i d e c l e a r - c u t e v i d e n c e to s u p p o r t f o r m a t i o n of ANG II in the p e r i p h e r a l c i r c u l a t i o n . A plausible reas~ is tb~t the r e s p o n s e is a f f e c t e d by the p h e n o m e n a of 0024-3205/90 $3.00 +.00 Copyright (c) 1990 Pergamon Press plc
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" t a c h y p h y l a x i s " ( 1 6 ) or a u t o p o t e n t i a t i o n ( 1 7 ) to ANG II. Therefore, we c o n d u c t e d e x p e r i m e n t s to q u a n t i t a t e the amount of ANG II in the D e r f u s a t e by HPLC and r a d i o i m m u n o a s s a y in order to e l u c i d a t e h o w the a l t e r n a t i v e and " c l a s s i c a l " p a t h w a y s c o n t r i b u t e to ANG II f o r m a t i o n in the p e r i p h e r a l c i r c u l a t i o n . Materials
and m e t h o d s
Chemicals. S y n t h e t i c A N G I, A N G II and h u m a n t r i d e c a p e p t i d e r e n i n s u b s t r a t e (13RS) and c h y m o s t a t i n w e r e p u r c h a s e d f r o m the P e p t i d e I n s t i t u t e (Osaka, Japan). A p r o t i n i n and c a p t o p r i l w e r e g i f t s k i n d l y p r o v i d e d b y B a y e r (Leverkusen, W e s t Germany) and S a n k y o P h a r m a c e u t i c a l Co. (Tokyo, Japan), r e s p e c t i v e l y . All o t h e r c h e m i c a l s w e r e of r e a g e n t grade. Hind]imb perfusion. E x p e r i m e n t s w e r e c a r r i e d out on m a l e W i s t a r rats (10-14 weeks of age) w h i c h w e r e a n e s t h e t i z e d and n e p h r e c t o m i z e d . A perfusion c a n n u l a was i n t r o d u c e d into the a b d o m i n a l aorta just b e l o w the left renal artery. A n o t h e r c a n n u l a was i n t r o d u c e d into the v e n a c a v a for c o l l e c t i o n of the r e t u r n e d p e r f u s a t e . Both hindquarters w e r e i s o l a t e d by t i g h t l i g a t i o n of the b o d y at the level at w h i c h the p e r f u s i o n c a n n u l a s w e r e i n t r o d u c e d . The p r e p a r a t i o n was p e r f u s e d w i t h m o d i f i e d ~ r e b s - H e n s e l e i t s o l u t i o n at 37°C, c o n t a i n i n g 1 g/L glucose, 5 g/L D e x t r a n T-70, and a m i n o acids(18) and g a s s e d w i t h O 9 / C O 2 ( 9 5 % / 5 % ) . The f l o w rate was kept at 4 ml/min, w h i c h r e s d l t e a in a p e r f u s i o n p r e s s u r e of 30 to 50 mmHg. P r i o r to each e x p e r i m e n t , the p r e p a r a t i o n was w a s h e d for 40-60 m i n to r e m o v e blood. The e f f l u e n t p e r f u s a t e was c o l l e c t e d at e a c h 7 m!n interval in i c e - c h i l l e d tubes. A N G I or 13RS was i n f u s e d for the first 2.5 min of each 7 min interval. D e t e r m i n a t i o n of A N G II. The c o l l e c t e d p e r f u s a t e was c e n t r i f u g e d at 1,000 r p m for 15 min to r e m o v e any t r a c e a m o u n t s of c o n t a m i n a t i n g b l o o d cells. The s u p e r n a t e was a p p l i e d to m e t h a n o l - a c t i v a t e d S e p - P a k C l R c a r t r i d g e s and e l u t e d w i t h 100% m e t h a n o l . The e l u a t e was then e g ~ p o r a t e d to dryness under reduced pressure. R e s i d u e s w e r e r e d i s s o l v e d in 20% a c e t o n i t r i l e in 0.05 M s o d i u m p h o s p h a t e b u f f e r (pH 5.5), and a p p l i e d to an HPLC c o l u m n (Nucleosil C , 0.4 x 20 cm) p r e e q u i l i b r a t e d with the same solution. ~e c o l u m n was e l u t e d i s o c r a t i c a l l y for i0 min, then e l u t e d w i t h a linear g r a d i e n t of a c e t o n i t r i ! e (20% to 35% in 0 . 