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Kallikrein-kinin system in plasma of poikilotherm vertebrates Dunn, Rex Stewart 1971

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THE KALLIKREIN-KININ SYSTEM IN PLASMA OF POIKILOTHERM VERTEBRATES b y REX DUNN B . S c , A c a d i a U n i v e r s i t y , 1965 M . S c , A c a d i a U n i v e r s i t y , 1967 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n t h e Department o f Z o o l o g y We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e ^ s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA O c t o b e r , 1970' In presenting this thesis in p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Z o o l o g y The University of B r i t i s h Columbia Vancouver 8, Canada Date  J u n e 1 4 /  1 9 7 1 ABSTRACT The l i t t l e s t u d i e d plasma k a l l i k r e i n - k i n i n s y s t e m o f p o i k i l o t h e r m v e r t e b r a t e s was i n v e s t i g a t e d i n s e v e r a l s p e c i e s o f f i s h , a m p h i b i a n s , and r e p t i l e s , and compared t o t h e w e l l - known mammalian enzyme s y s t e m . I t was f o u n d t h a t t h e p l a s m a s o f a l l f i s h and a m p h i b i a n s t e s t e d d i f f e r e d f r o m r e p t i l i a n and mammalian plasma i n t h e i r i n a b i l i t y t o r e l e a s e a k i n i n - l i k e f a c t o r when r e a c t e d w i t h t r y p s i n o r g l a s s , and no e v i d e n c e was o b t a i n e d t o s u g g e s t t h a t t h e s e p l a s m a s c o n t a i n e n zymic m a c h i n e r y which can p r o d u c e a k i n i n . However, i t was shown t h a t h e a t - d e n a t u r e d plasma f r o m t h e s e a n i m a l s d i d d e v e l o p b i o l o g i c a l a c t i v i t y when t r e a t e d w i t h hog p a n c r e a s k a l l i k r e i n , an enzyme s p e c i f i c f o r r e l e a s i n g k i n i n s . Thus, t h e e q u i  v a l e n t o f a k i n i n o g e n m i g h t e x i s t i n t h e s e p l a s m a s . S i n c e t u r t l e plasma p r o d u c e d a k i n i n b y endogenous en zymes, d e t a i l e d s t u d i e s o f t h i s s y s t e m were c o n d u c t e d . By a v a r i e t y o f c r i t e r i a , e nzymic mechanisms f o r k i n i n p r o d u c t i o n i n t h i s plasma were c l o s e l y s i m i l a r t o t h o s e o f mammalian plasma. However, p u r i f i c a t i o n o f t h e t u r t l e k i n i n r e l e a s e d by endogenous enzymes, f o l l o w e d by p h a r m a c o l o g i c a l and chem i c a l t e s t s showed t h a t t h i s k i n i n was c h e m i c a l l y d i f f e r e n t f rom b r a d y k i n i n , i t s mammalian c o u n t e r p a r t . Data o b t a i n e d from amino a c i d a n a l y s i s o f t h e p e p t i d e , and from c e r t a i n p h a r m a c o l o g i c a l t e s t s , s t r o n g l y s u g g e s t e d t h a t t h e s t r u c t u r e o f t u r t l e k i n i n i s 6 - t h r - b r a d y k i n i n ; i . e . , t h a t a t h r e o n i n e r e s i d u e has been s u b s t i t u t e d f o r a s e r i n e . The p o s s i b l e s i g  n i f i c a n c e o f t h i s f i n d i n g i s d i s c u s s e d . P r e l i m i n a r y s t u d i e s o f 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 f b r a d y k i n i n on a s p e c t s o f b l o o d p r e s s u r e and f l o w i n t h e t u r t l e i t s e l f a r e d e s c r i b e d . I n t r a - a r t e r i a l i n j e c t i o n s o f b r a d y k i n i n o v e r a wide r a n g e o f doses always p r o d u c e d a p r e s s o r r e s p o n s e which c o u l d be g r e a t l y r e d u c e d b y a d r e n e r g i c b l o c k a d e . T h i s i s i n c o n t r a s t w i t h the e f f e c t o f ,the p e p t i d e i n mammals, where t h e r e i s t y p i c a l l y a h y p o t e n s i v e r e s p o n s e w h i c h c a n n o t be r e d u c e d by a d r e n e r g i c b l o c k a d e . The s i g n i f i c a n c e o f t h i s d i f f e r  ence i s d i s c u s s e d , and a p p r o a c h e s t o f u t u r e i n v e s t i g a t i o n s a r e s u g g e s t e d . i i i TABLE OF CONTENTS Page A b s t r a c t 1 L i s t ' o f T a b l e s v i i i L i s t o f F i g u r e s x D e f i n i t i o n s and A b b r e v i a t i o n s x v i Acknowledgements x v i i i C h a p t e r 1. I n t r o d u c t i o n 1 C h a p t e r 2. G e n e r a l Methods 8 A. S i l i c o n i n g P r o c e d u r e 8 B. Rat U t e r u s B i o a s s a y 8 C. S o u r c e o f A n i m a l s 11 D. B l o o d S a m p l i n g 12 E. S a m p l i n g o f K i n i n - P r o d u c i n g R e a c t i o n s 12 F. P r e p a r a t i o n o f Sephadex G-25 f o r G e l F i l t r a t i o n C hromatography 14 G. P r e p a r a t i o n o f CM-Sephadex f o r Ion Exchange Chromatography 16 H. E s t i m a t i o n o f "Lowry" P e p t i d e 17 C h a p t e r 3. C o m p a r a t i v e S t u d i e s on F i s h , Amphibian, R e p t i l i a n and Mammalian Plasmas 19 A. D e n a t u r e d Plasma 19 1. E f f e c t o f t r y p s i n on whole b l o o d f r o m f i s h and mammals 19 2. E f f e c t o f t r y p s i n and hog p a n c r e a s k a l l i - k r e i n on v e r t e b r a t e plasmas 23 i v Page B. I n t a c t Plasma 31 1. K i n i n a s e s i n f i s h , a m p h i b i a n , r e p t i l i a n , and mammalian plasmas 31 2. R e l a t i v e e f f e c t i v e n e s s o f g l a s s s u r f a c e , t r y p s i n , and hog p a n c r e a s k a l l i k r e i n i n r e l e a s i n g k a l l i k r e i n f r o m u n d e n a t u r e d v e r t e b r a t e plasmas 37 3. E f f e c t o f g l a s s + Hageman F a c t o r i n r e l e a s i n g k i n i n f r o m u n d e n a t u r e d t r o u t , f r o g and Amphiuma pl a s m a s 40 C. D i s c u s s i o n and C o n c l u s i o n s 42 C h a p t e r 4. The K a l l i k r e i n - K i n i n System i n T u r t l e Plasma 47 A. P r e l i m i n a r y S t u d i e s 49 1. P r o d u c t i o n o f t u r t l e k i n i n i n t h e a b s e n c e o f a k i n i n a s e i n h i b i t o r 49 2. E f f e c t o f mammalian k i n i n a s e i n h i b i t o r s on t u r t l e k i n i n a s e » 52 3. E f f e c t o f plasma d i l u t i o n on t u r t l e k i n i n p r o d u c t i o n 56 B. E f f e c t o f G l a s s S u r f a c e A r e a on T u r t l e K i n i n P r o d u c t i o n 59 C. A c t i v a t i o n and Decay o f Plasma K a l l i k r e i n 64 1. P r e p a r a t i o n o f S u b s t r a t e 1 - d e p l e t e d p lasma 64 2. A c t i v a t i o n o f plasma k a l l i k r e i n i n t u r t l e and r a t plasma 70 3. Decay o f pl a s m a k a l l i k r e i n *. 74 D. E f f e c t o f Temperature on K i n i n P r o d u c t i o n 76 V Page E. Kininogens i n Turtle Plasma 78 1. Effect of trypsin concentration on t u r t l e k i n i n release i n intact plasma 80 2. Effect of trypsin on Substrate 1-depleted plasma 83 F. Discussion and Conclusions 89 Chapter 5. Chemical and Pharmacological Tests on Purified Turtle Kinin 90 A. P u r i f i c a t i o n of Glass Released Turtle Kinin ... 90 1. Method 90 a. Release of k i n i n 91 b. Extraction procedure 92 c. Column chromatography 93 i . Gel f i l t r a t i o n 93 i i . Ion exchange 94 2. Results 95 3. Discussion and Conclusions 99 B. Pharmacological Studies 100 1. Methods 101 a. General 101 b. Bioassays 102 i . Rat uterus sensitized with chymotrypsin 102 i i . Relaxation of the rat duodenum 102 v i Page i i i . C o n t r a c t i o n o f t h e g u i n e a p i g i l e u m 103 i v . D e p r e s s i o n o f r a t b l o o d p r e s s u r e 103 v. D e p r e s s i o n o f r a b b i t b l o o d p r e s s u r e 105 2. R e s u l t s and D i s c u s s i o n 107 C. C h e m i c a l S t u d i e s 110 1. M i s c e l l a n e o u s T e s t s 110 a. B o i l i n g 110 b . T r y p s i n 110 c. B u t a n o l e x t r a c t i o n I l l 2. Paper Chromatography I l l 3. G e l F i l t r a t i o n on Sephadex G-25 115 4. Ion Exchange Chromatography on CM- Sephadex 115 5. Amino A c i d A n a l y s i s 121 D. D i s c u s s i o n and C o n c l u s i o n s 129 C h a p t e r 6. P r e l i m i n a r y S t u d i e s o f t h e E f f e c t o f B r a d y k i n i n on B l o o d P r e s s u r e i n t h e T u r t l e . . 132 A. Method 133 B. R e s u l t s 135 C. D i s c u s s i o n and C o n c l u s i o n s 137 C h a p t e r 7. Summary,and G e n e r a l D i s c u s s i o n 142 L i t e r a t u r e C i t e d 151 V 1 X Page A p p e n d i x A 159 A p p e n d i x B 160 A p p e n d i x C 161 A p p e n d i x D 163 A p p e n d i x E 165 A p p e n d i x F 166 v i i i L I S T OF TABLES T a b l e Page 1 Summary o f method u s e d i n w i t h d r a w i n g b l o o d 13 2 K i n i n o g e n i n f i s h and mammalian b l o o d , as d e t e r m i n e d b y i n c u b a t i o n w i t h t r y p s i n 22 3 Mean p e r c e n t o f k i n i n - l i k e a c t i v i t y r e l e a s e d f r o m i n t a c t and d e n a t u r e d p l a s m a o f s e v e r a l s p e c i e s b y g l a s s s u r f a c e , t r y p s i n , and hog p a n c r e a s k a l l i k r e i n 41 4 K i n i n p r o d u c t i o n i n r a t and t u r t l e p lasma a t d i f f e r e n t t e m p e r a t u r e s , b y 50 cm g l a s s / ml plasma 77 5 The p u r i f i c a t i o n o f g l a s s r e l e a s e d t u r t l e k i n i n 96 6 P h a r m a c o l o g i c a l s t u d i e s ; t h e r a t i o s o f d i f f e r e n t b i o l o g i c a l a c t i v i t i e s o f t u r t l e k i n i n , a s s a y e d a g a i n s t s y n t h e t i c b r a d y -i x T a b l e Page 7 M o l a r r a t i o s o f amino a c i d s i n p u r i f i e d t u r t l e k i n i n and i n s y n t h e t i c b r a d y k i n i n h y d r o l y z e d f o r 24 and 36 h o u r s 125 8 R e l a t i o n o f b i o l o g i c a l a c t i v i t y o f b r a d y k i n i n and t u r t l e k i n i n t o nM o f k i n i n measured by amino a c i d a n a l y s i s 12 7 9 D e c r e a s e i n b l o o d p r e s s u r e o f 6 t u r t l e s o v e r t h e 5 - 1 0 h o u r e x p e r i m e n t a l p e r i o d 136 X LIST OF FIGURES F a c i n g F i g u r e Page 1 Diagrammatic summary o f the i n t e r r e l a t i o n  s h i p s of k i n i n o g e n , k i n i n s , and k i n i n - c o n t a i n i n g fragments 4 2 Schematic r e p r e s e n t a t i o n o f the e f f e c t of g l a s s s u r f a c e , t r y p s i n , and g l a n d u l a r k a l l i  k r e i n on major enzymic events i n v o l v e d i n k i n i n r e l e a s e i n mammalian plasma 5 3 a E f f e c t of t r y p s i n and hog pancreas k a l l i  k r e i n i n r e l e a s i n g k i n i n from h e a t e d dog, r a t and t u r t l e plasmas 26 3 b E f f e c t of t r y p s i n and hog pancreas k a l l i  k r e i n i n r e l e a s i n g k i n i n from heated Amphiuma, f r o g and t r o u t plasmas 27 4 R e l a t i v e amounts of k i n i n r e l e a s e d from heated Amphiuma and r a t plasma by i n c u b a  t i o n w i t h t r y p s i n and hog pancreas k a l l i  k r e i n 32 5 a I n a c t i v a t i o n o f s y n t h e t i c b r a d y k i n i n by i n t a c t u n d i l u t e d r a t and t u r t l e plasma 35 x i F a c i n g F i g u r e Page 5 b I n a c t i v a t i o n o f s y n t h e t i c b r a d y k i n i n i n i n t a c t u n d i l u t e d Amphiuma, f r o g and t r o u t p lasmas 36 6 E f f e c t o f g l a s s s u r f a c e i n r e l e a s i n g k i n i n f r o m t u r t l e p l a s m a i n t h e a b s e n c e o f k i n i n - a s e i n h i b i t o r 51 7 E f f e c t o f mammalian k i n i n a s e i n h i b i t o r s on the r a t e o f i n a c t i v a t i o n .of b r a d y k i n i n i n u n d i l u t e d t u r t l e plasma 54 8 P r o d u c t i o n o f k i n i n f r o m t u r t l e p l a s m a b y g l a s s s u r f a c e , i n t h e p r e s e n c e o f d i f f e r e n t k i n i n a s e i n h i b i t o r s 55 9 E f f e c t o f d i l u t i o n o f plasma on r a t e and y i e l d o f t u r t l e k i n i n p r o d u c t i o n 58 10 The e f f e c t o f g l a s s s u r f a c e a r e a on t u r t l e k i n i n p r o d u c t i o n 61 11 E f f e c t o f g l a s s s u r f a c e a r e a on maximum v e l o c i t y o f t u r t l e k i n i n p r o d u c t i o n 63 x i i F a c i n g F i g u r e Page 12 Rate o f d e p l e t i o n o f s u b s t r a t e f o r g l a s s a c t i v a t e d enzymes ( S u b s t r a t e 1) i n t u r t l e p lasma, b y 50 and 100 cm g l a s s / m l p l a s m a .... 67 13 E f f e c t o f a 2 h o u r i n c u b a t i o n o f samples a f t e r r e m o v a l o f g l a s s on t h e p r o p o r t i o n o f k i n i n r e l e a s e d , f r o m t u r t l e p lasma b y 100 and 50 cm g l a s s / m l plasma 69 14 a The r e l e a s e o f a c t i v e k a l l i k r e i n f r o m r a t and t u r t l e p r e k a l l i k r e i n p r e p a r a t i o n s a c t i v a t e d w i t h 200 cm g l a s s / m l 72 14 b Decay o f a c t i v e t u r t l e k a l l i k r e i n 72 15 E f f e c t o f t e m p e r a t u r e on g l a s s t r i g g e r e d k i n i n p r o d u c t i o n i n r a t and t u r t l e plasma 79 16 Comparison o f r a t e and y i e l d o f k i n i n p r o  d u c t i o n i n u n h e a t e d t u r t l e p l a s m a b y two c o n c e n t r a t i o n s o f t r y p s i n 82 17 Comparison o f r a t e and y i e l d o f k i n i n p r o  d u c t i o n i n u n h e a t e d and h e a t e d t u r t l e plasma, by t r y p s i n 84 x i i i F a c i n g F i g u r e Page 18 R e l a t i v e e f f e c t s o f t r y p s i n and g l a s s i n r e l e a s i n g k i n i n f r o m i n t a c t t u r t l e and r a t p l a s m a s 87 19 P u r i f i c a t i o n o f g l a s s r e l e a s e d t u r t l e k i n i n on a 90 x 2.5 cm Sephadex G-25 g e l f i l t r a t i o n column 97 20 P u r i f i c a t i o n o f g l a s s r e l e a s e d t u r t l e k i n i n on a 20 x 0.9 cm CM-Sephadex i o n exchange column 98 21 Responses t y p i c a l o f s y n t h e t i c b r a d y k i n i n and p u r i f i e d t u r t l e k i n i n , i n s i x b i o a s s a y s 106 22 Paper chromatography o f L y s - b r a d y k i n i n , t u r t l e k i n i n , and b r a d y k i n i n i n b u t a n o l - a c e t i c a c i d - w a t e r ; a 12 h o u r r u n 113 23 Paper chromatography o f L y s - b r a d y k i n i n , t u r t l e k i n i n , and b r a d y k i n i n i n b u t a n o l - a c e t i c a c i d - w a t e r ; a 24 h o u r r u n 114 x i v P a c i n g F i g u r e Page 24 E l u t i o n p a t t e r n o f s y n t h e t i c b r a d y k i n i n on a 95 x 2.5 cm Sephadex G-25 g e l f i l t r a t i o n column 116 25 E l u t i o n o f s y n t h e t i c b r a d y k i n i n and t u r t l e k i n i n f r o m s e p a r a t e CM-Sephadex columns d e v e l o p e d w i t h a c o n c e n t r a t i o n g r a d i e n t t o 0.5 M ammonium a c e t a t e b u f f e r , pH — 5.0 ... 119 26 a S e p a r a t i o n o f s y n t h e t i c b r a d y k i n i n f r o m s y n t h e t i c l y s - b r a d y k i n i n on a CM-Sephadex column d e v e l o p e d w i t h a c o n c e n t r a t i o n g r a d i e n t t o 1.0 M ammonium a c e t a t e , pH = 5.0 120 26 b The e l u t i o n p a t t e r n o f t u r t l e k i n i n f r o m a CM-Sephadex column d e v e l o p e d w i t h 1.0 M ammon ium a c e t a t e , as i n F i g u r e 26 a 120 2 7 E l u t i o n p a t t e r n o f a m i x t u r e o f s y n t h e t i c b r a d y k i n i n and p u r i f i e d t u r t l e k i n i n f r o m a CM-Sephadex column d e v e l o p e d w i t h a s h a l l o w c o n c e n t r a t i o n g r a d i e n t 122 X V F a c i n g F i g u r e Page 28 C o p i e s o f t r a c e s f r o m amino a c i d a n a l y s i s o f h y d r o l y s a t e s o f s y n t h e t i c b r a d y k i n i n , b r a d y k i n i n + t h r e o n i n e , p u r i f i e d t u r t l e k i n i n , and t u r t l e k i n i n + s e r i n e 129 2 9 a T y p i c a l r e s p o n s e s o f t u r t l e b l o o d p r e s s u r e and f l o w t o i n t r a - a r t e r i a l i n j e c t i o n s o f s y n t h e t i c b r a d y k i n i n and a d r e n a l i n e 138 29 b Reduced r e s p o n s e s o f t h e same a n i m a l t o h i g h e r d o s e s o f b r a d y k i n i n and a d r e n a l i n e , a f t e r a d m i n i s t r a t i o n o f phenoxybenzamine and p r o p a n o l o l 138 30 E f f e c t o f a d r e n e r g i c b l o c k a d e on t h e p r e s s o r r e s p o n s e t o b r a d y k i n i n i n 3 d i f f e r e n t t u r t l e s 139 x v i DEFINITIONS AND ABBREVIATIONS D e f i n i t i o n s Due t o t h e c o m p l e x i t y o f t h e f a c t o r s i n v o l v e d i n t h e k a l l i k r e i n - k i n i n system, t h e n o m e n c l a t u r e o f i t s v a r i o u s com po n e n t s has o f t e n b e en ambiguous and r e d u n d a n t . R e c e n t l y , M.E. Webster (1966) c h a i r e d a committee w h i c h recommended c e r t a i n terms and d e f i n i t i o n s i n an e f f o r t t o c l a r i f y t h e s i t u a  t i o n . These terms, which have b e e n a d o p t e d by most w o r k e r s , w i l l be used i n t h i s t h e s i s as d e f i n e d below: K i n i n s : A g e n e r a l term i n d i c a t i n g a p o l y p e p t i d e which r e s e m b l e s b r a d y k i n i n i n i t s s t r u c t u r e and p h a r m a c o l o g i c a l a c t i v i t y . B r a d y k i n i n : R e f e r s t o t h e n o n a p e p t i d e , A r g - P r o - P r o - G l y - P h e - S e r - P r o - P h e - A r g , as i s o l a t e d b y E l l i o t t e t a l (1961). L y s - b r a d y k i n i n : R e f e r s t o t h e d e c a p e p t i d e L y s - b r a d y k i n i n formed by the a c t i o n o f g l a n d u l a r k a l l i k r e i n s on k i n i n o g e n (Werle and T r a u t s c h o l d , 1963). K a i l i d i n : A commonly used t e r m f o r l y s - b r a d y k i n i n . K i n i n o g e n : R e f e r s t o a l l b i o l o g i c a l l y i n a c t i v e p r o t e i n s t h a t r e l e a s e a k i n i n when r e a c t e d w i t h a p p r o p r i a t e p r o t e o l y t i enzymes. K i n i n o g e n a s e : R e f e r s t o any enzyme t h a t l i b e r a t e s a k i n i f rom an i n a c t i v e p r o t e i n s u b s t r a t e ; e.g. k a l l i k r e i n s , t r y p s i n X V X 1 p e p s i n , snake venom. K a l l i k r e i n : A t y p e o f n a t u r a l l y o c c u r r i n g k i n i n o g e n a s e w hich r a p i d l y and s p e c i f i c a l l y l i b e r a t e s a k i n i n f r o m k i n i n - ogen. These a r e i d e n t i f i e d by s p e c i e s and s o u r c e ; e.g., hog p a n c r e a s k a l l i k r e i n . P r e k a l l i k r e i n : R e f e r s t o t h e i n a c t i v e p r e c u r s o r o f a k a l l i k r e i n . K i n i n a s e : A g e n e r a l term r e f e r r i n g t o enzymes t h a t i n  a c t i v a t e k i n i n s . A b b r e v i a t i o n s Used BK - b r a d y k i n i n G l y g l y c i n e Lys-BK - l y s - b r a d y k i n i n H i s - h i s t i d i n e K - k a l l i d i n l i e - i s o l e u c i n e HPK hog p a n c r e a s k a l l i k r e i n Leu - l e u c i n e T - t r y p s i n L y s l y s i n e BK-eq b r a d y k i n i n e q u i v a l e n t Met m e t h i o n i n e A l a a l a n i n e Pro - p r o l i n e A r g a r g i n i n e Phe - p h e n y l a l a n i n e Asp a s p a r t i c a c i d S e r - s e r i n e Cys c y s t i n e T y r - t y r o s i n e C y t c y s t e i c a c i d T h r t h r e o n i n e G l u - g l u t a m i c a c i d V a l v a l i n e XV 111 ACKNOWLEDGEMENTS Many p e o p l e i n t h e D e p a r t m e n t o f Z o o l o g y a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a gave f r e e l y o f t h e i r t i m e and k n o w l e d g e t o a i d me d u r i n g t h e c o u r s e o f t h i s work. I am e s p e c i a l l y g r a t e f u l t o Dr. A.M. P e r k s , who s u g g e s t e d t h e r e s e a r c h t o p i c , and who o f f e r e d h e l p f u l c r i t i c i s m d u r i n g t h e p r e p a r a t i o n o f t h e m a n u s c r i p t , and t o M r s . E. V i z s o l y i , whose a s s i s t a n c e i n many b i o a s s a y p r o c e d u r e s was i n v a l u a b l e . I am e n d e b t e d t o my w i f e and t o t h e N a t i o n a l R e s e a r c h C o u n c i l o f Canada f o r p e r s o n a l f i n a n c i a l s u p p o r t . The c o s t s o f t h e r e s e a r c h i t s e l f were b o r n e b y D r . P e r k s ' R e s e a r c h Coun c i l g r a n t . 1 CHAPTER 1. INTRODUCTION I n 1949 Rocha e S i l v a d e s c r i b e d a p o t e n t h y p o t e n s i v e p o l y p e p t i d e , w h i c h was r e l e a s e d f r o m mammalian p l a s m a b y s n a k e venom and b y t r y p s i n . The f a c t o r was named b r a d y k i n i n . S i n c e t h i s t i m e , p l a s m a k i n i n s and t h e enzymes w h i c h r e l e a s e them f r o m t h e i r a l p h a - g l o b u l i n p r e c u r s o r i n t h e b l o o d have b e e n t h e s u b j e c t o f numerous s t u d i e s , and t h e h i s t o r y and c u r r e n t s t a t e o f t h e a r t h a v e b e e n p r e s e n t e d i n d e t a i l i n many r e v i e w s and s y m p o s i a (see S c h a c h t e r , 1 9 6 9 ) . The s t r u c t u r e o f p u r i f i e d mammalian b r a d y k i n i n was d e t e r  mined b y s y n t h e s i s ( B o i s s o n a s , Guttmann, and J a q u e n o d , 1960) and b y amino a c i d a n a l y s i s o f t r y p s i n r e l e a s e d k i n i n ( E l l i o t t , H o r t o n , and L e w i s , 1 9 6 1 ) . L a t e r work h a s shown t h a t c e r t a i n g l a n d u l a r k a l l i k r e i n s a n d / o r a c i d t r e a t m e n t a l s o r e l e a s e l y s - b r a d y k i n i n (Webster and P i e r c e , 1963) and m e t - l y s - b r a d y k i n i n ( E l l i o t t , 1963; S c h r o d e r , 1964) f r o m p l a s m a g l o b u l i n s . B o t h o f t h e s e p e p t i d e s h a v e a l s o b e e n i d e n t i f i e d i n human u r i n e (Miwa, E r d o s , and S e k i , 1 9 6 9 ) . A l l t h r e e k i n i n s a r e b i o l o g i  c a l l y a c t i v e and i t i s now r e c o g n i z e d t h a t t h e h y p o t e n s i v e , t h e r m o l a b i l e and l a r g e m o l e c u l a r w e i g h t g l a n d u l a r k a l l i k r e i n s d e s c r i b e d by F r e y and h i s c o w o r k e r s i n t h e 1 9 3 0 1 s ( r e v i e w e d by S c h a c h t e r , 1969) e x e r t t h e i r p h a r m a c o l o g i c a l e f f e c t b y r e  l e a s i n g t h e s e k i n i n s f r o m a s p e c i f i c s u b s t r a t e i n b l o o d p l a s m a . 2 7A v a r i e t y o f o t h e r t r e a t m e n t s o f mammalian plasma k i n i n  ogen r e s u l t i n t h e p r o d u c t i o n o f y e t o t h e r b r a d y k i n i n - c o n t a i n - i n g f r a g m e n t s i n a d d i t i o n t o v a r y i n g p r o p o r t i o n s o f t h e plasma k i n i n s a l r e a d y d e s c r i b e d . The complex i n t e r r e l a t i o n s h i p s b e  tween t h e v a r i o u s t r e a t m e n t s and t h e p e p t i d e s r e l e a s e d have been r e v i e w e d b y P i e r c e (1968) and Hamberg (1969), and a r e summarized i n F i g u r e 1. W h i l e t h e s e s t u d i e s have c o n t r i b u t e d t o an u n d e r s t a n d i n g o f s t r u c t u r a l f e a t u r e s o f plasma k i n i n s and k i n i n o g e n s , t h e y a r e o f l i t t l e v a l u e i n u n d e r s t a n d i n g a p h y s i o  l o g i c a l r o l e f o r k i n i n s , s i n c e most o f t h e s e a c t i v e p e p t i d e s a r e c o n v e r t e d t o b r a d y k i n i n i n n a t i v e plasma, and t h e endogen ous plasma k a l l i k r e i n s p r o d u c e m o s t l y b r a d y k i n i n t o b e g i n w i t h ( P i e r c e , 1968). T h r o u g h o u t the s t u d y p r e s e n t e d h e r e , emphasis has been p l a c e d on k i n i n p r o d u c t i o n by a c t i v a t i o n o f t h e n a t u r a l plasma k a l l i k r e i n s p r e s e n t i n plasma, s i n c e t h i s p r o c e s s may i m i t a t e p h y s i o l o g i c a l r e l e a s e . S t u d i e s w i t h enzymes o t h e r t h a n t h o s e p r e s e n t i n t h e b l o o d , such as t r y p s i n o r g l a n d u l a r k a l l i k r e i n s , were i n c l u d e d o n l y f o r c o m p a r i s o n . W h i l e t h e r e i s d i s a g r e e m e n t on d e t a i l s o f t h e i n t e r a c t i o n o f many k i n i n o g e n a s e s w i t h d i f f e r  e n t t y p e s o f k i n i n o g e n , most workers would a g r e e t h a t the d i a  gram i n F i g u r e 2 e x p r e s s e s t h e major e v e n t s which o c c u r when k i n i n i s r e l e a s e d f r o m i n t a c t plasma by a g l a s s s u r f a c e , t r y p s i n o r - g l a n d u l a r k a l l i k r e i n . F i g u r e 1 S e r G l y - A r g - M e t - L y s - B K Lys-BK Plasma C a r b o x y p e p t i d a s e N o r C a r b o x y p e p t i d a s e C 1 INACTIVE PEPTIDES + ARGININE F i g u r e 1. Diagrammatic summary o f t h e i n t e r r e l a t i o n s h i p s o f k i n i n o g e n , k i n i n s , and k i n i n - c o n t a i n i n g f r a g m e n t s . From P i e r c e , 1968. Figure 2 . Schematic representation of the effect of glass surface, trypsin, and glandular k a l l i k r e i n on major enzymatic events involved i n k i n i n release i n . mammalian plasma. 4 F i g u r e 2 KININASES Mammalian Plasma Kallikrein-Kinin System 5 I t i s g e n e r a l l y a g r e e d t h a t t r y p s i n , a p o w e r f u l k i n i n o g e n - a s e , w i l l r e l e a s e maximum amounts o f k i n i n from a l l k i n i n o g e n s known t o o c c u r i n mammalian t i s s u e s ( S c h a c h t e r , 1969). On t h e o t h e r hand, plasma k a l l i k r e i n d e g r a d e s o n l y a p o r t i o n o f t h e t o t a l k i n i n o g e n . F o r c o n v e n i e n c e , t h i s f r a c t i o n o f t h e k i n i n o g e n w i l l be r e f e r r e d t o as s u b s t r a t e 1 ( J a c o b s e n 1966 a ) . T h e r e i s i n c r e a s i n g e v i d e n c e f o r t h e e x i s t e n c e o f numer ous m o l e c u l a r s p e c i e s o f k i n i n o g e n s , k a l l i k r e i n s , and k i n i n a s e s ( r e v i e w e d b y Schachter, 1969) . T h i s k a l l i k r e i n - k i n i n s y s t e m i s t h e r e f o r e a complex "damped c a s c a d e " pathway s i m i l a r t o t h e b l o o d c l o t t i n g r e a c t i o n s (as des/cribed by Hemker and Hemker, 1969), and s h a r i n g w i t h them t h e i n i t i a l s t e p o f Hageman F a c  t o r a c t i v a t i o n ( M a r g o l i s , 1958; Temme, e t a l , 1969). Non-Mammalian K i n i n s K i n i n - l i k e p o l y p e p t i d e s have now been i s o l a t e d f r o m a number o f non-mammalian s o u r c e s . These have been r e v i e w e d i n d e t a i l by pdsano (1968) and S c h a c h t e r (1968, 1969). B r i e f l y , wasp venoms (Vespa sp. and P o l i s t e s sp.) c o n t a i n a t l e a s t t h r e e k i n i n s , one i d e n t i f i e d as g l y - b r a d y k i n i n (Prado e t a l , 1966). B r a d y k i n i n i t s e l f has been i s o l a t e d f r o m f r o g (Rana t e m p o r a r i a ) s k i n i n c o n c e n t r a t i o n s as h i g h as 200-250 ug/g f r e s h t i s s u e ( A n a s t a s i , Erspamer, and B e r t a c c i n i , 1965). T h r e e p h a r m a c o l o g i c a l l y a c t i v e p e p t i d e s have r e c e n t l y been found i n the s k i n o f a s m a l l B r a z i l i a n a m p hibian, J?. r o h d e i , one o f 6 w h i c h h a s t h e s t r u c t u r e ; b r a d y k i n i n - I l e - T y r - H S O ^ . I t i s r e  m a r k a b l e t h a t t h e f u n d a m e n t a l b r a d y k i n i n c h a i n i s an i n t e g r a l p a r t o f a l l non-mammalian k i n i n s whose s t r u c t u r e s h a v e b e e n e s t a b l i s h e d . The a c t i v e r e s e a r c h p r e s e n t l y b e i n g c a r r i e d o u t on a w i d e v a r i e t y o f i n s e c t and r e p t i l i a n venoms i s l i k e l y t o i d e n t i f y a n i n c r e a s i n g number o f v a s o a c t i v e , b r a d y k i n i n - c o n t a i n i n g p e p t i d e s . However, l i t t l e i s known o f p l a s m a k i n i n s o r endogenous enzymes t h a t r e l e a s e them i n non-mammalian v e r t e b r a t e s . I t h a s b e e n shown t h a t o r n i t h o k a l l i k r e i n f r o m t h e b i r d p a n c r e a s r e  l e a s e s f r o m b i r d p l a s m a a p o l y p e p t i d e w h i c h i s h y p o t e n s i v e on b i r d b l o o d p r e s s u r e , b u t i s i n a c t i v e i n mammals ( S c h a c h t e r , 1 9 6 9 ) . The s t r u c t u r e o f t h i s f a c t o r d i f f e r s s i g n i f i c a n t l y f r o m known k i n i n s ( W e r l e , e t a l , 1 9 6 7 ) . I t i s n o t c l e a r w h e t h e r b i r d p l a s m a c a n r e l e a s e enzymes w h i c h p r o d u c e t h i s o r n i t h o k i n i n . However, E r d o s , Miwa and Graham (1967) h a v e shown t h a t i f Hage- man F a c t o r was s u p p l i e d , g l a s s b e a d s r e l e a s e d f r o m duck p l a s m a a k i n i n i n d i s t i n g u i s h a b l e f r o m b r a d y k i n i n . B r a d y k i n i n i t s e l f i s , h owever, i n a c t i v e on f o w l b l o o d p r e s s u r e ( E r d o s , 1 9 6 6 ) . D a t a on t h e o c c u r r e n c e o f k i n i n s and k a l l i k r e i n s i n p o i k - i l o t h e r m s i s v e r y s c a r c e . D i n i z and C a r v a l h o (1963) r e p o r t e d a s i n g l e r e l a t i v e l y l o w v a l u e f o r t r y p s i n - r e l e a s e d k i n i n f r o m d e n a t u r e d t o a d p l a s m a . W h i l e t h e p r e s e n t s t u d y was i n p r o g r e s s 7 t h r e e v e r y b r i e f , though p e r t i n i e n t , p a p e r s have a p p e a r e d : E r d o s e t a l (1967) r e p o r t e d t h e l i b e r a t i o n o f plasma k i n i n s b y g l a s s a c t i v a t i o n i n a l l i g a t o r and t u r t l e p l a s m a s ; V o g e l e t a l (1969) s t u d i e d t h e r e s p o n s e o f f i s h b l o o d p r e s s u r e t o v a r i o u s k i n i n s and k a l l i k r e i n s and c o n c l u d e d t h a t t h e r e were no h i n t s o f a k i n i n - l i b e r a t i n g s y s t e m c o m p a r a b l e t o t h a t o f mammals; A.C. A l b a L a v r a s e t a l (1969) came t o a s i m i l a r c o n  c l u s i o n f o r snake plasma, b u t d i d r e p o r t t h a t hog p a n c r e a s k a l l i k r e i n r e l e a s e d f r o m t h i s plasma a h y p e r t e n s i v e smooth muscle s t i m u l a t i n g a g e n t d i f f e r e n t f r o m b r a d y k i n i n . These i m p o r t a n t s t u d i e s w i l l be d i s c u s s e d i n l a t e r c h a p t e r s . 8 CHAPTER 2. GENERAL METHODS S e v e r a l g e n e r a l t e c h n i q u e s and p r o c e d u r e s were f o l l o w e d t h r o u g h o u t t h i s work, and t h e s e a r e d e t a i l e d below: A. S i l i c o n i n g P r o c e d u r e K i n i n s i n s o l u t i o n l o s e a c t i v i t y when i n c o n t a c t w i t h g l a s s ( M a r g o l i s and B i s h o p , 1963) , p r e s u m a b l y t h r o u g h a d s o r p  t i o n t o n e g a t i v e s i t e s on i t s s u r f a c e . T h i s l o s s , as w e l l as unwanted k a l l i k r e i n a c t i v a t i o n , c a n be g r e a t l y r e d u c e d b y p r e t r e a t m e n t o f t h e g l a s s s u r f a c e w i t h s i l i c o n e . A l l g l a s s  ware t o come i n c o n t a c t w i t h k i n i n s was t h e r e f o r e washed, ex posed f o r 10 seconds t o a 1% s o l u t i o n o f S i l i c l a d ( C l a y Adams, N.Y.), r i n s e d w i t h d i s t i l l e d w ater, and d r i e d f o r 20 m i n u t e s a t 100°C, b e f o r e use i n any e x p e r i m e n t . B. Rat U t e r u s B i o a s s a y T h e r e a r e many b i o a s s a y s w h i c h m i g h t be used t o measure k i n i n p r o d u c t i o n ( E r d o s , 1966), and s e n s i t i v e t e c h n i q u e s f o r radioimmunoassay a r e b e i n g d e v e l o p e d (Spragg, Haber, and A u s t e n , 1966; Talmo, Haber, and A u s t e n , 1969). However, t h e r a t u t e r u s b i o a s s a y i s s t i l l t h e most a c c u r a t e and s e n s i t i v e method f o r r o u t i n e e s t i m a t i o n o f plasma k i n i n a c t i v i t y . The a s s a y i s s i m p l e t o c a r r y o u t , w i l l measure a c o n c e n t r a t i o n o f b r a d y k i n i n as low as 50 pg/ml, and a l l o w s c o m p u t a t i o n o f p o t e n c y f o r an unknown w i t h 95% c o n f i d e n c e l i m i t s between 9 + 10 and 20%. The r a t u t e r u s a s s a y was t h e r e f o r e used t o s t u d y k i n e t i c s o f k i n i n r e l e a s e f r o m d e n a t u r e d and i n t a c t p l a s m a . I t was a l s o a b a s i s f o r p h a r m a c o l o g i c a l t e s t s , and was used t o f o l l o w k i n i n a c t i v i t y t h r o u g h p u r i f i c a t i o n , and s e v e r a l c h e m i c a l t e s t s . Female v i r g i n r a t s ( W i s t a r s t r a i n ) 180-220 g i n w e i g h t were u s e d . O n l y r a t s i n f u l l o e s t r u s ( i n d i c a t e d b y v a g i n a l smear) were s e l e c t e d . The a n i m a l was k i l l e d b y a s h a r p b l o w t o t h e back o f t h e head. The u t e r u s was exposed b y a s a g i t t a l i n c i s i o n i n t h e a b d o m i n a l w a l l , r a p i d l y d i s s e c t e d f r e e o f m e s e n t e r y and f a t t y t i s s u e , and p l a c e d i n warm (30°C) b u f f e r e d s a l i n e , w h i c h was M u n s i c k ' s m o d i f i c a t i o n o f Van D y k e - H a s t i n g s S o l u t i o n (Munsick, 1960). The c o m p o s i t i o n o f t h i s f l u i d , w h ich s u p p o r t e d t h e t i s s u e t h r o u g h o u t t h e b i o a s s a y , i s g i v e n below: C h e m i c a l Amount/L C h e m i c a l Amount/L N a C l 6.704 g g l u c o s e 0.5 g KC1 0.459 g *ph o s p h a t e b u f f e r 10 ml NaHC0 3 2.5 90 g Na p h e n o l - C a C l 2 0.5 ml, 1 M s u l f o n e p h t h - s o l u t i o n a l e i n 0.054 g *...Phosphate b u f f e r c o n s i s t e d o f s o l u t i o n s o f Na2HP04 (22.714 g/L) and Na H2PO4 (6.349 g o f h y d r a t e d form/L) combined so t h a t the f i n a l pH was 7.40. One h o r n o f t h e u t e r u s was t h e n p l a c e d i n a 5 ml or g a n b a t h c o n t a i n i n g Van D y k e - H a s t i n g s s o l u t i o n . The o r g a n b a t h and 10 a c o n n e c t i n g 2 L r e s e r v o i r were i n d i v i d u a l l y g a s s e d w i t h a m i x t u r e o f 5% C 0 2 and 95% 02, wh i c h k e p t t h e pH a t 7.4, and b o t h were i n a water b a t h r e g u l a t e d t o ± 0.1°C, and t h e a s s a y t e m p e r a t u r e s e t between 30 and 33°C. The p o s t e r i o r end o f t h e u t e r u s was s e c u r e d t o a g l a s s m u s c l e hook a t t h e b o t t o m o f t h e or g a n b a t h , and t h e o v a r y was c o n n e c t e d t o a w r i t i n g l e v e r b y s u r g i c a l t h r e a d under a t e n s i o n o f 1 - 2 g. The s o l u t i o n s t o be a s s a y e d were added t o t h e o r g a n b a t h by means o f g l a s s p i p e t t e s o r H a m i l t o n g l a s s s y r i n g e s , i n a volume n e v e r e x c e e d i n g 0.2 ml, and t h e r e s u l t i n g c o n t r a c t i o n s were r e c o r d e d on smoked pa p e r on a kymograph. A f t e r each r e s p o n s e had r e a c h e d maximum c o n t r a c t i o n , t h e b a t h was f l u s h e d w i t h an e x c e s s o f b u f f e r f r o m t h e r e s e r v o i r , and t h e t i s s u e was a l l o w e d t o r e c o v e r f o r a t l e a s t 5 m i n u t e s . The smoked p a p e r w i t h r e c o r d e d t r a c e s was d i p p e d i n s h e l l a c and t h e r e s p o n s e s l a t e r measured t o t h e n e a r e s t mm. E s t i m a t i o n o f a c t i v i t y f o r a l a r g e number o f samples (e.g., e l u a t e s f r o m c h r o m a t o g r a p h y columns) was b a s e d on l o g d o s e - r e s p o n s e c u r v e s , o r on s i n g l e f o u r p o i n t a s s a y g r o u p s . Where maximum a c c u r a c y was d e s i r e d , f i v e f o u r p o i n t groups c o n s i s t i n g o f matched r e s p o n s e s t o h i g h and low doses o f s t a n  d a r d and unknown, were o b t a i n e d a c c o r d i n g t o t h e method o f H o l t o n (1948). A l l a s s a y s used s y n t h e t i c b r a d y k i n i n (Sandoz, 0.1 mg/ml) as s t a n d a r d , and r e s u l t s t h r o u g h o u t were e x p r e s s e d as ug (or ng) b r a d y k i n i n - e q u i v a l e n t (BK-eq) p e r ml f l u i d . 