0 5 M s o d i u m p h o s p h a t e buffer, pH 5.5) for 20 min at a f l o w rate of 1 m l / m i n and m o n i t o r e d at 214 nm. The c o l u m n was s t a n d a r d i z e d w i t h s y n t h e t i c ~ N G I, ANG II and 13RS. A n g i o t e n s i n I I - c o n t a i n i n g f r a c t i o n s w e r e c o l l e c t e d and p r o c e s s e d for r a d i o i m m u n o a s s a y of ANG II. The a n t i b o d y against a n g i o t e n s i n II r a i s e d in male w h i t e rabbit was u s e d at 1 , 0 0 0 , 0 0 0 dilution(ref.10). The cross r e a c t i v i t y of the a n t i s e r u m w i t h ANGI apd 13 RS w e r e 0.3% a~d 0.1%, r e s p e c t i v e l y . The r e c o v e r y of ANG d u r i n g the e x p e r i m e n t a l p r o c e d u r e s was d e t e r m i n e d by a d d i t i o n of s y n t h e t i c ANG II into the p e r f u s a t e b e f o r e S e p - P a k C18 e x t r a c t i o n . S t a t i s t i c a l analyses. T h e e x p e r i m e n t a l r e s u l t s are p r e s e n t e d as m e a n ± S.D. E f f e c t s of i m h i b i t o r s w e r e a n a l y s e d by t w o - t a i l e d S t u d e n t ' s t - t e s t for p a i r e d or u n p a i r e d ~ata, w i t h p < 0 . 0 5 b e i n g taken as ~ i g n i f i c a n t . T u k e y ' s m e t h o d was u s e d to control the e r r o r a s s o c i a t e d w i t h
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4.
Experimental ?rotecols. E x p e r i m e n t I: R a t s w e r e i n f u s e d w i t h ANG I (i or 2 ~g, N=8) or ]3RS fl0 or 20 IJg, N=5) at 14 min i n t e r v a l s . A f t e r this c o n t r o l experiment, similar infusions were repeated during continuous i n f u s i o ~ of a p r o t i n i n . E x p e r i m e n t 2: Rats w e r e i n f u s e d w i t h A N G I (I or 2 ug, N=6) or 13RS (10 or 20 ug, N=6) by the same p r o c e d u r e s as in E x p . l d u r i n g c o n t i n u o u s i n f u s i o n of c a p t o p r i l a l o n e or c a p t o p r i l p l u s aprotinin. E x p e r i m e n t 3: E f f e c t s of c o n t i n u o u s i n f u s i o n of c h y m o s t a t i n on the f o r m a t i o n of A N G II f r o m 20 Hg of 13RS or 2 ~g of ANG I w e r e e x a m i n e d (N=5). E x p e r i m e n t 4: ANG II f o r m a t i o n f r o m e q u i m o l a r a m o u n t s (about 3.8 nmol) of the s u b s t r a t e s (5 pg of A N G I, 6.2 ~g 13RS) in the p r e s e n c e of c a p t o p r i l was d e t e r m i n e d b e f o r e and d u r i n g c o n t i n u o u s i n f u s i o n of c h y m o s t a t i n (N=5). W e u s e d t h r e e e n z y m e i n h i b i t o r s - c a p t o p r i l , a p r o t i n i n and c h y m o s t a t J n - w h i c h are k n o w n to be p o t e n t i n h i b J t o r s of ACE, k a ] l i k r e i n and c h y m o t r y p s i n , r e s p e c t i v e l y . These inhibitors were u s e d to d e t e r m i n e w h i c h e n z y m e was r e s p o n s i b l e for the f o r m a t i o n cf ANG II in the system, b e c a u s e k a l l i k r e i n - l i k e and c h y m o t r y p s i n - s e n s i t i v e e n z y m e s have b e e n s u g g e s t e d to f o r m A N G II by us ( 1 0 , 1 1 ) a n d o t h e r s (12,13). The final c o n c e n t r a t i o n s of the e n z y m e i ~ h i b J t ~ r s , c a p t o p r i l , a p r o t i n i n and c h y m o s t a t i n w e r e i0 M, 2x10 M and 4 . 