11 C. S o u r c e o f A n i m a l s a) F i s h . The f o l l o w i n g a n i m a l s were o b t a i n e d b y o t t e r t r a w l n e a r F r i d a y Harbour, U.S.: s t a r r y f l o u n d e r ( P l a t i c h t h y s s t e l l a t u s ) , b u t t e r s o l e ( I s o p s e t t a i s o l e p i s ) , lemon s o l e ( P a r o p h r y s v e t u l u s ) , cod (Gadus m a c r o c e p h a l u s ) , s k a t e s (Raja b i n o c u l a t a and R a j a r h i n a ) , d o g f i s h ( S q u a l u s s u c k l e y i ) and r a t f i s h ( H y d r o l a g u s c o l l i e i ) . T r o u t (Salmo g a i r d n e r i i ) were o b t a i n e d f r o m a c o m m e r c i a l s u p p l i e r i n P o r t C o q u i t l a m , B.C. b) A m p h i b i a n s . F r o g s (Rana c a t e s b i e n a ) were o b t a i n e d f r o m C o l l e g e B i o l o g i c a l S u p p l y , S e a t t l e , and m a i n t a i n e d a t 10-15°C i n t h e l a b o r a t o r y . Amphiuma t r i d a c t y l u m was o r d e r e d from N o r t h C a r o l i n a B i o l o g i c a l S u p p l y Co., Durham, N o r t h C a r o  l i n a , and k e p t i n l a r g e a q u a r i a a t room t e m p e r a t u r e u n t i l u s e . c ) R e p t i l e s . T u r t l e s (Pseudemys s c r i p t a e l e g a n s ) were o b t a i n e d from C o l l e g e B i o l o g i c a l S u p p l y , and k e p t i n m o i s t t a n k s a t 10-15°C u n t i l u s e . d) Mammals. S e a l b l o o d (Phoca v i t u l i n a r i c h a r d i ) was o b t a i n e d f r o m c a p t i v e a n i m a l s a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a . K i l l e r whale b l o o d ( O r c i n u s o r c a ) was t a k e n from Skana, a t t h e V a n c o u v e r P u b l i c Aquarium. Dog b l o o d was d o n a t e d by a g o l d e n r e t r i e v e r k e p t i n t h e P h y s i o l o g y Department, a t t h e U n i v e r s i t y o f B r i t i s h C olumbia, and w h i t e r a t s ( W i s t a r s t r a i n ) , r a b b i t s and g u i n e a p i g s were o b t a i n e d f r o m t h e v i v a r i u m 12 a l s o a t U.B.C. D. B l o o d S a m p l i n g B l o o d f r o m a l l a n i m a l s was w ithdrawn i n t o s t e r i l e , c h i l l e d p l a s t i c s y r i n g e s (Tomac) p r e v i o u s l y r i n s e d i n a 10 mg% h e p a r i n - s a l i n e s o l u t i o n . B a s i c t e c h n i q u e s used f o r each s p e c i e s a r e g i v e n b e l o w i n T a b l e 1. A l l a n i m a l s l i s t e d i n t h e T a b l e were d e s t r o y e d a f t e r r e m o v a l o f b l o o d , e x c e p t f o r t h e s e a l , dog, and k i l l e r whale. E. S a m p l i n g o f K i n i n - P r o d u c i n g R e a c t i o n s A l t h o u g h k i n i n r e l e a s i n g methods v a r i e d between e x p e r i  ments, c e r t a i n b a s i c t e c h n i q u e s were u s e d t o sample a l l k i n i n p r o d u c i n g r e a c t i o n s i n t h e v a r i o u s p l a s m a s . Plasma, whether h e a t e d o r i n t a c t , was a l w a y s d i l u t e d w i t h 2 volumes o f t h e a p p r o p r i a t e s a l i n e (Appendix B ) . A t z e r o time, t h e k i n i n o g e n a s e o r a c t i v a t o r was added t o t h e m i x t u r e , which h a d a t o t a l volume of 5-10 ml. A t t h e d e s i r e d t i m e i n t e r v a l s s m a l l samples ( u s u a l l y 0.5 o r 1.0 ml) were removed f r o m t h e i n c u b a t e i n a 1 ml s t e r i l e p l a s t i c s y r i n g e and r a p i d l y i n j e c t e d i n t o 2 volumes o f d i s  t i l l e d water or t h e a p p r o p r i a t e s a l i n e p r e h e a t e d t o 100°C i n a s i l i c o n e d g l a s s t e s t t u b e . The f i n a l d i l u t i o n o f t h e plasma was 1/9. These samples were k e p t i n a b o i l i n g w a t e r b a t h f o r a f u r t h e r 10 m i n u t e s , t h e n removed and s t o r e d a t 4°C u n t i l a s s a y on the i s o l a t e d r a t u t e r u s . T h i s t e c h n i q u e had c e r t a i n a d v a n t a g e s f o r t h i s s t u d y : T a b l e 1. Summary o f methods used i n w i t h d r a w i n g b l o o d . S p e c i e s S t a r r y f l o u n d e r B u t t e r s o l e Lemon s o l e R a t f i s h T r o u t Cod D o g f i s h S k a t e s Amphiuma F r o g T u r t l e S e a l K i l l e r whale Dog Rat S o u r c e o f b l o o d D o r s a l a o r t a , n e a r a n a l f i n V olume/animal(ml) 2-6 C a r d i a c p u n c t u r e * S y s t e m i c a r t e r y n e a r h e a r t C a r d i a c p u n c t u r e * L e f t ' a o r t a D o r s a l a o r t a C a u d a l a r t e r y L e g v e i n * D o r s a l a o r t a 5-10 10-30 2-5 10-70 20 20 20 7-15 A n a e s t h e t i c MS-222 none MS-222 none MS-222 E t h e r Nembutal none E t h e r *...These b l o o d v e s s e l s were exposed b y m i d - v e n t r a l i n c i s i o n s , and s u b s e q u e n t d i s s e c  t i o n . A s q u a r e a b o u t 6 x 6 cm was c u t i n t h e t u r t l e p l a s t r o n , u s i n g a D e s o u t t e r A u t o p s y saw. U n d e r l y i n g m uscle was c a r e f u l l y trimmmed away u s i n g a s c a l p e l , and t h e s q u a r e d i s c a r d e d . A l l o f t h e s e v e s s e l s were clamped, t h e n c a n n u l a t e d w i t h the syrxnge d i s t a l t o t h e clamp. The a r t e r y clamp was t h e n removed, and t h e b l o o d withdrawn. 14 1. The r a p i d i n a c t i v a t i o n a l l o w e d s e r i a l samples t o be t a k e n a t time i n t e r v a l s s h o r t enough ( t o 30 s e c o n d i n t e r v a l s ) t o a l l o w t h e k i n e t i c s o f k i n i n p r o d u c t i o n t o be s t u d i e d . 2. The t r e a t m e n t a c h i e v e d a p a r t i a l p u r i f i c a t i o n , s i n c e much p r o t e i n was p r e c i p i t a t e d , w h i l e t h e k i n i n r e m a i n e d i n t h e aqueous s u p e r n a t a n t s a v e d f o r a s s a y . 3. The samples o b t a i n e d can be t e s t e d d i r e c t l y on t h e r a t u t e r u s , whereas o t h e r methods o f h a l t i n g s u c h r e a c t i o n s , b y t h e u s e o f a c e t o n e o r e t h y l a l c o h o l , f o r example, r e q u i r e t h a t t h e s e i n t e r f e r i n g s u b s t a n c e s be e l i m i n a t e d b e f o r e a s s a y . The l a t t e r time consuming s t e p s c o u l d be a v o i d e d i n t h e p r e s e n t s t u d y , t h e r e b y a l l o w i n g t h e "author t o deal;-with t h e numerous s m a l l samples o b t a i n e d . F. P r e p a r a t i o n o f Sephadex G-25 f o r G e l F i l t r a t i o n Chromatography 96 g (dry weight) o f Sephadex G-25 beads ( f i n e g r a d e , l o t # 9053) were s t i r r e d s l o w l y w i t h 3 volumes o f d i s t i l l e d w a ter, and a l l o w e d t o s w e l l f o r t h r e e h o u r s . The m a g n e t i c s t i r r e r was t h e n s h u t o f f and t h e g e l a l l o w e d t o s e t t l e . The s u p e r n a t a n t and f i n e s were d e c a n t e d and. r e p l a c e d w i t h 3 volumes o f 0.02 M ammonium a c e t a t e b u f f e r , pH 5.0 (Appen d i x B ) . T h i s m i x t u r e was t h e n s t i r r e d , a l l o w e d t o s e t t l e , and was d e c a n t e d and re-washed w i t h t h e b u f f e r a t l e a s t 3 more ti m e s o v e r a 5 h o u r p e r i o d . A g e l s l u r r y i n the same b u f f e r was t h e n a p p l i e d w i t h 15 c o n s t a n t s t i r r i n g t o a 100 x 2.5 cm s i l i c o n e d g l a s s column f i l l e d w i t h 0.02 M ammonium a c e t a t e b u f f e r . A s m a l l wad o f g l a s s wool weighed down w i t h a few g l a s s beads p r e v e n t e d t h e g e l f r o m r u n n i n g o u t o f t h e column. A v e r y s l o w f l o w t h r o u g h t h e column, l e s s t h a n 5 ml/hour, was m a i n t a i n e d t h r o u g h o u t most o f t h e b u i l d i n g p r o c e s s . A f t e r a l l o f t h e g e l had s e t t l e d the column was 90-96 cm i n h e i g h t . A f t e r b u i l d i n g was com p l e t e , a s m a l l d i s c o f f i l t e r p a p e r was p l a c e d on t o p o f t h e g e l and b u f f e r was a l l o w e d t o f l o w t h r o u g h t h e column a t a b o u t 12 ml/hour, f o r a t l e a s t 24 h o u r s . The e l u a t e was p e r i o d i c a l l y m o n i t o r e d f o r c o n d u c t i v i t y and pH, and compared t o t h e o r i g i n a l b u f f e r . B e f o r e any e x p e r i m e n t a l m a t e r i a l was a p p l i e d , t h e column was t e s t e d f o r homo g e n e i t y and r e s o l u t i o n b y a p p l y i n g a s o l  u t i o n o f B l u e D e x t r a n 2000 (Pharmacia) and 1.0 g Na C l i n 20 ml o f t h e same b u f f e r . These two s u b s t a n c e s a r e i d e a l f o r c a l  i b r a t i o n , s i n c e B l u e D e x t r a n i s t o t a l l y e x c l u d e d f r o m t h e g e l , w h i l e N a C l p e n e t r a t e s f r e e l y . T h i s m a t e r i a l was washed t h r o u g h w i t h 0.02 M b u f f e r , and c o l l e c t e d i n 100 d r o p f r a c t i o n s w i t h a f r a c t i o n c o l l e c t o r (LKB). The B l u e D e x t r a n i n t e n s i t y i n t h e e l u a t e was e s t i m a t e d b y eye, o r measured on a Unicam s p e c t r o  photometer (#17914) a t A = 625 nM. S a l t c o n c e n t r a t i o n was mea s u r e d as c o n d u c t i v i t y a t 20°C on a CDM-2d c o n d u c t i v i t y meter 16 (Radiometer, Copenhagen). A f t e r c a l i b r a t i o n , t h e column was washed w i t h s e v e r a l volumes o f 0.02 M b u f f e r , p r i o r t o a p p l i c a t i o n o f t h e e x p e r i - men t a 1 s ample. G. P r e p a r a t i o n o f CM-Sephadex f o r Ion Exchange Chromatography 4 g ( d r y w e i g h t ) o f CM-Sephadex g e l (medium g r a d e , l o t #6071) were p l a c e d i n 100 ml d i s t i l l e d w a t e r and s t i r r e d f o r 3 h o u r s . The s l u r r y was t h e n f i l t e r e d on a Buchner f u n n e l w i t h Whatman #1 f i l t e r p a p e r , and washed t h r o u g h w i t h a t l e a s t 500 ml o f a number o f s o l u t i o n s , i n t h e f o l l o w i n g o r d e r : 0.5 N NaOH, d i s t i l l e d w ater, 0.5 N H C l , d i s t i l l e d water, 0.5 N NH^OH, e x c e s s d i s t i l l e d w ater, f o l l o w e d b y 1.5 L 0.02 M ammonium a c e t a t e b u f f e r . A s l u r r y o f t h e g e l i n 50 ml o f b u f f e r was a p p l i e d w i t h c o n s t a n t s t i r r i n g t o a 25 ml s i l i c o n e d b u r e t t e p r e v i o u s l y f i l l e d w i t h b u f f e r . G l a s s wool p l a c e d j u s t above t h e s t o p c o c k p r e v e n t e d t h e g e l f r o m r u n n i n g o u t o f t h e column. A f t e r t h e column had b u i l t t o a h e i g h t o f 15-20 cm, t h e r e m a i n i n g s l u r r y was d i s c a r d e d , and 0.02 M b u f f e r was a l l o w e d t o f l o w t h r o u g h t h e g e l f o r a t l e a s t 24 h o u r s . The i o n i c s t r e n g t h and pH o f the e l u a t e was p e r i o d i c a l l y c h e cked a g a i n s t t h e o r i g i n a l b u f f e r t o e n s u r e t h a t e q u i l i b r i u m had o c c u r r e d % K i n i n - c o n t a i n i n g samples were a p p l i e d s l o w l y , i n volumes r a n g i n g from 2 t o 2 7 ml, and were washed on w i t h a t l e a s t 30 17 ml o f 0.02 M b u f f e r . The column was t h e n s u b j e c t e d t o an i o n i c s t r e n g t h g r a d i e n t , f r o m 0.02 up t o 1.0 M ammonium a c e t a t e , pH = 5.0. A 50 ml m i x i n g chamber was c o n n e c t e d t o t h e column, b o t h e q u i l i b r a t e d i n 0.02 M b u f f e r . The g r a d i e n t was i n i t i a t e d b y c o n n e c t i n g t o t h e m i x i n g chamber a r e s e r v o i r c o n t a i n i n g t h e h i g h s t r e n g t h b u f f e r , w h i c h g r a d u a l l y r e p l a c e d t h e 0.02 M b u f f e r i n t h e m i x i n g chamber as b u f f e r was e l u t e d f r o m t h e column. H. E s t i m a t i o n o f "Lowry" P e p t i d e The e s t i m a t i o n o f t o t a l p e p t i d e c o n c e n t r a t i o n was h a s e d on t h e c o l o r i m e t r i c method o f Lowry, Rosebrough, F a r r and R a n d a l l (1951) . 1. Reagents. A. 2% Na 2C03 i n 0.1 N NaOH B. 0.5% Cu S O ^ / S E v j O i n 1% p o t a s s i u m t a r t a r a t e C. I N F o l i n - C i o c a l t e a u P h e n o l r e a g e n t 2. S t a n d a r d s . A s e r i e s o f p r o t e i n s o l u t i o n s c o n  t a i n i n g 10, 25, 50, 150, 200 and 300 ug/ml, were p r e p a r e d b y d i l u t i o n f r o m a 50 mg% B o v i n e Serum A l b u m i n S t o c k ( f r a c t i o n v, Armour P h a r m a c e u t i c a l s ) . 3. Measurement o f p e p t i d e c o n t e n t . 0.2 ml o f t h e unknown and s t a n d a r d s were p i p e t t e d i n t o a s e r i e s o f 2 ml t e s t t u b e s . To each sample 1 ml o f r e a g e n t C was added, shaken, and a l l o w e d t o s t a n d f o r 15-30 m i n u t e s . 0.1 ml o f F o l i n C i o c a l t e a u 18 r e a g e n t was t h e n added v e r y r a p i d l y and mixed i m m e d i a t e l y . A f t e r a p e r i o d o f 40-60 m i n u t e s th e c o l o u r was f u l l y d e v e l o p e d , and t h e samples were r e a d a g a i n s t a b l a n k on a Unicam SP 600 s p e c t r o p h o t o m e t e r a t A - 750 nM, u s i n g t h e r e d f i l t e r p h o t o c e l l . The c o n c e n t r a t i o n o f p r o t e i n ("Lowry" p e p t i d e ) i n t h e e x p e r i  m e n t a l samples could, t h e n be r e a d i n ug/ml f r o m t h e s t a n d a r d c u r v e . 19 CHAPTER 3. COMPARATIVE STUDIES ON FISH, AMPHIBIAN, REPTILIAN AND MAMMALIAN PLASMAS A. D e n a t u r e d Plasma The f i r s t l o g i c a l s t e p i n c h a r a c t e r i z i n g a k a l l i k r e i n - k i n i n s y s t e m i s t o d e t e r m i n e whether a k i n i n o g e n i s p r e s e n t ; i . e . , whether smooth muscle s t i m u l a t i n g a c t i v i t y c an be d e v e l o p  ed i n t h e t e s t plasma b y t r e a t m e n t w i t h s t r o n g k i n i n o g e n a s e s . T h i s i s most e a s i l y done u s i n g h e a t - d e n a t u r e d plasma, s i n c e t h i s t r e a t m e n t e l i m i n a t e s n a t u r a l k i n i n a s e a c t i v i t y w h i c h would d e s t r o y any k i n i n p r o d u c e d . The e n t i r e k a l l i k r e i n - k i n i n system, i f i t e x i s t s , i s r e d u c e d t o a s u s p e n s i o n o f k i n i n o g e n , which may be t r e a t e d w i t h k i n i n o g e n a s e s such as t r y p s i n , o r w i t h t h e more s p e c i f i c k a l l i k r e i n s , such as hog p a n c r e a s k a l l i k r e i n . The method t h e r e f o r e b y p a s s e s t h e pre-enzyme t o enzyme con  v e r s i o n s n o r m a l l y r e q u i r e d t o r e l e a s e k i n i n i n n a t i v e plasma. These i n i t i a l s t u d i e s on d e n a t u r e d plasma were c a r r i e d o u t i n two s t a g e s : 1) t h e e f f e c t o f t r y p s i n on whole b l o o d o f f i s h and mammals, and 2) the e f f e c t o f t r y p s i n and hog p a n c r e a s k a l l i k r e i n on f i s h , a m p h ibian, r e p t i l i a n and mammalian plasmas. 1. E f f e c t o f t r y p s i n on whole b l o o d f r o m f i s h and mammals A s u r v e y o f the k i n i n o g e n c o n t e n t o f s e v e r a l f i s h plasmas was c a r r i e d o u t u s i n g t h e method o f B r o c k l e h u r s t and Z e i t l i n (1967), which a l l o w s s e p a r a t e d e t e r m i n a t i o n o f k i n i n o g e n and f r e e k i n i n . B l o o d was c o l l e c t e d from f i s h and from mammalian 20 c o n t r o l s ( s e a l and k i l l e r whale) as d e s c r i b e d p r e v i o u s l y (p 13) and i m m e d i a t e l y i n j e c t e d i n t o 3 volumes o f c h i l l e d 95% e t h a n o l . These samples c o u l d be s t o r e d i n p o l y p r o p y l e n e t u b e s a t 0°C f o r a t l e a s t a week, w i t h no l o s s o f k i n i n o g e n . I n t h e l a b o r a t o r y t h e s e samples were c e n t r i f u g e d , washed w i t h 8 0% e t h a n o l , homogenized, r e s u s p e n d e d i n p h o s p h a t e b u f f e r , pH = 7 . 4 (Appen d i x B ) , and i n c u b a t e d w i t h t r y p s i n a t 37°C f o r 30 m i n u t e s . These i n c u b a t e s were t h e n p l a c e d i n a b o i l i n g w a t e r b a t h f o r 10 m i n u t e s t o e n s u r e i n a c t i v a t i o n o f a l l t r y p s i n . B i o l o g i c a l a c t i v i t y o f t h e samples was a s s a y e d on t h e i s o l a t e d r a t u t e r u s u s i n g s y n t h e t i c b r a d y k i n i n as s t a n d a r d . C o n t r o l s t o wh i c h no t r y p s i n h a d been added were al w a y s i n c l u d e d , and t e s t e d f o r a c t i v i t y . An a t t e m p t was made t o e s t i m a t e l e v e l s o f f r e e k i n i n p r e s e n t i n t h e b l o o d a t t h e time o f s a m p l i n g . The e t h a n o l e x t r a c t and t h e washings o b t a i n e d f r o m t h e p r o c e d u r e d e s c r i b e d above were p o o l e d , and p l a c e d i n a b o i l i n g w a t e r b a t h f o r 10 m i n u t e s . The sample was t h e n r e d u c e d t o d r y n e s s i n a f l a s h e v a p o r a t o r , and t a k e n up i n warm (60°C) s a t u r a t e d N a C l s o l u t i o n a c i d i f i e d t o pH 1.5 w i t h HC1. T h i s s o l u t i o n was t w i c e e x t r a c t e d i n a s e p a r a t i n g f u n n e l w i t h 2 volumes o f r e d i s t i l l e d 1-N-but- a n o l . The b u t a n o l was p o o l e d and e v a p o r a t e d t o d r y n e s s under o r e d u c e d p r e s s u r e i n a w a t e r b a t h a t 50 C. These samples were s t o r e d d r y a t - 20°C u n t i l a s s a y on the r a t u t e r u s 21 i n t h e p r e s e n c e o f a t r o p i n e (1 ug/ml) and b r o m o l y s e r g i c a c i d d i e t h y l a m i d e (0-5 u g / m l ) . F u l l d e t a i l s o f t h e method a r e g i v e n i n A p p e n d i x C. R e s u l t s . T a b l e 2 summarizes r e s u l t s f o r t r y p s i n t r e a t e d b l o o d . No v a l u e s f o r f r e e k i n i n a r e g i v e n , s i n c e no a c t i v i t y was d e t e c t e d i n any sample, mammalian o r f i s h . I t i s e v i d e n t t h a t t r y p s i n r e l e a s e d no k i n i n - l i k e a c t i v i t y f r o m f i s h b l o o d . One sample o f s t a r r y f l o u n d e r b l o o d d i d a p p e a r t o show a low oxy t o c i c a c t i v i t y , b u t t h i s was t h e r e s u l t o f an e a r l y e x p e r i m e n t and c o u l d n o t be r e p e a t e d i n s e v e r a l t r i a l s . T h e r e f o r e t h e s i g n i f i c a n c e o f t h i s p o s i t i v e r e s u l t was d u b i o u s , and i t can be c o n c l u d e d , t e n t a t i v e l y , t h a t f i s h b l o o d t r e a t e d as d e s c r i b e d above does n o t a c t as a s u b s t r a t e f o r t h e k i n i n o g e n a s e t r y p s i n , as i t does i n mammals. V a l u e s obtained, f o r s e a l and k i l l e r whale b l o o d a r e t h e f i r s t r e p o r t e d f o r t h e s e s p e c i e s , and a r e i n t h e range t y p i c a l o f mammals ( D i n i z and C a r v a h l o , 1963). These p o s i t i v e r e s u l t s were o b t a i n e d i n e x p e r i m e n t s c o n d u c t e d i n p a r a l l e l w i t h t h o s e on f i s h b l o o d , t h e r e b y u n d e r l i n i n g t h e v a l i d i t y o f t h e n e g a t i v e d a t a o b t a i n e d f o r f i s h . C o n t r o l samples f r e e o f t r y p s i n n e v e r c o n t a i n e d o x y t o c i c a c t i v i t y . The absence o f d e t e c t a b l e amounts o f f r e e k i n i n i n mammalian samples i s n o t s u r p r i s i n g , s i n c e normal l e v e l s a r e e x p e c t e d t o T a b l e 2. K i n i n o g e n i n f i s h and mammalian b l o o d , as d e t e r m i n e d by i n c u b a t i o n w i t h t r y p s i n . S p e c i e s * K i n i n o g e n (ug/m l p l a s m S , BK-eq) Mean S e a l 0. 94, 0. 70, 1.56 1-07 K i l l e r whale 3. 75, 2.50, 3.00 3.08 T r o u t 0.06, 0.01, 0.01, 0.01, 0.01, 0.01 none S t a r r y f l o u n d e r 0.18, 0.01, 0.01, 0.01, 0.01 d o u b t f u l B u t t e r s o l e 0.01, 0.01, 0.01, 0.01 none Lemon s o l e 0.01, 0.01 none Cod 0.01, 0.01 none S k a t e (R. r h i n a ) 0.01, 0.01 none Skate (R. b i n o c u l a t a ) 0.01, 0.01 none R a t f i s h 0.01, 0.01 none D o g f i s h 0.01, 0.01 none * . . . s c i e n t i f i c names a r e g i v e n on page 11. C...where b i o l o g i c a l a c t i v i t y was o b s e r v e d , v a l u e s were c o n - v e r t e d f r o m ug/ml b l o o d , t o ug/ml plasma, BK-eq, u s i n g hema- t o c r i t r e a d i n g s . 23 be e x t r e m e l y low (e.g., a b o u t 2.8 ng/ml b l o o d i n man, B r o c k l e - h u r s t and Z e i t l i n , 1967). 2. E f f e c t o f t r y p s i n and hog p a n c r e a s k a l l i k r e i n on v e r t e b r a t e p l a s m a s . W h i l e t h e method o f B r o c k l e h u r s t and Z e i t l i n d e s c r i b e d p r e v i o u s l y has d i s t i n c t a d v a n t a g e s f o r f i e l d work and i s u s e  f u l i n s e p a r a t i n g f r e e k i n i n f r o m t h a t h e l d i n t h e k i n i n o g e n p r e c u r s o r , i t has s e r i o u s d i s a d v a n t a g e s . The method i n v o l v e s r e a c t i n g t r y p s i n w i t h a homogenized sample o f whole b l o o d p r o  t e i n s which i n c l u d e r u p t u r e d b l o o d c e l l s , and a l l p a r t i c u l a t e m a t t e r i n t h e o r i g i n a l b l o o d . I t i s d i f f i c u l t t o r e l a t e r e s u l t s o b t a i n e d t o e v e n t s w h i c h m i g h t o c c u r p h y s i o l o g i c a l l y , p a r t i c  u l a r l y when e x a m i n i n g p r e v i o u s l y u n t e s t e d b l o o d . I n a d d i t i o n , t h e k i n i n o g e n has been d e n a t u r e d , p r e c i p i t a t e d , and r e s u s p e n d e d . W h i l e t h i s p r o c e d u r e a p p a r e n t l y does n o t a f f e c t t h e r e p r o d u c i  b i l i t y o f r e s u l t s i n mammalian plasmas, i t may a r t i f i c i a l l y i n c r e a s e t h e amount o f s u b s t r a t e a v a i l a b l e t o k i n i n o g e n a s e s (Hamberg and Rocha e S i l v a , 1957). F u r t h e r e x p e r i m e n t s w i t h t r y p s i n and k i n i n o g e n were r e  d e s i g n e d t o a p p r o a c h p h y s i o l o g i c a l c o n d i t i o n s more c l o s e l y . B l o o d plasma was used as a s o u r c e o f k i n i n o g e n . D e n a t u r a t i o n o f the plasma was a c c o m p l i s h e d by h e a t i n g o n l y t o 65°C, r a t h e r than by b o i l i n g w i t h e t h a n o l . F i n a l l y , the more s p e c i f i c hog p a n c r e a s k a l l i k r e i n (HPK) was used, a l o n g w i t h t r y p s i n , t o r e l e a s e p o s s i b l e k i n i n s . 24 E x p e r i m e n t 1 Method. B l o o d was o b t a i n e d f r o m t h e t e s t a n i m a l s as d e s  c r i b e d p r e v i o u s l y ( C h a p t e r 2) and c e n t r i f u g e d a t 2000 g f o r 40 m i n u t e s , a t 4°C. The plasma was d e c a n t e d , h e a t e d t o 65°C f o r 30 m i n u t e s , d i l u t e d w i t h t h e a p p r o p r i a t e s a l i n e (Appendix B) t o which HPK o r t r y p s i n had b een added, as f o l l o w s : plasma - 2 ml b u f f e r e d s a l i n e , pH = 7.4 4 ml *HPK o r t r y p s i n - 2 mg T o t a l volume - 6 ml *...Hog p a n c r e a s k a l l i k r e i n had 10 K.U./mg A l l i n c u b a t i o n s w i t h t h e s e mammalian enzymes were c a r r i e d o u t i n s i l i c o n e d g l a s s t e s t t u b e s i n a water b a t h a t 37°C ± 0.1°C. The pH o f t h e b u f f e r e d s a l i n e s was 7.4, and t h a t o f i n t a c t plasma, about 7.8. The pH o f t h e i n c u b a t e was about 7.6. Both t r y p s i n and HPK f u n c t i o n e f f i c i e n t l y o v e r t h i s r a n g e o f pH. The f o l l o w i n g c o n t r o l s were always i n c l u d e d : a) plasma (heated) - 1 ml b) plasma (heated) - 1 ml b u f f e r e d s a l i n e - 2 ml b u f f e r e d s a l i n e - 2 ml T o t a l volume - 3 ml HPK o r t r y p s i n , p r e v i o u s l y b o i l e d f o r 10 m i n u t e s - 1 mg T o t a l volume - 3 ml A t the d e s i r e d time i n t e r v a l s the i n c u b a t e s d e s c r i b e d 25 above were sampled as d e s c r i b e d i n C h a p t e r 2, c o o l e d , and a s s a y e d on t h e r a t u t e r u s b i o a s s a y . R e s u l t s . Samples f r o m t h e c o n t r o l s l i s t e d above n e v e r e x h i b i t e d a p r o d u c t i o n o f k i n i n a c t i v i t y . However, a few mammalian co n  t r o l s c o n t a i n e d a low l e v e l o f a c t i v i t y , b u t t h i s was u n l i k e l y t o be due t o k i n i n , s i n c e i t was i n d e p e n d e n t o f enzyme t r e a t  ment, and was r e d u c e d o r a b o l i s h e d by BOL (0.5 ug/ml) and a t r o p i n e (1.0 u g / m l ) . A l l o t h e r plasmas n e v e r had a c t i v i t y i n t h e c o n t r o l s , t h u s e n s u r i n g , a) t h a t a c t i v i t y d i d n o t a r i s e s p o n t a n e o u s l y , b u t was t h e r e s u l t o f t h e enzyme's a c t i o n on t h e h e a t e d plasma, and b) t h a t t h e enzymes ( t r y p s i n and HPK) were t o t a l l y i n a c t i  v a t e d by 10 m i n u t e s b o i l i n g , and. were i n t h e m s e l v e s t o t a l l y i n a c t i v e on t h e i s o l a t e d r a t u t e r u s . W i t h r e s p e c t t o t h e r e l a t i v e e f f i c i e n c y o f t r y p s i n and HPK i n r e l e a s i n g k i n i n a c t i v i t y , t h e v e r t e b r a t e plasmas t e s t e d were c l e a r l y d i v i d e d i n t o two g r o u p s , as shown i n F i g u r e 3a & b Group 1 - Mammalian and t u r t l e p l a s m a s , which r e l e a s e d l a r g e amounts o f a c t i v i t y t o b o t h enzymes, b u t always most t o t r y p s i n , and, Group 2 - Amphibian and f i s h p lasmas, which r e l e a s e d l i t t l e , i f any, a c t i v i t y t o t r y p s i n , b u t which always d e v e l o p e d s i g n i f i c a n t b i o l o g i c a l a c t i v i t y i n t h e p r e s e n c e o f HPK. In c o n s i d e r i n g t h e s e r e s u l t s i t may be i m p o r t a n t t o be Figure 3 a. E f f e c t of t r y p s i n (T) and hog pancreas k a l l i k r e i n (HPK) i n r e l e a s i n g k i n i n from dog, r a t and t u r t l e o plasmas. Plasma was denatured by heating to 65 C fo r 30 minutes, a f t e r d i l u t i o n 1 :2 with s a l i n e . Trypsin was used, at 1 mg/ml undiluted plasma, and hog pancreas k a l l i k r e i n at 10 k.u./ml undiluted plasma. B i o l o g i c a l a c t i v i t y released i s expressed as ug/ml undiluted plasma, BK-eq, as determined on the r a t uterus bioassay. 26 F i g u r e 3 a. F i g u r e 3 b. E f f e c t o f t r y p s i n (T) and hog p a n c r e a s k a l l i k r e i n (HPK) i n r e l e a s i n g k i n i n f r o m Amphiuma, f r o g and t r o u t p l a s m as. C o n d i t i o n s o f i n c u b a t i o n as d e s c r i b  ed i n F i g u r e 3 a. 27 F i g u r e 3 b. AMPHIUMA Incubation Time, Min FROG 1 • z TROUT • *. HO 20 30 I n c u b a t i o n T i m e / M i n 28 be aware o f d i f f e r e n c e s i n t h e a c t i o n o f t r y p s i n and HPK on a g i v e n s u b s t r a t e . T r y p s i n has been shown t o r e l e a s e k i n i n f r o m a l l t y p e s o f k i n i n o g e n , w h i l e HPK may a c t on o n l y one (e.g., Habermann, 1966). A l s o , p a n c r e a t i c k a l l i k r e i n r e l e a s e s l y s - b r a d y k i n i n f r o m d e n a t u r e d mammalian k i n i n o g e n , w h i l e t r y p s i : r e l e a s e s e x c l u s i v e l y b r a d y k i n i n ( S c h a c h t e r , 1969). S i n c e l y s - b r a d y k i n i n i s a b o u t 2/3 as a c t i v e as b r a d y k i n i n on t h e r a t u t e r u s (Sturmer and Berde, 1963), v a l u e s g i v e n f o r a c t i v i t y r e l e a s e d might be c o n s i d e r a b l y h i g h e r i n terms o f m o l a r amounts o f k i n i n l i b e r a t e d . The r e l e a s e o f k i n i n f r o m h e a t e d r a t plasma by HPK ( F i g  u r e 3 a) was an u n e x p e c t e d r e s u l t , s i n c e s e v e r a l w o r k e r s ( F a s c i o l o and H a l v o r s e n , 1946; Vogt, 1966; J a c o b s e n , 1966b) have s t a t e d t h a t t h i s k a l l i k r e i n r e l e a s e s no k i n i n f r o m r a t ' plasma. However, J a c o b s e n (1966 b) and G a u t v i k and Rugstad (1967) have shown t h a t r a t plasma r e l e a s e s l y s - b r a d y k i n i n on i n c u b a t i o n w i t h r a t s a l i v a , and c o n c l u d e t h a t a k i n i n o g e n i s p r e s e n t (termed s u b s t r a t e 2) which w i l l y i e l d k i n i n t o a t l e a s t some g l a n d u l a r k a l l i k r e i n . S i n c e HPK c o n c e n t r a t i o n s used i n the p r e s e n t s t u d y were g r e a t e r t h a n t h o s e used by most w o r k e r s , i t seems p o s s i b l e t h a t t h i s h i g h e r c o n c e n t r a t i o n o f enzyme might degrade t h e " s u b s t r a t e 2". T h i s would e x p l a i n the a p p a r e n t c o n t r a d i c t i o n . I t i s a l s o p o s s i b l e t h a t the HPK p r e p a r a t i o n ( N u t r i t i o n a l 29 B i o c h e m i c a l s Corp.) used i n t h e s e e x p e r i m e n t s d i f f e r e d s l i g h t l y f r o m t h a t u s e d by o t h e r w o r k e r s ( u s u a l l y P a d u t i n , B a y e r ) , o r t h a t t h e r e a r e i n t r a - s p e c i f i c v a r i a t i o n s i n k i n i n o g e n l i n k  age 'among s t r a i n s o f l a b o r a t o r y r a t s . W i t h r e g a r d t o t h e a c t i v i t y r e l e a s e d b y HPK i n g r o u p #2 plasmas, i t i s p o s s i b l e t h a t enzymes o t h e r t h a n t h o s e w h i c h p r o d u c e l y s - b r a d y k i n i n i n mammalian plasma might be r e s p o n s i b l e f o r t h e a p p e a r a n c e o f t h i s a c t i v i t y , s i n c e t h e r e a r e a t l e a s t 5 a c t i v e k a l l i k r e i n s and two p r e k a l l i k r e i n s i n hog p a n c r e a s e x t r a c t s ( r e v i e w e d by S c h a c h t e r , 1969). I t i s a f u r t h e r p o s s i b i l  i t y t h a t a p r o t e a s e i m p u r i t y c o u l d c o n t r i b u t e t o r e s u l t s i n plasmas f r o m b o t h g r o u p s . In any c a s e , t h e major c o n c l u s i o n drawn f r o m t h e e x p e r i  ment ( i . e . , t h e s e p a r a t i o n o f t h e plasmas i n t o two groups) i s u n a l t e r e d . E x p e r i m e n t 2 A l t h o u g h t h e p r e v i o u s e x p e r i m e n t s u g g e s t e d t h a t t r y p s i n and HPK a c t on d i f f e r e n t s u b s t r a t e s i n a m p h i b i a n and f i s h p l asmas, t h e p o s s i b i l i t y r e m a i n ed t h a t t r y p s i n i t s e l f m i g h t i n a c t i v a t e t h e o x y t o c i c f a c t o r i t r e l e a s e d i n t h e s e p l a s m a s . T h e r e f o r e , f u r t h e r e x p e r i m e n t s were c a r r i e d o u t t o t e s t whether HPK c o u l d r e l e a s e t h i s a c t i v e p r i n c i p l e * f r o m plasma p r e v i o u s l y t r e a t e d w i t h t r y p s i n . 30 Method. 