1 x 1 0 M, r e s p e c t i v e l y . C a p t o p r i l in this c o n c e n t r a t i o n has b e e n r e p o r t e d to i n h i b i t lung A C E ( 1 9 ) . The a m o u m t of a p r o t J n i n was the same as that u t i l i z e d in a p r e v i o u s e x p e r i m e n t ( 1 0 ) in w h i c h it showed ~ull i n h i b i t i o n of t i s s u e ka]]ikrein. The c o n c e n t r a t i o n of c h y m o s t a t i n in this e x p e r i m e n t was o n e - t e n t h of t h a t u s e d in an e x p e r i m e n t r e p o r t e d b y O k u n i s h i et al(!3), b e c a u s e of its i n s o l u b i l i t y in a q u e o u s s o l u t i o n . Results The p e p t i d e r e c o v e r y from the e x t r a c t i o n p r o c e d u r e s in the p r e s e n t e x p e r i m e n t was e x a m i n e d u s i n g s y n t h e t i c A N G II. W h e n the p e p t i d e was p e r f u s e d ms w i t h ANG I and 13RS, a p p r o x i m a t e l y 25% of the i n f u s e d a m o u n t was d e t e c t e d by r a d i o i m m u n o a s s a y . This r e c o v e r y was not a f f e c t e d s i g n i f i c a n t l y by any of the t h r e e ]nhibitors utilized. W h e n s y m t h e t i c A N G II was a d d e d to the p e r f u s a t e just b e f o r e S e o - P a k e x t r a c t i o n , the r e c o v e r y was 88.7 ± 5.~%. R e t e n t i o ~ times of s y t h e t i c ANG II, A N G I and ]3RS w e r e 14.7 min, 26.4 mJ~ and 30.6 min, r e s p e c t i v e l y , w i t h the H P L C c o n d i t i o n s shown in " M a t e r i a l s and M e t h o d s " . The e l u a t e was c o l l e c t e d for ANG II r a d J o i m m u n o a s s a y from ]4 t h r o u g h ]6 min. F r a g m e n t s of A N G p e p t i d e s , H i s - L e u , A s p - A r g - V a l - T y r and I l e - H i s - P r o - P h e w e r e not e l u t e d ~n th~s p e r i o d . The e f f e c t s of a p r o t i n i n on the a m o u n t of ANG II r e c o v e r e d in the a b s e n c e or p r e s e n c e of c a p t o p r i l w h e n A N G I or 13RS was ~ n f u s e d are s n m m a r i z e d Jn T a b l e I. D a t a are shown as the a m o u n t of ANG TT d e r i v e d f r o m 1 pg of e a c h s u b s t r a t e . E f f e c t s of
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a p r o t i n i n d i d not r e a c h a s t a t i s t i c a l l y s i g n i f i c a n t l e v e l , b u t t h e r e was a t e n d e n c y for r e d u c t i o n in A N G II f o r m a t i o n f r o m 1 3 R S in t h e p r e s e n c e of c a p t o p r i l . Conversely, captopril suppressed s i g n i f i c a n t l y the f o r m a t i o n of A N G II f r o m A N G I, b u t not t h a t f r o m 13RS. TABLE
I
E f f e c t of A p r o t ~ n i n on the A m o u n t of A n g i o t e n s i n II R e c o v e r e d in the A b s e n c e a n d P r e s e n c e of C a p t o p r i l Inhibitor Captopril
-
Aprotinin
+
-
+
-
+
4.5±4.6* 7.3±2.4
3.1±2.2" 5.7±1.5
Substrate ANG I 13RS
95.1±41.8 5.5± 2.8
113.4±55.6 5 . 4 ± 2.1
ANG II/ug substrate, mean ± SD * p < 0 . 0 5 c o m p a r e d w i t h v a l u e s in the
ng
absence
of c a p t o p r i l
C h y m o s t a t i n i n h i b i t e d A N G II f o r m a t i o n f r o m 1 3 R S ( 2 . 9 ± 1 . 9 to 0 . 6 ± 0 . 3 ng A N G I I / ~ g 13RS, p < 0 . 0 2 5 ) , w h e r e a s no s u p p r e s s i o n w a s o b s e r v e d w h e n A N G I s e r v e d as the s u b s t r a t e ( i i i . 2 ± 3 3 . 6 to 1 0 4 . 8 ± 6 1 . 5 ng A N G I I / ~ g A N G I, NS).