2 ml samples o f plasma were h e a t e d , d i l u t e d w i t h s a l i n e o and i n c u b a t e d a t 37 C w i t h e x t h e r t r y p s x n o r HPK, as d e s c r x b e d i n E x p e r i m e n t 1 (p 2 4 ) . Plasma f r o m Amphiuma (group #2) and f r o m r a t s (group #1) were used i n t h e s e e x p e r i m e n t s . A f t e r 30 m i n u t e s , 2 samples were t a k e n from t h e s e t u b e s , one f o r immediate a s s a y , t h e o t h e r f o r r e i n c u b a t i o n w i t h t h e a l t e r n a  t i v e enzyme, e i t h e r HPK o r t r y p s i n , as f o l l o w s : h e a t e d plasma 2 ml b u f f e r e d s a l i n e , pH = 7.4 4 ml t r y p s i n (or HPK) 2 m ? T o t a l volume 6 ml i o i n c u b a t e d a t 37 C f o r 30 mxnutes b o i l e d f o r 10 m i n u t e s •> 1 ml removed f o r a s s a y 1 ml removed, d i l u t e d w i t h 2 ml s a l i n e , r e i n c u b a t e d w i t h 0.33 mg HPK (1.0 mg/ml plasma) a t 37°C, f o r 30 m i n u t e s . b o i l e d f o r 10 m i n u t e s I 4 ml d i s c a r d e d 3 ml sample f o r a s s a y The f i n a l d i l u t i o n o f t h e plasma was 1/9, f o r a l l samples. R e s u l t s . F i g u r e 4 i s a h i s t o g r a m summarizing the r e s u l t s o f 31 t h e s e r e c i p r o c a l e x p e r i m e n t s . As e x p e c t e d f r o m E x p e r i m e n t 1, HPK r e l e a s e d more a c t i v i t y f r o m Amphiuma plasma t h a n d i d t r y p s i n , a n d t r y p s i n r e l e a s e d t h e most f r o m r a t p l a s m a . However, t h e r e i n c u b a t i o n w i t h HPK r e l e a s e d c o n s i d e r a b l y more a c t i v i t y t h a n d i d t h e i n i t i a l i n c u b a t i o n w i t h t r y p s i n i n t h e same plasma. T h i s o b s e r v a t i o n s u p p o r t s t h e c o n c l u s i o n t h a t HPK u t i l i z e s a s u b s t r a t e i n Amphiuma plasma which i s u n a v a i l  a b l e t o t r y p s i n . B. I n t a c t Plasma In o r d e r t o d e t e r m i n e whether o t h e r components o f a plasma k a l l i k r e i n - k i n i n s y s t e m b e s i d e s k i n i n o g e n m i g h t be p r e s e n t i n p o i k i l o t h e r m v e r t e b r a t e s , e x p e r i m e n t s were a l s o c a r r i e d o u t u s i n g u n d e n a t u r e d , o r i n t a c t p l a s m a . 1. K i n i n a s e s i n f i s h , a m p h i b i a n , r e p t i l i a n and mammalian plasmas Compared t o o t h e r p h a r m a c o l o g i c a l l y a c t i v e p e p t i d e s , b r a d y k i n i n and l y s - b r a d y k i n i n a r e t h e most q u i c k l y i n a c t i v a t e d i n mammalian b l o o d plasma ( E r d o s , 1966), and t h i s h o m e o s t a t i c mechanism i s p r o b a b l y a major f a c t o r i n d e t e r m i n i n g t h e d u r  a t i o n o f t h e i r a c t i o n . I n mammals, a t l e a s t two k i n i n a s e s a r e r e s p o n s i b l e f o r t h i s i n a c t i v a t i o n ; plasma k i n i n a s e I (Carboxy- 8 9 p e p t i d a s e N), w h i c h s p l i t s t h e Phe - A r g bond o f b r a d y k i n i n (Erdos and S l o a n e , 1962), and plasma k i n i n a s e I I , which s p l i t s 7 o the Pro -Phe bond (Yang and E r d o s , 1967). These enzymes can F i g u r e 4. R e l a t i v e amounts o f k i n i n r e l e a s e d f r o m h e a t e d Amphiuma and r a t plasma i n 30 m i n u t e s o f i n c u b a t i o n o a t 37 C w i t h t r y p s i n (T; 1 mg/ml p l a s m a ) ; w i t h t r y p  s i n f o l l o w e d b y hog p a n c r e a s k a l l i k r e i n (HPK; 10 K.U./ml p l a s m a ) ; w i t h hog p a n c r e a s k a l l i k r e i n ; and w i t h hog p a n c r e a s k a l l i k r e i n f o l l o w e d by t r y p s i n . V a l u e s a r e b a s e d on two e x p e r i m e n t s , and t h e numbers i n each b a r a r e a c t u a l means, i n ug BK-eq/ml plasma. g u r e 4 Amphiuma 100h 50H c u Q_ 100K < IT) o T;HPK o o o Rat HPK HPK; T 33 be t o t a l l y i n h i b i t e d b y a v a r i e t y o f c h e l a t i n g a g e n t s (Erdos, 1966) . K i n i n a s e a c t i v i t y i n t h e t e s t plasmas was compared t o c h a r a c t e r i z e t h e n a t u r e o f t h i s i m p o r t a n t component o f t h e k a l l i k r e i n - k i n i n s y s t e m i n t h e s e a n i m a l s . The r e l a t i v e e f f e c t o f 8 - h y d r o x y q u i n o l i n e s u l f a t e (8-HQSO4) on k i n i n a s e a c t i v i t y was a l s o compared i n t h e d i f f e r e n t p l asmas, s i n c e a),mammalian k i n i n a s e s a r e t o t a l l y i n h i b i t e d by low d o s e s o f t h i s compound, t h u s p r o v i d i n g a n o t h e r c r i t e r i o n f o r c o m p a r i s o n , and b ) , f u r  t h e r s t u d y o f k i n i n r e l e a s e i n i n t a c t plasma would depend upon the i d e n t i f i c a t i o n and use o f an e f f e c t i v e k i n i n a s e i n h i b i t o r . Method. K i n i n a s e a c t i v i t y was s t u d i e d d i r e c t l y i n t h e u n d i l u t e d plasmas by f o l l o w i n g t h e d i s a p p e a r a n c e o f t h e o x y t o c i c a c t i v i t y o f a r t i f i c i a l l y added s y n t h e t i c b r a d y k i n i n , 1 ug/ml pla s m a . A t z e r o t i m e 1 ug o f b r a d y k i n i n i n 0-01 ml was added by means o f a 25 u l H a m i l t o n s y r i n g e t o 1 ml o f plasma i n a s i l i c o n e d g l a s s t e s t t u b e . A t the d e s i r e d t i m e s , 0.2 ml o f the i n c u b a t e was removed i n a s t e r i l e 1 ml p l a s t i c s y r i n g e and r a p i d l y i n j e c t e d i n t o 1 ml o f d i s t i l l e d w a t er i n a b o i l i n g water b a t h . A f t e r 10 m i n u t e s i n t h e b a t h t h e s e tubes were removed, c o o l e d , and a s s a y e d on the i s o l a t e d r a t u t e r u s , as d e s c r i b e d i n C h a p t e r 2. The e f f e c t o f 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 the k i n i n a s e i n h i b i t o r 8-HQSO4 on the k i n e t i c s o f b r a d y k i n i n i n a c t i v a t i o n was a l s o s t u d i e d i n each plasma. E x p e r i m e n t a l tubes were s e t up 34 and sampled e x a c t l y as d e s c r i b e d above, e x c e p t t h a t t h e r e  q u i r e d amount o f 8-HQSO^ i n 0.2 ml o f s a l i n e was mixed w i t h the plasma p r i o r t o t h e a d d i t i o n o f s y n t h e t i c b r a d y k i n i n . C o n t r o l i n c u b a t e s c o n t a i n i n g b r a d y k i n i n and s a l i n e (but no plasma) were i n c l u d e d i n e ach e x p e r i m e n t . T h i s p r e c a u t i o n e n s u r e d t h a t t h e d i s a p p e a r a n c e o f b r a d y k i n i n i n t h o s e i n c u b a t e s w h ich c o n t a i n e d plasma was n o t due t o a d s o r p t i o n on t h e g l a s s s u r f a c e , n o r t o s p o n t a n e o u s breakdown i n t h e s a l i n e . R e s u l t s . The g r a p h s i n F i g u r e 5 a & b show the amount o f b r a d y  k i n i n r e m a i n i n g i n the plasmas a t t h e d i f f e r e n t s a m p l i n g t i m e s . In a l l plasmas b r a d y k i n i n was r a p i d l y i n a c t i v a t e d i n t h e a b sence o f 8 - H Q S O 4 . However, i n r a t and t u r t l e plasma ( F i g u r e 5 a ) 0.5 mg 8-HQS04/ml u n d i l u t e d plasma was s u f f i c i e n t t o c o m p l e t e l y i n h i b i t k i n i n a s e a c t i v i t y , whereas up t o 8 t i m e s t h i s amount o f t h e i n h i b i t o r (4 mg/ml plasma) o n l y p a r t i a l l y i n h i b i t e d k i n i n a s e s i n Amphiuma, f r o g and t r o u t plasmas ( F i g  u r e 5 b ) . S a l i n e c o n t r o l s n e v e r showed any l o s s o f k i n i n a c t i v i t y o v e r th e s a m p l i n g p e r i o d . A l l plasmas t e s t e d had c o n s i d e r a b l e k i n i n a s e a c t i v i t y . However, w i t h r e s p e c t t o t h e e f f i c i e n c y o f 8 - H Q S O 4 as an i n  h i b i t o r t h e s e plasmas were once more c l e a r l y d i v i d e d i n t o two g r o u p s : Group 1. - Rat and t u r t l e plasmas, where k i n i n a s e a c t i v  i t y was c o m p l e t e l y i n h i b i t e d by low l e v e l s o f 8-HQSO4, and F i g u r e 5a. I n a c t i v a t i o n o f s y n t h e t i c b r a d y k i n i n (1 ug/ml) by i n t a c t , u n d i l u t e d r a t and t u r t l e plasma i s i n d i c a t e d by the c u r v e s w i t h s o l i d d o t s . The e f f e c t o f 0.5 mg/ml 8-HQSO^ on b r a d y k i n i n i n a c t i v a t i o n i s g i v e n by c u r  v e s w i t h h o l l o w d o t s , and a c t i v i t y i n c o n t r o l i n  c u b a t e s (1 ug b r a d y k i n i n i n s a l i n e ) i s i n d i c a t e d by s o l i d s q u a r e s . A l l i n c u b a t e s were a t 20°C. V a l u e s a r e means from two e x p e r i m e n t s . 35 F i g u r e 5 a . Incubat ion Time, M i n Incubation Time, Min Figure 5 b. Inactivation of synthetic bradykinin (1 ug/ml plasma) in Amphiuma, frog and trout plasmas. Legend for curves, and conditions of incubation are described in Figure 5 a. 36 Figure 5 b. 37 Group 2. - F r o g , Amphiuma and t r o u t plasmas, where k i n i n  ase a c t i v i t y was o n l y p a r t i a l l y r e d u c e d by much h i g h e r c o n c e n  t r a t i o n s o f t h e compound. The r e s u l t s u g g e s t s a t l e a s t two e x p l a n a t i o n s : F i r s t l y , t h e enzymes which c o n t r i b u t e t o b r a d y k i n i n a s e a c t i v i t y i n Group 2 plasmas may d i f f e r q u a l i t a t i v e l y f r o m t h o s e i n Group 1; S e c o n d l y , d i f f e r e n c e s c o u l d be due t o t h e d i f f e r e n t c h e m i c a l e n v i r o n m e n t s i n t h e s e p l a s m a s . Most o f the k i n i n a s e i n h i b i t o r s d e s c r i b e d t o d a t e a r e c h e l a t i n g a g e n t s w h i c h i n h i b i t m e t a l l o - enzymes i n g e n e r a l (Erdos and Wohler, 1963). I t has been s u g g e s t e d t h a t t h e a c t i o n o f t h e s e s t r u c t u r a l l y d i f f e r e n t i n h i b i t o r s may r e s t on t h e b i n d i n g o f a m e t a l c o f a c t o r (Erdos and W o h l e r ) . I n t h i s c a s e , t h e i o n i c e n v i r o n m e n t i n t h e plasma would be a v e r y i m p o r t a n t f a c t o r i n d e t e r m i n i n g t h e e f f i c i e n c y o f i n h i b i t o n . I t i s t h e r e f o r e a p o s s i b i l i t y t h a t d i f f e r e n c e s i n c o n c e n t r a t i o n s o f c e r t a i n i o n s (perhaps d i v a l e n t c a t i o n s ) i n t r o u t , Amphiuma and f r o g plasmas m i g h t a c c o u n t f o r t h e i n e f f e c t i v e n e s s o f 8-HQSO4 as a k i n i n a s e i n h i b i t o r i n t h e s e a n i m a l s . 2. R e l a t i v e e f f e c t i v e n e s s o f g l a s s s u r f a c e , t r y p s i n , and hog p a n c r e a s k a l l i k r e i n i n r e l e a s i n g k i n i n f r o m un- d e n a t u r e d v e r t e b r a t e p l a s m a s . As has been mentioned p r e v i o u s l y , an e f f e c t i v e k i n i n a s e i n h i b i t o r i s e s s e n t i a l t o any s t u d y o f k i n i n p r o d u c t i o n i n i n t a c t plasma. S t u d i e s d e s c r i b e d i n the p r e v i o u s e x p e r i m e n t 38 i n d i c a t e d t h a t 8-HQSO was an e f f e c t i v e i n h i b i t o r i n some 4 plasmas, and a t l e a s t p a r t i a l l y e f f e c t i v e i n a l l . T h e r e f o r e a c t i v a t i o n o f endogenous k a l l i k r e i n - k i n i n systems b y g l a s s s u r f a c e (as i n F i g u r e 2) c o u l d a t l e a s t be a t t e m p t e d . The o b j e c t o f t h e s e p r e l i m i n a r y e x p e r i m e n t s was s i m p l y t o d e t e r m i n e whether g l a s s a c t i v a t i o n would r e s u l t i n t h e appe a r a n c e o f k i n i n - l i k e a c t i v i t y v i a t h e endogenous enzymes i n lower v e r t e b r a t e plasmas, as i t does i n mammals. The magnitude o f t h e s e r e s p o n s e s would be compared t o k i n i n p r o  d u c t i o n f r o m t h e same plasma by t r y p s i n and HPK. 2 Method. M a r g o l i s (1963) i n d i c a t e d t h a t 50 cm o f g l a s s s u r f a c e p e r ml o f u n d i l u t e d plasma r a p i d l y r e l e a s e d k i n i n f r o m human plasma a t room t e m p e r a t u r e , and t h i s amount o f aci d - w a s h e d g l a s s b a l l o t i n i ( J e n c o n 1 s No. 8, 0.6 g = 50 cm ) was used i n t h i s e x p e r i m e n t . Plasma was d i l u t e d 1:2 w i t h t h e a p p r o p r i a t e s a l i n e (Appendix B) c o n t a i n i n g 8-HQSO^ i n a 50 ml p o l y p r o p y l e n e t e s t tube. The tube was clamped i n t o an E b e r b a c h water b a t h a t 20°C, and shaken c o n t i n u o u s l y . A t z e r o time, t h e g l a s s beads were added from a 5 ml g l a s s b e a k e r . A t 0, 2, 5, 10, 30, and 60 minutes o f i n c u b a t i o n 0.5 ml samples were removed, i n j e c t e d i n t o 1 ml o f b o i l i n g water, and s t o r e d f o r a s s a y . E x a c t amounts used i n the p r e p a r a t i o n s were as f o l l o w s : 33 Group 1 ( r a t , t u r t l e ) G r oup 2 (Amphiuma, f r o g and t r o u t ) p l a s m a - 2 ml - 2 ml b u f f e r e d s a l i n e - 4 ml 4 ml ' 8-HQS04 - 2 mg - 8 mg g l a s s b e a ds - 1.2 g - 1.2 g T o t a l f l u i d v o lume - 6.0 m l - 6.0 m l I n c u b a t i o n s w i t h HPK and t r y p s i n were c o n d u c t e d i n a w a t e r b a t h a t 37°C, as f o l l o w s : Group 1 Group 2 p l a s m a - 2 m l - 2 m l b u f f e r e d s a l i n e - 4 ml - 4 m l 8-HQS04 - 2 mg - 8 mg T r y p s i n o r HPK - 2 mg - 2 mg T o t a l f l u i d v o lume - 6.0 ml 6.0 ml T r e a t m e n t o f g r o u p s 1 and 2 d i f f e r e d o n l y i n t h e amount o f k i n i n a s e i n h i b i t o r . Two t y p e s o f c o n t r o l i n c u b a t e s were i n  c l u d e d w i t h e x p e r i m e n t s on ea c h p l a s m a . These were p r e p a r e d e x a c t l y as d e s c r i b e d a b o ve, e x c e p t t h a t , a) 8-HQSO4 was o m i t t e d , o r b) g l a s s o r enzyme was o m i t t e d . R e s u l t s . HPK r e l e a s e d no d e t e c t a b l e k i n i n - l i k e a c t i v i t y f r o m any o f t h e s e i n t a c t p l a s m a s , w h i c h was* i n c o n t r a s t t o p r e v i o u s r e s u l t s w i t h d e n a t u r e d p l a s m a . E a r l i e r e x p e r i m e n t s i n d i c a t e d t h a t t h e HPK p r e p a r a t i o n had some i n t r i n s i c k i n i n a s e a c t i v i t y 40 ( F i g u r e s 3 a & b) wh i c h was n o t i n h i b i t e d by 8-HQSO4, b u t n o t enough t o e x p l a i n t h e complete l a c k o f k i n i n a c t i v i t y i n i n c u b a t e s from a l l s a m p l i n g t i m e s . T h e r e a r e a t l e a s t two p o s s i b l e e x p l a n a t i o n s f o r t h i s f a i l u r e t o d e v e l o p f r e e k i n i n ; a) t h a t 8-HQS0 4 m i g h t have i n t e r f e r e d w i t h t h e a c t i o n o f HPK, and b) t h a t t h e r e a r e n a t u r a l i n h i b i t o r s o f HPK i n t h e s e plasmas which were d e s t r o y e d b y h e a t i n g i n p r e v i o u s e x p e r i m e n t s . A l l o t h e r r e s u l t s a r e summarized i n T a b l e 3. V a l u e s a r e e x p r e s s e d as p e r c e n t s , b ased on maximum a c t i v i t i e s o b t a i n e d i n a g i v e n e x p e r i m e n t on one plasma sample, u s u a l l y a t 10 o r 30 minutes of i n c u b a t i o n . 3. E f f e c t o f g l a s s + Hageman F a c t o r i n r e l e a s i n g k i n i n from u n d e n a t u r e d t r o u t , f r o g and Amphiuma plasmas M a r g o l i s (1960) has p r e s e n t e d good e v i d e n c e t h a t g l a s s beads a c t i v a t e and a d s o r b Hageman F a c t o r i n mammalian plasma, t h e r e b y i n i t i a t i n g t h e k a l l i k r e i n - k i n i n r e a c t i o n s . B i r d plasma does n o t r e l e a s e a k i n i n when a c t i v a t e d w i t h g l a s s , b u t E r d o s e t a l (1967) found t h a t duck plasma would r e l e a s e a k i n i n i n d i s t i n g u i s h a b l e f r o m b r a d y k i n i n when a c t i v a t e d w i t h g l a s s which had p r e v i o u s l y been r o t a t e d i n mammalian plasma. These workers c o n c l u d e d t h a t duck plasma l a c k s Hageman F a c t o r , b u t p o s s e s s e s a l l o t h e r components o f a k a l l i k r e i n - k i n i n system. The t r o u t , f r o g and Amphiuma plasmas t e s t e d i n the p r e s e n t 41 T a b l e 4. Mean p e r c e n t o f k i n i n - l i k e a c t i v i t y r e l e a s e d f r o m i n t a c t and d e n a t u r e d plasma o f s e v e r a l s p e c i e s b y g l a s s s u r f a c e , t r y p s i n , and hog p a n c r e a s k a l l i k r e i n . I n t a c t Plasma D e n a t u r e d Plasma *Mean maximal a c t i v i t y / m l S p e c i e s G l a s s T r y p s i n T r y p s i n HPK plasma Dog 52 (2)£ 100 (2) 100 (2) 56(2) 3.1(4) Rat 83 (6) 100(6) 100(4) 38 (4) 3.0 (10) T u r t l e 68(7) 100 (7) 100 (3) 19(3) 1. 9(10) Amphiuma - - 6(3) 100(3) 2. 9 (3) F r o g - - 2(3) 100 (3) 0.2 (3) T r o u t - - - 100 (3) 0.4(3) * . . . v a l u e s i n t h i s column a r e e x p r e s s e d as ug/ml plasma, BK-eq. v a l u e s i n p a r e n t h e s e s i n d i c a t e t h e number o f e x p e r i m e n t s upon which each mean i s b a s e d . 42 s t u d y r e l e a s e d no k i n i n - l i k e a c t i v i t y when t r e a t e d w i t h g l a s s . To d e t e r m i n e w h e t h e r t h i s f a i l u r e c o u l d be due t o a l a c k o f Hageman F a c t o r , as i t i s i n b i r d s , e x p e r i m e n t s s i m i l a r t o t h o s e d e s c r i b e d b y E r d o s e t a l were c a r r i e d o u t w i t h t h e s e p l a s m a s . Method. The method u s e d was e x a c t l y as d e s c r i b e d f o r g l a s s b e a ds (p 38) e x c e p t t h a t t h e a c i d - w a s h e d b a l l o t i n i were f i r s t r o t a t e d i n 10 ml o f f r e s h r a t p l a s m a f o r 10 m i n u t e s , w h i c h was ample t i m e f o r a d s o r p t i o n o f Hageman F a c t o r ( M a r g o l i s , 1 9 6 0 ) . The r a t p l a s m a was t h e n p o u r e d o f f , and t h e b e a d s were washed 5 t i m e s w i t h s a l i n e p r i o r t o t h e i r a d d i t i o n t o t h e t e s t p l a s m a s . R e s u l t s . None o f t h e t e s t p l a s m a s d e v e l o p e d k i n i n - l i k e a c t i v i t y i n r e s p o n s e t o t h e t r e a t m e n t d e s c r i b e d a b o v e . C. D i s c u s s i o n and C o n c l u s i o n s The f i s h , a m p h i b i a n , r e p t i l i a n and mammalian p l a s m a s s u b j e c t e d t o t h e b a s i c q u a l i t a t i v e t e s t s d e s c r i b e d i n t h i s c h a p t e r had o n l y two c h a r a c t e r i s t i c s o f t h e mammalian k a l l i k r e i n - k i n i n s y s t e m ( F i g u r e 2) i n common; a l l h a d k i n i n a s e a c t i v i t y , and a l l r e l e a s e d b i o l o g i c a l a c t i v i t y when h e a t e d p l a s m a was i n c u b a t e d w i t h h og p a n c r e a s k a l l i k r e i n . I n a l l o t h e r t e s t s , r e s p o n s e s o f t h e d i f f e r e n t p l a s m a s c l e a r l y s e p a r a t e d them i n t o two g r o u p s , as summarized b e l o w : TEST .RESPONSE Group 1 ( T u r t l e and Group 2 ( F i s h and mammalian p l a s m a s ) a m p h i b i a n p l a s m a s 1. a. T r y p s i n on M a x i m a l a c t i v i t y Low l e v e l s o f h e a t e d p l a s m a a c t i v i t y 43 TEST RESPONSE Group 1 Group 2 b. HPK on heated plasma Sub-maximal a c t i v i t y Maximal a c t i v i t y 2. 8-HQS04 on k i n i n a s e i n i n t a c t plasma T o t a l i n h i b i t i o n w i t h 0-5 mg/ml plasma Only p a r t i a l i n  h i b i t i o n w i t h 4.0 mg/ml plasma 3. a. T r y p s i n on i n t a c t plasma Maximal a c t i v i t y No a c t i v i t y b. G l a s s beads on i n t a c t plasma Sub-maximal a c t i v i t y No a c t i v i t y Two c o n c l u s i o n s may be drawn from the r e s u l t s of these p r e l i m i n a r y s t u d i e s : a) No evidence was o b t a i n e d t o support the h y p o t h e s i s t h a t a k a l l i k r e i n - k i n i n system analagous t o t h a t of the mammal e x i s t s i n the plasma o f those f i s h and amphibians t e s t e d : b) T u r t l e plasma c o n t a i n s a k a l l i k r e i n - k i n i n system com p a r a b l e i n those c h a r a c t e r i s t i c s examined t o t h a t o f the mammal With r e g a r d to the r e p t i l e s , evidence o b t a i n e d i n t h i s s tudy i s supported by the work of Erdos, Miwa and Graham (1967) which was p u b l i s h e d d u r i n g the course o f these i n v e s t i g a t i o n s . These workers s t u d i e d g l a s s r e l e a s e d k i n i n - l i k e a c t i v i t y i n plasma o f the common snapping (Chelydra s e r p e n t i n a ?) and e l e g a n t s l i d e r (Pseudemys s c r i p t a elegans ?) t u r t l e s , and o f the a l l i g a t o r , u s i n g 1, 10 p h e n a n t h r o l i n e as k i n i n a s e i n h i b i t o r .Basic chemical and p h a r m a c o l o g i c a l p r o p e r t i e s o f the a c t i v e p r i n c i p l e l e d the authors to suggest t h a t the r e p t i l i a n k i n i n 44 was bradykinin and/or l y s - b r a d y k i n i n . In any case, these plasmas c l e a r l y belong i n Group 1, as described above. Recent observations of Lavras, et a l (1969) on plasma of the snake Bothrops jararaca i n d i c a t e that not a l l r e p t i l i a n plasma f i t i n t o t h i s category. These workers found no evidence for an endogenous k a l l i k r e i n - k i n i n system capable of glass a c t i  v a t i o n , and no k i n i n was generated by the a c t i o n of t r y p s i n . I t was also demonstrated that HPK did release a f a c t o r which was oxytocic on the i s o l a t e d r a t uterus, but that t h i s substance was d i f f e r e n t from mammalian k i n i n s , since i t was hypertensive (rather than hypotensive) on mammalian blood pressure. Plasma of t h i s r e p t i l e therefore has c h a r a c t e r i s t i c s ascribed to Group 2 (p 43) . The r o l e of the k a l l i k r e i n - k i n i n system i n blood pressure regulation i n a shark (Scyliorhinus s t e l l a r i s ) and a t e l e o s t (Silurus glanis) was examined by Vogel et a l (1969). Bradykinin, Lys-bradykinin, hog pancreas k a l l i k r e i n , f i s h pancreas k a l l i  k r e i n , and t r y p s i n had no e f f e c t on f i s h blood pressure, and the authors have suggested that a k a l l i k r e i n - k i n i n system does not e x i s t i n the animals. Data obtained i n the present study are not i n c o n f l i c t with these r e s u l t s , or t h i s conclusion, since Vogel et a l did not investigate.smooth muscle s t i m u l a t i n g agents released by HPK from f i s h plasma. Although the HPK released f a c t o r may not be a k i n i n , the observation that the enzyme can l i b e r a t e a b i o l o g i c a l l y a c t i v e 45 p r i n c i p l e i n f i s h , a mphibian, and snake plasmas e n c o u r a g e s s p e c u l a t i o n on the p o s s i b i l i t y t h a t t h e r e c o u l d be a p h y s i o  l o g i c a l r e l e a s e and r o l e f o r the f a c t o r . However, f u r t h e r r e s e a r c h on the c h e m i c a l n a t u r e o f t h e agent, i t s pharmaco l o g i c a l e f f e c t s , and p o s s i b l e mechanisms o f i n v i v o r e l e a s e (e.g., the e f f e c t o f g l a n d u l a r e x t r a c t s on plasma o f each s p e c i e s ) must be c a r r i e d o u t i n a l l s p e c i e s b e f o r e any such p o s s i b i l i t y can be m e a n i n g f u l l y d i s c u s s e d . Any d e t a i l e d c o n s i d e r a t i o n o f e v o l u t i o n o f plasma k a l l i  k r e i n - k i n i n systems would be premature a t t h i s time, s i n c e so few v e r t e b r a t e s have been examined However, c o n s i d e r i n g the e v i d e n c e p r e s e n t l y a v a i l a b l e , t h e f o l l o w i n g p i c t u r e emerge; when major c l a s s e s a r e ranked a c c o r d i n g t o t h e s y s t e m each possesses: B i r d s * O r n i t h o k a l l i k r e i n - k i n i n system C K a l l i k r e i n - k i n i n system Mammals K a l l i k r e i n - k i n i n s y stem R e p t i l e s ( t u r t l e , a l l i g a t o r ) I K a l l i k r e i n - k i n i n system F i s h , Amphibians, R e p t i l e s (snakes) No d i r e c t e v i d e n c e f o r a k a l l i k r e i n - k i n i n system. *...Werle e t a l , 1966. C. - . T h i s k a l l i k r e i n - k i n i n system l a c k s Hageman F a c t o r . 46 I t s h o u l d be emphasized t h a t the l i n e s between gro u p s i n t h e above d i a g r a m do n o t i m p l y e v o l u t i o n o f one enzyme system t o a n o t h e r by any d i r e c t means. 47 CHAPTER 4. THE K A L L I K R E I N - K I N I N SYSTEM I N TURTLE PLASMA E x p e r i m e n t s on f i s h , a m p h i b i a n and r e p t i l i a n p l a s m a s d e s c r i b e d i n C h a p t e r 3 e s t a b l i s h e d t u r t l e p l a s m a as one o f t h e few w h i c h m i g h t h a v e an endogenous k a l l i k r e i n - k i n i n s y s t e m c o m p a r a b l e t o t h e w e l l - s t u d i e d mammalian s y s t e m . F u r t h e r i n v e s t i g a t i o n s were t h e r e f o r e e x t e n d e d t o t h e t u r t l e Pseudemys s c r i p t a e l e g a n s , t o c h a r a c t e r i z e more p r e c i s e l y t h e mechanisms o f t h e r e l e a s e o f t h i s k i n i n , and t o compare t h i s p r o c e s s w i t h t h e r e l e a s e o f b r a d y k i n i n i n mammalian p l a s m a . A w i d e v a r i e t y o f t r e a t m e n t s w i l l r e s u l t i n k i n i n p r o  d u c t i o n f r o m p l a s m a ( E r d o s , 1 9 6 6 ) , b u t few c o u l d be r e g a r d e d as c o n c e i v a b l e p h y s i o l o g i c a l e v e n t s . S i n c e g l a s s s u r f a c e i n i t i a t e s k i n i n p r o d u c t i o n b y a c t i v a t i o n o f t h e a n i m a l ' s own enzymes, w h i l e c a u s i n g a minimum o f s i d e e f f e c t s , i t may i m i t a t e p h y s i o l o g i c a l r e l e a s e o f k i n i n much more c l o s e l y t h a n w o u l d a t f i r s t a p p e a r . F o r t h i s r e a s o n t h e method o f g l a s s a c t i v a t i o n was u s e d t o s t u d y k i n i n p r o d u c t i o n . D e t a i l s o f c u r r e n t t h e o r i e s on g l a s s a c t i v a t i o n o f Hageman F a c t o r and human p r e k a l l i k r e i n have b e e n r e v i e w e d b y W e b s t e r (1968) . T h e r e a r e o b v i o u s d i f f i c u l t i e s i n k i n e t i c s t u d i e s o f a r e a c t i o n whose f i n a l p r o d u c t must be measured b y b i o a s s a y . A l t h o u g h t h e r a t u t e r u s a s s a y i s r e m a r k a b l y s e n s i t i v e , and i n  d i s p e n s a b l e i n m e a s u r i n g l o w c o n c e n t r a t i o n s o f k i n i n s , d a t a 48 are l e s s a c c u r a t e and more d i f f i c u l t t o o b t a i n than i n e q u i v a l e n t s t u d i e s on r e a c t i o n s whose p r o g r e s s can be measured s p e c t r o p h o t o m e t r i c a l l y . , I n t e r p r e t a t i o n of r e s u l t s i s c o m p l i c a t e d by the presence of i n a c t i v a t o r s of enzymes and p r o d u c t s , and by the p l u r a l  i t y of enzyme-substrate i n t e r a c t i o n s which c o n t r i b u t e t o a g i v e n c o n c e n t r a t i o n o f k i n i n . More p r e c i s e i n f o r m a t i o n on p a r t i c u l a r p o i n t s i n the system, such as k a l l i k r e i n - k i n i n o g e n i n t e r a c t i o n s , can be o b t a i n e d u s i n g p u r i f i e d p r e p a r a t i o n s of k i n i n o g e n s (e.g., Kato and S u z u k i , 1 9 6 8 ) , plasma k a l l i k r e i n s (e.g., Colman, M a t t l e r , and Sherry, 1 9 6 9 ) , p r e k a l l i k r e i n s (e.g., McConnell and Mason, 1970) or g l a n d u l a r k a l l i k r e i n s (e.g., Takami, 1969) . However, i t was the purpose of t h i s study t o examine the d i f f e r e n t a s p e cts of k i n i n p r o d u c t i o n i n v o l v i n g the e n t i r e k a l l i k r e i n - k i n i n system, under c o n d i t i o n s which might o b t a i n _in v i v o . There may be s e a s o n a l changes i n h e m a t o c r i t (Hutton, 1960) and b l o o d c o a g u l a t i o n (Jacques, 1963) i n J?. s c r i p t a e legans, which c o u l d r e s u l t i n s e a s o n a l changes i n concen t r a t i o n s of components i n the plasma k a l l i k r e i n - k i n i n system. Since t h i s study was p r i m a r i l y d i r e c t e d towards c h a r a c t e r i z i n g the q u a l i t a t i v e mechanisms of the system, no s e a s o n a l or e x p e r i m e n t a l l y induced q u a n t i t a t i v e changes of t h i s nature Were e x p l o r e d . T u r t l e s were ke p t a t n a t u r a l p h o t o p e r i o d and 49 c o n s t a n t t e m p e r a t u r e (15°C) i n the l a b o r a t o r y ; t h e a n i m a l s were sampled t h r o u g h o u t th e y e a r , and no c o n s i s t e n t d i f f e r  e n ces i n k i n i n p r o d u c t i o n were n o t e d . A. p r e l i m i n a r y S t u d i e s . These s t u d i e s were c o n d u c t e d t o d e t e r m i n e t h e e f f e c t on t u r t l e k i n i n p r o d u c t i o n o f v a r y i n g i m p o r t a n t c o n d i t i o n s o f i n c u b a t i o n s , such as the amount and t y p e o f k i n i n a s e i n h i b i t o r , the d i l u t i o n o f plasma, o r t h e amount o f g l a s s s u r f a c e . Data from t h e s e e x p e r i m e n t s would a l l o w s e l e c t i o n o f s t a n d a r d c o n  d i t i o n s f o r r e l e a s e , e n a b l i n g the d i r e c t c o m p a r i s o n o f the c h a r a c t e r i s t i c s o f r e l e a s e w i t h mammalian plasma. I t would a l s o a l l o w the d e s i g n o f an e x p e r i m e n t s u i t a b l e f o r t h e p r o  d u c t i o n o f t h e l a r g e amount o f k i n i n r e q u i r e d f o r f u t u r e p u r i  f i c a t i o n o f t h e p e p t i d e . 1. P r o d u c t i o n of t u r t l e k i n i n i n the absence o f a k i n i n a s e i n h i b i t o r . A l t h o u g h d e t a i l e d s t u d y o f t u r t l e k i n i n r e l e a s e would i n v o l v e t h e use o f a k i n i n a s e i n h i b i t o r , and adop t i o n o f c o n v e n i e n t s a m p l i n g t e c h n i q u e s w i t h d i l u t e d plasma, i t i s n o n e t h e l e s s i m p o r t a n t t o examine the r e l e a s e o f t u r t l e k i n i n i n u n m o d i f i e d plasma, p a r t i c u l a r l y s i n c e t h i s i s t h e c l o s e s t a p p r o x i m a t i o n t o _in v i v o a c t i v a t i o n o f t h e enzyme system. Method. U n d i l u t e d plasma o b t a i n e d i n the u s u a l manner (Chapter 2) was p l a c e d i n 5 ml l o t s i n 10 ml p o l y p r o p y l e n e t e s t t u b e s , 50 i n an E b e r b a c h water b a t h a t 20°C, and a c t i v a t e d w i t h 6.0 g 2 a c i d washed g l a s s beads p e r tube (50 cm /ml p l a s m a ) . A t 1, 2, 5, 10, 20, and 30 mi n u t e s samples were removed and h e a t e d as . d e s c r i b e d i n C h a p t e r 2 (p 12) . C o n t r o l l o t s c o n t a i n i n g no g l a s s were i n c u b a t e d and sampled i n t h e same way. R e s u l t s . I n d i v i d u a l v a l u e s f o r f i v e e x p e r i m e n t s on plasmas o f d i f f e r e n t t u r t l e s a r e p r e s e n t e d i n F i g u r e 6. Samples t a k e n b e f o r e 2 m i n u t e s , and a f t e r 10 mi n u t e s o f i n c u b a t i o n n e v e r showed k i n i n a c t i v i t y . Some plasmas a c t i v a t e d w i t h g l a s s d i d n o t c o n t a i n m e a s u r a b l e t u r t l e k i n i n a t any s a m p l i n g time, w h i l e o t h e r s d i d d e v e l o p up t o 55 ng/ml, b r a d y k i n i n e q u i v a l e n t . C o n t r o l s w i t h no g l a s s n e v e r d e v e l o p e d a c t i v i t y o v e r t h e s a m p l i n g p e r i o d U n m o d i f i e d t u r t l e plasma can d e v e l o p low l e v e l s o f k i n i n a c t i v i t y , and i t i s c l e a r t h a t the plasma c o n t a i n s an a c t i v e k i n i n a s e , as s u g g e s t e d by e a r l i e r e x p e r i m e n t s w i t h b r a d y k i n i n ( F i g u r e 5 a ) . I n mammals, t h e t h r e s h o l d dose f o r a d e p r e s s o r r e s p o n s e i s u s u a l l y n o t l e s s t h a n 250 ng/kg ( E r d o s , 1966). I f t u r t l e s a r e s i m i l a r , t h e n a t t h i s pH (about 7.8) and o t e m p e r a t u r e (20 C) i t s h o u l d t h e r e f o r e r e q u i r e a v e r y s t r o n g s t i m u l u s f o r the c o n c e n t r a t i o n o f k i n i n t o b u i l d up t o a p o i n t where a major c a r d i o v a s c u l a r e f f e c t c o u l d be o b s e r v e d . I t s h o u l d be emphasized t h a t t h e r e i s a t p r e s e n t no e v i d e n c e on the p h y s i o l o g y or pharmacology o f k i n i n s i n r e p t i l e s . F i g u r e 6. E f f e c t o f g l a s s s u r f a c e (50 cm z/ml plasma) i n r e l e a s i n g k i n i n f r o m t u r t l e plasma c o n t a i n i n g no k i n i n a s e i n h i b i t o r . R e s u l t s o f 5 s e p a r a t e o e x p e r i m e n t s a t 20 C a r e shown. D o t t e d l i n e i n  d i c a t e s t h e l i m i t o f s e n s i t i v i t y f o r t h e r a t u t e r  us b i o a s s a y . A c t i v i t y i s e x p r e s s e d as ng/ml plasma, BK-eq. 5 1 Figure 6 52 2 . E f f e c t o f mammalian k i n i n a s e i n h i b i t o r s on t u r t l e k i n i n a s e . T h i s e x p e r i m e n t was d e s i g n e d t o f i n d a compound which c o u l d e f f e c t i v e l y i n h i b i t k i n i n a s e s i n t u r t l e plasma, and t h e r e f o r e would a l l o w t h e r e l e a s e o f t u r t l e k i n i n . The compounds 2 - m e r c a p t o e t h a n o l and 3 - m e r c a p t o p r o p r i o n i c a c i d (Sigma) have been shown t o be p o t e n t i a t o r s o f t h e _in v i v o e f f e c t s o f b r a d y k i n i n i n mammals (Erdos and Wohler, 1963), presumably t h r o u g h k i n i n a s e i n h i b i t i o n . These compounds were t e s t e d i n t h i s s t u d y i n the hope t h a t t h e same i n h i b i t o r m i g h t be u s e d i n f u t u r e _in v i v o and in v i t r o e x p e r i m e n t s . D e r i v a t i v e s o f 8 - h y d r o x y q u i n o l i n e a r e e f f e c t i v e i n h i b i t o r s i n v i t r o (Erdos, 1966; Rocha e S i l v a , 1963), as a r e m e t a l s a l t s o f EDTA ( M a r g o l i s , 1963; E r d o s , 1966), a l t h o u g h b o t h a r e i n e f f e c t i v e p o t e n t i a t o r s o f _in v i v o b r a d y k i n i n e f f e c t s (Erdos and Wohler, 1963); t h e s e compounds were a l s o t e s t e d . A l l compounds were t e s t e d i n two ways; a) i n e x p e r i m e n t s t o d e t e r m i n e t h e e f f e c t o f 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 i n  h i b i t o r on t h e r a t e o f i n a c t i v a t i o n o f b r a d y k i n i n i n i n t a c t plasma; and b) i n e x p e r i m e n t s t o d e t e r m i n e t h e e f f e c t o f 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 i n h i b i t o r on the k i n e t i c s o f t u r t l e k i n i n r e l e a s e b y g l a s s a c t i v a t i o n . Method, a) The method f o r t r a c i n g the .rate o f b r a d y k i n i n des t r u c t i o n was e x a c t l y as d e s c r i b e d i n C h a p t e r 3, p 33. I n h i b i t o r s were added t o t h e plasma i n volumes n e v e r e x c e e d i n g 53 0.2 ml o f s a l i n e c a r r i e r . b) Plasma d i l u t e d 1:2 w i t h t u r t l e s a l i n e was a c t i  v a t e d w i t h 50 cm 2 g l a s s s u r f a c e / m l plasma, and sampled as d e s c r i b e d i n C h a p t e r 2. I n h i b i t o r s were added t o t h e t u r t l e s a l i n e p r i o r t o d i l u t i o n o f t h e plasma. R e s u l t s . F i g u r e 7 i n d i c a t e s t h a t 3 - m e r c a p t o p r o p r i o n i c a c i d and Na-EDTA i n doses up t o 4 mg/ml were i n e f f e c t i v e i n de c r e a s i n g t h e r a t e o f b r a d y k i n i n i n a c t i v a t i o n , w h i l e 2-mercap- t o e t h a n o l was p a r t i a l l y e f f e c t i v e . However, 8-HQSO4 i n doses above 0.5 mg/ml plasma was n o r m a l l y s u f f i c i e n t t o g i v e t o t a l p r o t e c t i o n t o b r a d y k i n i n (as d i s c u s s e d i n C h a p t e r 3, p 3 4 ) . These r e s u l t s were m i r r o r e d i n t h e e f f e c t s o f t h e s e same compounds on t u r t l e k i n i n p r o d u c t i o n , i n F i g u r e 8. I n t h e s e e x p e r i m e n t s , 8-HQSO4 (1 mg/ml plasma) gave t h e h i g h e s t v a l  ues f o r t u r t l e k i n i n a t any i n c u b a t i o n t ime, and a f t e r 1 h o u r ( i n one c a s e 2 ho u r s ) o f i n c u b a t i o n w i t h t h i s i n h i b i t o r , t h e c o n c e n t r a t i o n o f t u r t l e k i n i n showed no d e c r e a s e . 