Chymostatir Captopril h\\\\\\\\\\\9
lOOlE •,=
~
B
"
.8
13RS Angl13RS Angl 6.2pg 5/Jg621Jg 5pg Substrate FIG.
1
E f f e c t of c h y m o s t a t i n on a n g i o t e n s i n II f o r m a t i o n in t h e D r e s e n c e of c a p t o p r i l (mean ± SD). 13RS, t r i d e c a p e p t i d e h u m a n r e n i n s u b s t r a t e ; A n g I, a n g i o t e n s i n I; M e a n s w i t h s a m e l e t t e r are n o t s i g n i f i c a n t l y d i f f e r e n t .
the
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339
In the p r e s e n c e of captopril, s i g n i f i c a n t l y m o r e A N G II w a s g e n e r a t e d from 13RS t h a n f r o m e q u i m o l a r a m o u n t s of A N G I. The a m o u n t s of ANG II f o r m e d f r o m b o t h s u b s t r a t e s in the p r e s e n c e of c a p t o p r i l was s u p p r e s s e d by c h y m o s t a t i n , a l t h o u g h s t a t i s t i c a l s i g n i f i c a n c e was not a c h i e v e d in the case of ANG I (FIG.l). Discussion The p r e s e n t s t u d y c o n f i r m e d the f o r m a t i o n of A N G II in the p e r i p h e r a l c i r c u l a t i o n , by the q u a n t i t a t i o n of A N G II in the p e r f u s a t e , as p r e v i o u s l y s u g g e s t e d by h i n d l i m b p e r f u s i o n e x p e r i m e n t s on rats(5) and dogs(6), and b y h u m a n f o r e a r m experiments(20). In the p r e s e n t e x p e r i m e n t , a r o u n d 1 0 - 1 5 % of i n f u s e d ANG I was c o n v e r t e d to a n g i e t e n s i n II w h e n m e a s u r e d as i m m u n o r e a c t i v e ANG II in the p e r f u s a t e a f t e r e x t r a c t i o n . This v a l u e is c o n s i s t e n t w i t h r e p o r t s in w h i c h the c o n v e r s i o n of A N G I to ANG II was e s t i m a t e d as 40%, and d e g r a d a t i o n of the A N G II thus f o r m e d was 65% i~ the p e r i p h e r a l t i s s u e ( 2 1 ) . In addition, it was p r o v e n that f o r m a t i o n of ANG II in this s y s t e m d e p e n d s on b o t h the v a s c u l a r r e n i n - a n g i o t e n s i n s y s t e m and an a l t e r n a t i v e p a t h w a y . Namely, an ACE inhibitor, c a p t o p r i l d i d not a f f e c t A N G II g e n e r a t i o n f r o m 13RS, w h i l s t the g e n e r a t i o n f r o m A N G I was s u p p r e s s e d b y a p p r o x i m a t e l y 90% or more. The i n c o m p l e t e i n h i b i t i o n of ANG II f c r m a t i o n from ANG I b y c a p t o p r i l does not s i m p l y i n d i c a t e that t h e r e is an a l t e r n a t i v e pathway, r a t h e r it m a y i n d i c a t e d i f f e r e n t i a l s e n s i t i v i t y of t i s s u e ACEs to the ACE J~hibitors. The e f f i c i e n c y of 13RS as a s u b s t r a t e for the f o r m a t i o n of A N G II was o n e - t w e n t i e t h of that of ANG I in the h i n d l i m b p e r f u s i o n system, b a s e d on m o l a r considerations. However, the c o n c e n t r a t i o n of 13RS in the p e r f u s a t e was not e x t r e m e l y high b u t was s i m i l a r t o that of the renin s u b s t r a t e c o n c e n t r a t i o n in n e p h r e c t o m i z e d animals(22). The human p l a s m a c o n c e n t r a t i o n of renin s u b s t r a t e has also b e e n r e p o r t e d as about 100 ng A n g I e q u i v a l e n t / m l , w h i c h is m u c h h i g h e r than the c o n c e n t r a t i o n of A n g I(23). Thus the reqllirement for h i g h e r c o n c e n t r a t i o n s of