2-mercap- t o e t h a n o l (2 mg/ml plasma) a l l o w e d c o n s i d e r a b l e k i n i n a c t i v i t y t o b u i l d up, b u t t h i s was always d e s t r o y e d i n p r o l o n g e d i n  c u b a t i o n s . I t i s p o s s i b l e t h a t v e r y h i g h c o n c e n t r a t i o n s o f t h i s s u b s t a n c e m i g h t g i v e complete p r o t e c t i o n t o b r a d y k i n i n and t u r t l e k i n i n , b u t m i g h t a l s o i n t e r f e r e w i t h o t h e r a s p e c t s o f k i n i n r e l e a s e . A c t i v a t i o n o f t u r t l e plasma i n th e p r e s e n c e o f Na-EDTA o r 3 - m e r c a p t o p r o p r i o n i c a c i d p r o d u c e d o n l y t r a c e s o f t u r t l e F i g u r e 7. E f f e c t o f mammalian k i n i n a s e i n h i b i t o r s on t h e r a t e o f i n a c t i v a t i o n o f b r a d y k i n i n ( l u g / m l plasma) i n u n d i l u t e d t u r t l e plasma, a t 20°C. V a l u e s a r e means f o r two e x p e r i m e n t s . The l e g e n d f o r t h e c u r v e s i s as f o l l o w s : A no k i n i n a s e i n h i b i t o r added v up t o 4 mg/ml 3 - m e r c a p t o p r o p r i o n i c a c i d • up t o 4 mg/ml Na-EDTA • 4 mg/ml 2 - m e r c a p t o e t h a n o l * 0.5 mg/ml 8-HQS0 4 X c o n t r o l s , c o n t a i n i n g o n l y s a l i n e and s y n t h e t i c b r a d y k i n i n , l u g / m l . 54 F i g u r e 7 V Figure 8. Production of k i n i n from t u r t l e plasma by glass sur face (50 cm2/ml plasma) at 20°C, in the presence of different kininase inhibitors. A c t i v i t y present in the samples i s expressed as ug/ml undiluted, plasma, BK-eq. The legend for the curves i s as follows: A 4 mg/ml 3-mercaptoproprionic acid A 4 mg/ml Na-EDTA • 2 mg/ml 2-mercaptoethanol * 1 mg/ml 8-HQS04 55 Figure 8 5 6 k i n i n a c t i v i t y no d i f f e r e n t f r o m p l a s m a a c t i v a t e d i n t h e ab sence o f any i n h i b i t o r ( F i g u r e 6 ) . T u r t l e plasma i s t h e r e f o r e s i m i l a r t o r a t and g u i n e a p i g plasma i n t h a t Na-EDTA i s i n  e f f e c t i v e , and d i f f e r e n t f r o m human ( M a r g o l i s , 1963) and sheep (Campbell e t a l , 1968) plasmas i n w h i c h EDTA i s p a r t i a l l y e f f e c t i v e . I t was c o n c l u d e d f r o m t h e e x p e r i m e n t s g i v e n h e r e t h a t t h e mercapto compounds, and Na-EDTA a r e u n s a t i s f a c t o r y i n v i t r o k i n i n a s e i n h i b i t o r s i n t u r t l e p l a s m a . However, t h i s does n o t e x c l u d e t h e p o s s i b i l i t y t h a t t h e s e c h e m i c a l s may p o t e n t i a t e _in v i v o e f f e c t s i n t h e t u r t l e . In r a t s , f o r ex ample, Ryan, R o b l e r o and S t e w a r t (1968) have shown t h a t 2- m e r c a p t o e t h a n o l may p o t e n t i a t e _in v i v o e f f e c t s o f b r a d y k i n i n b y i n h i b i t i n g p o w e r f u l k i n i n a s e s s i t u a t e d n o t i n t h e plasma, b u t i n t h e v a s c u l a r e n d o t h e l i u m i n t h e l u n g . 3. E f f e c t o f plasma d i l u t i o n on t u r t l e k i n i n p r o d u c t i o n . F o r most i n c u b a t i o n s i t was f o u n d t o be c o n v e n i e n t t o d i l u t e plasma 1:2 w i t h t u r t l e s a l i n e (Appendix B ) . T h i s p r o c e d u r e a l l o w e d t h e h e a t e d samples u s e d i n some e x p e r i m e n t s t o r e m a i n i n a l i q u i d form, whereas u n d i l u t e d ' plasma formed a g e l when h e a t e d above 65°C. S i n c e i t i s p o s s i b l e t h a t t h e k i n e t i c s ' . ' f k i n i n p r o d u c t i o n c o u l d be s i g n i f i c a n t l y a l t e r e d b y d i l u t i o n o f the components from p h y s i o l o g i c a l c o n c e n t r a t i o n s , e x p e r i m e n t s were c a r r i e d o u t t o t e s t the e f f e c t o f v a r y i n g d i l u t i o n o v e r 57 t h e range o f d i l u t i o n s t o be u s e d i n f u t u r e e x p e r i m e n t s . Method. U s i n g 1 mg 8-HQS0 4/ ml u n d i l u t e d plasma, a t 20°C, 2 samples o f plasma f r o m one a n i m a l were i n c u b a t e d w i t h 50 cm gla^ss s u r f a c e , as f o l l o w s : Plasma S a l i n e 8-HQS0 4 G l a s s Beads a. 2 ml none 2 mg 2.4 g b. 2 ml 4 ml 2 mg 2.4 g c. 1 ml 9 ml 1 mg 1.2 g A t T = 0, 10, 50, and 120 m i n u t e s samples were removed, i n  a c t i v a t e d b y b o i l i n g , and s t o r e d f o r a s s a y . R e s u l t s . R e s u l t s f o r each d i l u t i o n a r e summarized i n F i g u r e 9, i n terms o f b r a d y k i n i n - e q u i v a l e n t / m l u n d i l u t e d plasma p r e s e n t a t each s a m p l i n g t i m e . Each b a r r e p r e s e n t s t h e mean o f t h r e e v a l u e s , and 95% c o n f i d e n c e l i m i t s a r e g i v e n . T h e r e was no s i g  n i f i c a n t d i f f e r e n c e between t r e a t m e n t s , and i t i s d o u b t f u l whether a l a r g e r sample s i z e would i n d i c a t e t h a t s i g n i f i c a n t d i f f e r e n c e s e x i s t i n a c t i v i t i e s p r e s e n t a t 10 mi n u t e s (a measure of r e a c t i o n r a t e ) o r a t 50 and 120 mi n u t e s (a measure o f maxi mum y i e l d ) . I n s i m i l a r e x p e r i m e n t s on human plasma, M a t g o l i s (1963) f o u n d t h a t d i l u t i o n h a d l i t t l e e f f e c t , a l t h o u g h a t v e r y h i g h d i l u t i o n s , up t o 1:100, b o t h r a t e and y i e l d o f k i n i n p r o  d u c t i o n were r e d u c e d . Throughout r e m a i n i n g e x p e r i m e n t s i t was assumed t h a t d i l u  t i o n o f plasma t o 1:2 d i d n o t cause major changes i n t h e e 9. E f f e c t o f d i l u t i o n o f plasma on r a t e and y i e l d o f t u r t l e k i n i n p r o d u c t i o n . Plasma was d i l u t e d w i t h b u f f e r e d s a l i n e as i n d i c a t e d i n t h e F i g u r e , and a c t i v a t e d w i t h 50 cm g l a s s s u r f a c e / m l u n d i l u t e d plasma, a t 20°C. A c t i v i t y a t each o f t h e s a m p l i n g t i m e s i s g i v e n as ug/ml u n d i l u t e d plasma, BK-eq. Each b a r r e p r e s e n t s t h e mean o f 3 e x p e r i m e n t s , and 95% c o n f i d e n c e l i m i t s a r e i n d i c a t e d . 58 F i g u r e 9 59 k i n e t i c s o f t u r t l e k i n i n r e l e a s e . B. E f f e c t o f G l a s s S u r f a c e A r e a on T u r t l e K i n i n P r o d u c t i o n S i n c e v a r i a t i o n i n g l a s s s u r f a c e a r e a c o u l d be e x p e c t e d t o a f f ; e c t k i n i n p r o d u c t i o n , e x p e r i m e n t s were c a r r i e d o u t t o d e t e r  mine t h e r e l a t i o n o f t h i s p a r a m e t e r t o t h e r a t e and y i e l d o f t h e k i n i n r e l e a s i n g r e a c t i o n s . T h ese e x p e r i m e n t s were a l s o d e s i g n e d t o t e s t t h e g l a s s - t r i g g e r e d r e a c t i o n s i n t u r t l e plasma a g a i n s t t h e model f o r k i n i n p r o d u c t i o n i n mammals ( F i g u r e 2 ) . A c c o r d i n g t o t h i s p r o p o s e d mechanism f o r a c t i v a t i o n o f t h e endogenous k a l l i k r e i n - k i n i n system, the amount o f p r e k a l l i k r e i n c o n v e r t e d t o k a l l i  k r e i n s h o u l d be d i r e c t l y p r o p o r t i o n a l t o t h e amount o f Hageman F a c t o r a c t i v a t e d , w h i c h i n t u r n i s p r o b a b l y d i r e c t l y r e l a t e d t o t h e number o f n e g a t i v e s i t e s on t h e a c t i v a t i n g m a t e r i a l ( M a r g o l i s , 1963: N o s s a l , e t a l , 1968) . I f t h e mammalian and t u r t l e systems were s i m i l a r , v a r i a t i o n s i n g l a s s s u r f a c e a r e a / ml o f plasma w o u l d be p r e d i c t e d t o change t h e r a t e b u t n o t t h e y i e l d o f k i n i n p r o d u c i n g r e a c t i o n s . Method. I n c u b a t e s were p r e p a r e d as f o l l o w s : G l a s s b a l l o t i n i Plasma S a l i n e 8-HQSO^ a. 0.24 g (5 cm 2/ml) 2 ml 4 ml 2 mg b. 0.96 g (20 cm 2/ml) 2 ml . 4 ml 2 mg c. 2.4 g (50 cm 2/ml) 2 ml 4 ml 2 mg d. 4.8 g (100 cm 2/ml) 2 ml 4 ml 2 mg These tubes were i n c u b a t e d a t 20°C i n t h e u s u a l manner 60 and sampled a t T = 0, 1, 2, 5, 10, 20, 30, and 50 m i n u t e s , a f t e r t h e a d d i t i o n o f the g l a s s . Each e x p e r i m e n t on t h e plasma o f one t u r t l e i n c l u d e d a l l f o u r i n c u b a t e s , as w e l l as c o n t r o l i n c u b a t e s c o n t a i n i n g no g l a s s . The whole e x p e r i m e n t was r e p e a t e d on plasma o f d i f f e r e n t t u r t l e s . R e s u l t s . Data from 3 e x p e r i m e n t s on d i f f e r e n t plasmas a r e g i v e n i n F i g u r e 10. These r e s u l t s a r e e x p r e s s e d as t h e mean a c t i v i t y p r e s e n t a t a p a r t i c u l a r s a m p l i n g t i m e . I t i s c l e a r t h a t t h e r a t e o f k i n i n p r o d u c t i o n was d i r e c t l y p r o p o r t i o n a l t o the amount o f g l a s s s u r f a c e added. Maximum l e v e l s o f k i n i n p r o  duced i n 50 m i n u t e s d i d n o t d i f f e r between i n c u b a t e s c o n t a i n - 2 i n g 100, 50, and 20 cm g l a s s s u r f a c e / m l . K i n i n p r o d u c e d by 2 5 cm g l a s s / m l d i d n o t appear t o have r e a c h e d maximum l e v e l s even a f t e r 50 m i n u t e s o f i n c u b a t i o n , b u t would p r o b a b l y have done so i n a l o n g e r i n c u b a t i o n p e r i o d . These r e s u l t s a r e e x a c t l y as would be p r e d i c t e d f r o m t h e mammalian model ( F i g  u r e 2) . M a r g o l i s and B i s h o p (1963) a l s o i n v e s t i g a t e d the r e l a t i o n  s h i p o f g l a s s s u r f a c e a r e a t o b r a d y k i n i n p r o d u c t i o n i n human plasma. S u r p r i s i n g l y , t h e y f o u n d t h a t 250 cm g l a s s / m l plasma gave a lower y i e l d t h a n 50 cm g l a s s / m l , and t h a t k i n i n was r e l e a s e d i n b o t h i n c u b a t e s a t the same r a t e . As s u g g e s t e d 2 by t h e s e w o r k e r s , t h e l o w e r y i e l d a t 250 cm c o u l d have been due t o a d s o r p t i o n o f k i n i n t o g l a s s s u r f a c e a t t h i s h i g h 10. The e f f e c t o f g l a s s s u r f a c e a r e a on t u r t l e k i n i n p r o d u c t i o n a t 20°C. Plasma d i l u t e d 1:2 w i t h s a l i n e 2 was a c t i v a t e d w i t h 5, 10, 50 and 100 cm g l a s s s u r f a c e / m l u n d i l u t e d p l a s m a . V a l u e s a r e g i v e n as ug/ml plasma, BK-eq, and a r e means o f 2 e x p e r i m e n t s . g l a r e 10 62 c o n c e n t r a t i o n . W i t h r e g a r d t o t h e s i m i l a r i t i e s f o u n d . i n r a t e s o f k i n i n p r o d u c t i o n , i t seems p o s s i b l e t h a t the h i g h e r i n c u b a  t i o n t e m p e r a t u r e (about 23°C) and t h e i n c o m p l e t e k i n i n a s e i n h i b i t i o n b y EDTA ( M a r g o l i s and B i s h o p , 19 63) o b s c u r e d th e r e l a t i o n between r a t e o f p r o d u c t i o n and g l a s s s u r f a c e a r e a t h a t s h o u l d e x i s t i n t h i s p lasma. Maximum v e l o c i t y o f k i n i n p r o d u c t i o n was e s t i m a t e d f r o m the s t r a i g h t p o r t i o n a t t h e b e g i n n i n g o f each c u r v e i n F i g u r e 10, and the r e l a t i o n between the l o g o f t h i s i n i t i a l v e l o c i t y and g l a s s s u r f a c e a r e a i s g i v e n i n F i g u r e 11. Over t h e ex p e r i m e n t a l range th e r e l a t i o n a p p r o x i m a t e s a s t r a i g h t l i n e , ' i n d i c a t i n g t h a t t h e k i n e t i c s i n v o l v e d a r e n o t f i r s t - o r d e r . T h a t i s , a d o u b l i n g o f t h e g l a s s s u r f a c e a r e a more t h a n d o u b l e s the r a t e o f k i n i n f o r m a t i o n , i m p l y i n g t h a t the t h e enzymic r e a c t i o n s i n v o l v e d have a " m u l t i p l i e r e f f e c t " , as can o c c u r i n "damped c a s c a d e " enzyme pathways (Hemker and Hemker, 1969) . The s l o p e o f t h e l i n e s u g g e s t s t h a t i t would, n o t p a s s t h r o u g h the o r i g i n , and t h a t a t 0 cm^/ml plasma, the v " m a x would be about 7 ng BK-eq/ml/min. T h i s c a n n o t be t h e c a s e , s i n c e c o n t r o l s w i t h no g l a s s d i d n o t d e v e l o p k i n i n a c t i v i t y . T h e r e f o r e , i t i s p r o b a b l e t h a t t h i s m u l t i p l i e r e f f e c t i s g r e a t e r when s m a l l amounts o f g l a s s s u r f a c e ( l e s s t h a n 5 cm 2/ml plasma) o r some o t h e r a c t i v a t o r a r e i n v o l v e d . F i g u r e 11. E f f e c t o f g l a s s s u r f a c e a r e a on maximum v e l o c i t y . o f t u r t l e k i n i n p r o d u c t i o n a t 20°C. V a l u e s f o r vmax a r e < ? i v e n a s ng/ml plasma/min, BK-eq, and were e s t i m a t e d f r o m t h e i n i t i a l s t r a i g h t p o r t i o n o f the c u r v e s shown i n F i g u r e 10. 63 F i g u r e 11 64 C. A c t i v a t i o n and Decay o f Plasma K a l l i k r e i n I t seems r e a s o n a b l e t o assume t h a t any plasma w h i c h r e l e a s e s a k i n i n on c o n t a c t w i t h g l a s s c o n t a i n s some t y p e o f k a l l i k r e i n i n a pre-enzyme s t a t e . However, the r e l a t i v e c o n c e n t r a t i o n s o f t h e pre-enzyme i n t h e plasma, i t s r a t e o f a c t i v a t i o n , and t h e r a t e o f decay o f t h e a c t i v a t e d enzyme a r e a l l i m p o r t a n t v a r i a b l e s i n t h e system, and c o u l d m a r k e d l y a f f e c t i t s a b i l i t y t o f u n c t i o n i n a p h y s i o l o g i c a l r o l e . These a s p e c t s o f k i n i n r e l e a s e were t h e r e f o r e s t u d i e d i n t u r t l e plasma, and compared d i r e c t l y t o the e q u i v a l e n t e v e n t s i n r a t plasma. The method u s e d i n t h i s s t u d y had f e a t u r e s i n common w i t h t h o s e d e s c r i b e d by M a r g o l i s (1960) and B r i s e i d e t a l (1968) f o r human plasma, and i t i s b a s e d on t h e p r e p a r a t i o n o f a plasma d e p l e t e d o f s u b s t r a t e f o r plasma k a l l i k r e i n , b u t which s t i l l c o n t a i n s p r e - k a l l i k r e i n . I t has been shown t h a t t r y p s i n r e l e a s e s more k i n i n from t u r t l e plasma than i s r e l e a s e d by g l a s s ( T a b l e 3 ) . F o r c o n v e n i e n c e , t h a t p o r t i o n o f the t o t a l k i n i n o g e n w h i c h r e  l e a s e s k i n i n t o g l a s s a c t i v a t e d enzymes w i l l be r e f e r r e d t o as S u b s t r a t e 1 (see C h a p t e r 1 ) . 