s u b s t r a t e for the a l t e r n a t i v e p a t h w a y d o e s not imply that it is p h y s i o l o g i c a l l y iDsignificant. It c o u l d be m o r e s i g n i f i c a n t w h e n ACE i n h i b i t o r s are a d m i n i s t e r e d . C h y m o s t a t i n s u p p r e s s e d ANG II f o r m a t i o n f r o m 13RS b y about 80%, w h i l s t no s u p p r e s s i o n was s h o w n w h e n A N G I s e r v e d as the s u b s t r a t e in the a b s e n c e of c a p t o p r i l . T h i s is t a k e n to suggest that c o n v e r s i o n of ANG I to A N G II d e p e n d s h e a v i l y on ACE. This was also s u p p o r t e d by the fact t h a t A N G II f o r m a t i o n f r o m ANG I was a l m o s t c o m p l e t e l y i n h i b i t e d by c a p t o p r i l . In o r d e r to d e t e r m i n e w h e t h e r or not the a l t e r n a t i v e p a t h w a y shows s u b s t r a t e d e p e n d e n c y , e q u i m o ] a r a m o u n t s of 13RS a n d A N G I w e r e u s e d as ~ u b s t r a t e s in the p r e s e n c e of captopril. Significantly m e r e ANG II was r e c o v e r e d when 13ES was u t i l i z e d . We u t i l i z e d this s y n t h e t i c RS as the model of the n a t u r a l substrate, a n g i o t e n s i n o g e n , as h~ve o t h e r i n v e s t i g a t o r s ( 5 , 7 ) . In summary, a n e w p u t a t i v e A N G I I - f o r m i n g p a t h w a y was d e m o n s t r a t e d in the rat p e r i p h e r a l v a s c u l a r bed. This pathway was s e n s i t i v e to c h y m o s t a t i n , but r e s i s t a n t to c a p t o p r i l . The s e n s i t i v i t y of the p r e s e n t s y s t e m to c h y m o s t a t i n seems to be s i m i l a r to that of the e n z y m e d e s c r i b e d b y O k u n i s h i et al(13).
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However, d i f f e r e n c e s in s u b s t r a t e s p e c i f i c i t y still r e m a i n obscure, since ANG I I - f o r m i n g a c t i v i t y was d e t e c t e d o n l y w i t h ANG I as a substrate. Further, it should be e l u c i d a t e d w h e t h e r the s n b s t r a t e d e p e n d e n c y of the p r e s e n t s y s t e m was due e i t h e r to its b i o c h e m i c a l nature or to its anatomical localization. It is p o s s i b l e that a c c e s s i b i l i t y of the e n z y m e causes s u b s t r a t e d e p e n d e n c y since the n e w enzyme r e c o g n i z e d by O k u n i s h i et al. is u b i q u i t o u s in the adventitia. ~ n o t h e r c a n d i d a t e in the p r e s e n t s y s t e m is the mast cell c h y m o t r y p s i n - l i k e enzyme r e p o r t e d by W i n t r o u b et ai(24). However, d e f i n i t i v e b i o c h e m i c a l c o m p a r i s o n s must await complete i s o l a t i o n and c h a r a c t e r i z a t i o n of these enzymes. It remains to be e l u c i d a t e d w h e t h e r the a l t e r n a t i v e p a t h w a y d e s c r i b e d here cleaves natural a n g i o t e n s i n o g e n and h o w it c o n t r i b u t e s to r e g u l a t i o n of the p e r i p h e r a l circulation. Acknowledgments T h i s w o r k was s u p p o r t e d in part by a G r a n t - i n - A i d f r o m the M i n i s t r y of Education, Science and C u l t u r e of Japan (No.62480221). We are grateful to Miss M i t s u k o O n i k u r a for technical a s s i s t a n c e and to Miss H i s a k o Oka for s e c r e t a r i a l work.
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