1. P r e p a r a t i o n o f S u b s t r a t e 1 - d e p l e t e d plasma. The o b j e c t o f t h i s e x p e r i m e n t was t o i n c u b a t e plasma w i t h g l a s s b a l l o t i n i f o r a p e r i o d j u s t l o n g enough t o a l l o w t h e r e l e a s e 65 o f a l l k i n i n i n S u b s t r a t e 1. T h i s plasma c o u l d t h e n be u s e d as a s o u r c e o f p r e k a l l i k r e i n . Method. T u r t l e plasma was i n c u b a t e d i n s e v e r a l e x p e r i m e n t s w i t h 50 and 100 cm g l a s s s u r f a c e / m l plasma, i n t h e absence o f a k i n i n a s e i n h i b i t o r . A t s e l e c t e d t i m e i n t e r v a l s samples o f t h i s plasma were s e p a r a t e d from t h e g l a s s and i n c u b a t e d a l o n e f o r a f u r t h e r two h o u r s a t 20°C; t h i s a l l o w e d time f o r a complete i n a c t i v a t i o n o f any k i n i n s p r o d u c e d , and f o r th e normal l o s s o f t h e k a l l i k r e i n s w h i c h had been r e s p o n s i b l e f o r d e v e l o p i n g them. A p o r t i o n o f each i n c u b a t e was t h e n removed, i t s enzymes were i n a c t i v a t e d i n b o i l i n g w ater, and th e p r o d u c t a s s a y e d as a c o n t r o l . The r e m a i n d e r o f t h e mix t u r e was u s e d t o measure t h e amount o f S u b s t r a t e 1 s t i l l p r e s e n t i n t h e s e g l a s s - d e p l e t e d samples. T h i s was d e t e r m i n e d by subsequent i n c u b a t i o n o f each sample w i t h t h e k i n i n a s e i n h i b i t o r 8-HQSO^ and f r e s h a cid-washed g l a s s f o r one h o u r a t 20°C. Samples from t h e s e t u b e s were b o i l e d and a s s a y e d i n the u s u a l manner. A l s o i n c l u d e d i n each e x p e r i m e n t was a c o n t r o l i n c u b a t e which was t r e a t e d e x a c t l y as d e s c r i b e d above, e x c e p t f o r t h e a d d i t i o n o f 8-HQSO4 i n the i n i t i a l i n c u b a t i o n . These samples always c o n t a i n e d some k i n i n a c t i v i t y . t D e t a i l s o f each e x p e r i  ment can be shown s c h e m a t i c a l l y as f o l l o w s : 66 Plasma S a l i n e G l a s s 8--HQS0 4 1. 5 ml 10 ml 50 cm /ml - 2 . 5 ml 10 ml 2 , , 100 cm /ml - 3 . , 5 ml 10 ml 2 100 cm /ml A. 2 ml removed from each i n  c u b a t e a t T = 0, 2, 5, 10, 20, and 30 m i n u t e s , and i n  c u b a t e d s e p a r a t e l y f o r 2 h o u r s a t 20°C. * Purpose To d e p l e t e Sub s t r a t e 1 by g l a s s a c t i v a t i o n . To a l l o w k i n i n and k a l l i k r e i n t o be d e s t r o y e d b y n a t u r a l mechanisms. C. 1.5 ml + 0.6 g g l a s s + 0.5 mg 8-HQS0 4 ( e x c e p t #3) . Shaken a t 20°C f o r 60 min. To c o n v e r t r e s i d u a l S u b s t r a t e 1 t o a c t i v e k i n i n b y g l a s s a c t i v a t i o n . B. 0.5 ml sam- D. 1.0 ml sampled p l e d and and a s s a y e d , a s s a y e d . *... A l t h o u g h k a l l i k r e i n c o n t i n u e s t o r e l e a s e k i n i n a f t e r r e m oval o f g l a s s , the e f f e c t i s s h o r t - l i v e d , as w i l l be shown i n s e c t i o n 3, t h i s c h a p t e r . R e s u l t s . A summary o f e x p e r i m e n t a l r e s u l t s g i v e n i n F i g u r e 12 shows the p r o g r e s s i v e d e c l i n e o f S u b s t r a t e 1 on i n c u b a t i o n w i t h g l a s s . F o r a l l i n c u b a t e s the a c t i v i t y c o n t a i n e d i n sample D (see above) a t T = 0 r e p r e s e n t e d 100% S u b s t r a t e 1. F o r F i g u r e 12. Rate o f d e p l e t i o n o f s u b s t r a t e f o r g l a s s a c t i v a t e d enzymes ( S u b s t r a t e 1) i n t u r t l e plasma, b y 50 and 2 100 cm g l a s s s u r f a c e / m l . D e t a i l s o f methods a r e g i v e n i n t h e t e x t , p 66. gure 12 P e r c e n t S u b s t r a t e 1 68 i n c u b a t e s #1 and #2 a c t i v i t y i n s u b s e q u e n t samples (D) was e x p r e s s e d as a p e r c e n t o f t h i s maximum a c t i v i t y . F o r i n c u b a t e #3, which c o n t a i n e d k i n i n a s e i n h i b i t o r , t h e k i n i n o g e n r e m a i n  i n g was c a l c u l a t e d as t h e maximum a c t i v i t y p o s s i b l e f o r t h a t plasma ( v a l u e o f D a t T = 0) minus t h e a c t i v i t y i n sample B a t a p a r t i c u l a r time, e x p r e s s e d as a p e r c e n t . I n c u b a t e s #1 and #2, w i t h o u t k i n i n a s e i n h i b i t o r , n e v e r had k i n i n a c t i v i t y a f t e r 2 h o u r s ' i n c u b a t i o n i n t h e absence o f g l a s s (sample B) . 2 10 m i n u t e s ' i n c u b a t i o n w i t h 100 cm g l a s s / m l plasma, o r 20 m i n u t e s ' i n c u b a t i o n w i t h 20 cm /ml was s u f f i c i e n t t o de grade v i r t u a l l y a l l S u b s t r a t e 1. 90% o f t h i s s u b s t r a t e was 2 d e p l e t e d by 5 m i n u t e s ' i n c u b a t i o n w i t h 100 cm , o r 10 mi n u t e s 2 w i t h 50 cm g l a s s / m l . T u r t l e plasma t r e a t e d i n t h i s f a s h i o n , i f s i m i l a r t o human plasma, s h o u l d s t i l l c o n t a i n l a r g e amounts of p r e - k a l l i k r e i n , c a p a b l e o f a c t i v a t i o n by a d d i t i o n o f f r e s h g l a s s s u r f a c e . I t i s a p p a r e n t f rom F i g u r e 12 t h a t t h e r e i s l i t t l e d i f f e r  ence i n r a t e s o f k i n i n o g e n d e p l e t i o n i n t h e p r e s e n c e o r ab sence o f 8-HQSO4. T h i s s u g g e s t s t h a t the amounts o f k i n i n a s e i n h i b i t o r used t h r o u g h o u t t h i s work d i d n o t s i g n i f i c a n t l y m o d i f y the k i n e t i c s o f t u r t l e k i n i n r e l e a s e . A l l t h r e e c u r v e s f o r k i n i n o g e n d e p l e t i o n a r e the i n v e r s e o f the c o r r e s p o n d i n g c u r v e s f o r k i n i n p r o d u c t i o n . F i g u r e 13 F i g u r e 13. E f f e c t o f a 2 hour i n c u b a t i o n o f samples a f t e r r e m o v a l o f g l a s s on the p r o p o r t i o n o f k i n i n r e - 2 l e a s e d f r o m t u r t l e plasma by 100 cm ( s o l i d d o ts) 2 and 50 cm ( h o l l o w d o t s ) / m l plasma. S o l i d l i n e s i n d i c a t e samples w h i c h were i n a c t i v a t e d immediate l y , and dashed l i n e s i n d i c a t e t h o s e samples which were i n c u b a t e d f o r 2 h o u r s a t 20°C a f t e r t h e r e  moval o f g l a s s . D e t a i l s a r e g i v e n i n the t e x t , p 68, 70. 69 F i g u r e 13 Percent Kinin Released cn o 70 compares t h e p r o p o r t i o n o f k i n i n p r o d u c e d b y t h e two methods, a t d i f f e r e n t t i m e i n t e r v a l s . S o l i d l i n e s a r e b a s e d on i n f o r m a  t i o n p r e s e n t e d i n F i g u r e 10. D a t a f o r p e r c e n t S u b s t r a t e 1 d e p l e t e d i n F i g u r e 12 w e re t r a n s l a t e d i n t o k i n i n p r o d u c e d , and a r e c o n n e c t e d by d a s h e d l i n e s i n F i g u r e 13. The c o n t r o l i n c u b a t e i n F i g u r e 12 ( w i t h 8-HQSO^) was n o t i n c l u d e d . I t i s c l e a r t h a t t h o s e s a m p l e s i n c u b a t e d f o r 2 h o u r s a f t e r r d m o v a l o f g l a s s ( dashed l i n e s ) c o n t a i n e d o n l y s l i g h t l y more a c t i v i t y t h a n t h o s e w h i c h were b o i l e d i m m e d i a t e l y upon r e m o v a l o f t h e sample ( s o l i d l i n e s ) . T h i s i m p l i e s a r a p i d i n a c t i v a t i o n o f p l a s m a k a l l i k r e i n ; o t h e r w i s e a much l a r g e r p o r t i o n o f t h e k i n i n o g e n w o u l d be d e g r a d e d a f t e r t h e r e m o v a l o f g l a s s , b y k a l l i k r e i n r e l e a s e d i n t h e i n i t i a l i n c u b a t i o n . 2. A c t i v a t i o n o f p l a s m a k a l l i k r e i n i n t u r t l e and r a t p l a s m a . The p u r p o s e o f t h i s e x p e r i m e n t was t o compare r e l a  t i v e amounts, and r a t e s o f a c t i v a t i o n , o f p r e k a l l i k r e i n i n t u r t l e and mammalian ( r a t ) p l a s m a . G l a s s a c t i v a t i o n o f p r e  k a l l i k r e i n p r e p a r a t i o n s was f o l l o w e d , b y p e r i o d i c a l l y r e  m o v i n g samples and r e a c t i n g them w i t h a s t a n d a r d amount o f s u b s t r a t e . I t was assumed t h a t t h e amount o f k i n i n t h u s p r o  duced w o u l d be p r o p o r t i o n a l t o t h e amount o f a c t i v e p l a s m a k a l l i k r e i n added t o t h e s u b s t r a t e . Method. The p r e k a l l i k r e i n p r e p a r a t i o n u s e d h e r e was S u b s t r a t e 71 d e p l e t e d plasma; i t was p r e p a r e d as d e s c r i b e d i n the p r e v i o u s e x p e r i m e n t . T u r t l e plasma was d e p l e t e d f o r 5 and 10 m i n u t e s 2 2 w i t h 100 cm g l a s s / m l , and r a t plasma was exposed t o 100 cm g l a s s / m l f o r 10 mi n u t e s o n l y . The amount o f S u b s t r a t e 1 r e  m a i n i n g i n t h e s e p r e p a r a t i o n s a f t e r d e p l e t i o n was d e t e r m i n e d by t h e p r o c e d u r e o u t l i n e d on page 66 (samples C and D). A s t a n d a r d s u b s t r a t e was p r e p a r e d b y d i l u t i n g i n t a c t t u r t l e o r r a t plasma 1:2 w i t h t h e a p p r o p r i a t e s a l i n e , w h i c h a l s o c o n t a i n e d 1 mg 8-HQSO^/ml plasma. A t T = 0, 10 ml o f each p r e k a l l i k r e i n p r e p a r a t i o n was a c t i v a t e d i n a 50 ml p o l y p r o p y - l e n e c e n t r i f u g e tube w i t h 16 g g l a s s beads (200 cm / m l ) . A t the d e s i r e d t i m e i n t e r v a l s 0-5 ml samples were withdrawn and r a p i d l y i n j e c t e d i n t o 1.5 ml o f the s u b s t r a t e p r e p a r a t i o n , i n 5 ml p l a s t i c t e s t tubes- These m i x t u r e s were i n c u b a t e d a t 20°C f o r 60 m i n u t e s t o e n s u r e c o m p l e t i o n o f t h e r e a c t i o n , t h e n sampled and a s s a y e d i n t h e u s u a l manner (Chapter 2 ) . The maximum y i e l d o f t h e s u b s t r a t e p r e p a r a t i o n s was d e t e r m i n e d i n each c a s e b y a s e p a r a t e i n c u b a t i o n w i t h g l a s s and k i n i n a s e i n h i b i t o r , f o r 60 m i n u t e s , a t 20°C. In c a s e s where the p r e k a l l i k r e i n p r e p a r a t i o n i t s e l f c o n t a i n e d r e s i d u a l S u b s t r a t e 1 (up t o 10% i n 5 m i n u t e - d e p l e t e d t u r t l e plasma) t h e maximum y i e l d o f the s u b s t r a t e was i n c r e a s e d t o i n c l u d e t h i s amount. R e s u l t s . Data from 8 e x p e r i m e n t s a r e g i v e n i n F i g u r e 14 a 14 a- The r e l e a s e o f a c t i v e k a l l i k r e i n f r o m r a t and t u r t l e p r e k a l l i k r e i n p r e p a r a t i o n s a c t i v a t e d w i t h 200 cm g l a s s s u r f a c e / m l plasma. R e s u l t s a r e e x p r e s s e d as p e r c e n t S u b s t r a t e 1 i n t h e s t a n d a r d s u b s t r a t e p r e p a r a t i o n s d e g r a d e d b y a l i q u o t s o f the a c t i v a t e d , k a l l i k r e i n a t d i f f e r e n t i n c u b a t i o n t i m e s . D e t a i l s o f t h e method a r e g i v e n on page 71. V a l u e s f o r r a t and t u r t l e (5 min) a r e means o f 2 e x p e r i m e n t s , and v a l u e s f o r t u r t l e , 10 min, a r e means o f 4 e x p e r i m e n t s . b. Decay o f a c t i v e t u r t l e k a l l i k r e i n . K a l l i k r e i n was r e l e a s e d from a p r e k a l l i k r e i n p r e p a r a t i o n b y a c t i v a t i o n w i t h 200 cm g l a s s / m l plasma f o r 10 m i n u t e s . D i s a p p e a r a n c e o f a c t i v i t y a f t e r r e moval o f t h e g l a s s was f o l l o w e d by r e a c t i n g samples w i t h s t a n d a r d amounts o f s u b s t r a t e . V a l u e s a r e means f o r two e x p e r i m e n t s . 72 F i g u r e 14 a I n c u b a t i o n Time, min 73 e x p r e s s e d as t h e p e r c e n t o f t h e t h e o r e t i c a l maximum a c t i v i t y - r e l e a s e d f r o m t h e substra-te b y each o f t h e a c t i v a t e d samples. C o n t r o l s w i t h n o n - a c t i v a t e d p r e k a l l i k r e i n and s u b s t r a t e plasma d i d n o t c o n t a i n k i n i n a c t i v i t y . M a r g o l i s (1958) and B r i s e i d e t a l (1968) f o u n d t h a t human plasma c o n t a i n e d p r e k a l l i k r e i n f a r i n e x c e s s o f t h e amount r e q u i r e d t o degrade a l l k i n i n o g e n p r e s e n t a t any t i m e . I t i s e v i d e n t from F i g u r e 14 a t h a t t h i s s i t u a t i o n a l s o e x i s t s i n b o t h r a t and t u r t l e plasma. A c t i v a t i o n by 200 cm g l a s s / m l plasma r a p i d l y r e l e a s e d k a l l i k r e i n , and a t 10 m i n u t e s t h e c o n  c e n t r a t i o n was s u f f i c i e n t t o degrade a l l S u b s t r a t e 1 i n the s u b s t r a t e samples. T h i s l e v e l o r g r e a t e r was m a i n t a i n e d f o r about 40 m i n u t e s , a f t e r w hich t h e r e was a r e l a t i v e l y s l o w d e c r e a s e i n k a l l i k r e i n a c t i v i t y . A f t e r 120 m i n u t e s t h e r e was s t i l l enough enzyme p r e s e n t , t o r e l e a s e about h a l f t h e a v a i l a b l k i n i n . The f a c t t h a t p r e k a l l i k r e i n was p r e s e n t i n e x c e s s i s u n d e r l i n e d by the f o l l o w i n g f a c t s : a) P a r t o f the s u p p l y had a l r e a d y been u s e d i n d e p l e t i n g S u b s t r a t e 1 i n t h e p r e k a l l i k r e i n p r e p a r a t i o n (see p 66); b) I n any t e s t i n c u b a t i o n t h e s u b s t r a t e p r e p a r a t i o n was d e r i v e d from t h r e e times as much plasma as t h e k a l l i k r e i n used t o a c t i v a t e i t ; c) H i g h c o n c e n t r a t i o n s o f a c t i v e k a l l i k r e i n e x i s t e d d e s p i t e s i m u l t a n e o u s i n a c t i v a t i o n . 74 Mammalian plasma has more than one t y p e o f k a l l i k r e i n h e l d i n an i n a c t i v e f o r m "(Schachter, 1969) . I f t u r t l e plasma i s s i m i l a r , i t i s p o s s i b l e t h a t t h e k a l l i k r e i n a c t i v i t y a s s a y e d i n , t h e above e x p e r i m e n t s i s due t o more t h a n one enzyme, and t h a t t h e r e l a t i v e p r o p o r t i o n o f t h e s e enzymes changes w i t h c o n t i n u e d a c t i v a t i o n . F o r example, i t c a n n o t be r u l e d o u t t h a t the k a l l i k r e i n r e s p o n s i b l e f o r k i n i n r e l e a s e a t 120 m i n u t e s might be e n t i r e l y d i f f e r e n t f r o m t h a t w hich c a u s e s the r a p i d k i n i n p r o d u c t i o n when g l a s s beads a r e added t o f r e s h plasma (e.g.. F i g u r e 1 0 ) . 3. Decay o f plasma k a l l i k r e i n . A c c o r d i n g t o S c h a c h t e r (1969), t h e plasma o r serum o f a l l v e r t e b r a t e s t e s t e d t o d a t e , as w e l l as t h e hemolymph o f some i n v e r t e b r a t e s , has t h e a b i l i  t y to i n a c t i v a t e o r i n h i b i t a l l v a r i e t i e s o f k a l l i k r e i n . T h e r e a r e a t l e a s t two o f t h e s e i n a c t i v a t o r s i n mammalian plasma, one o f which may be an enzyme (Werle and Schmal, 1968). The e f f i c i e n c y o f t h e s e i n h i b i t o r s i s o b v i o u s l y an i m p o r t a n t v a r i a b l e i n a k a l l i k r e i n - k i n i n system, s i n c e t h e c o n c e n t r a t i o n o f k a l l i k r e i n i n t h e plasma a t any time w i l l depend on t h e r a t e s o f a c t i v a t i o n and des t r u c t i o n o f t h e enzyme. I t was the o b j e c t o f t h i s e x p e r i m e n t t o c h a r a c t e r i z e t h e decay o f t u r t l e plasma k a l l i k r e i n , i n the absence o f s i m u l t a n e o u s p r o  d u c t i o n o f the enzyme. Method. S u b s t r a t e 1 d e p l e t e d plasma was p r e p a r e d as a s o u r c e 75 o r p r e k a l l i k r e i n , f r e e o f e x t r a k i n i n o g e n . - 5 ml were r e - a c t i v a t - 2 ed w i t h 8 g g l a s s beads (200 cm /ml plasma) f o r 10 m i n u t e s . T h i s e n s u r e d t h a t s u f f i c i e n t k a l l i k r e i n would be r e l e a s e d t o p roduce maximum amounts o f k i n i n from th e s u b s t r a t e ( a c c o r d  i n g t o F i g u r e 14 a) . A t z e r o t ime, 4 ml o f t h e s u p e r n a t a n t plasma were removed i n a 5 ml p l a s t i c s y r i n g e and p l a c e d i n a 50 ml p o l y p r o p y l e n e c e n t r i f u g e t u b e . A t t h e d e s i r e d time i n t e r v a l s 0.5 ml samples were withdrawn from t h i s m i x t u r e i n s t e r i l e 1 ml s y r i n g e s and added t o 1.5 ml samples o f s u b s t r a t e (as i n e x p e r i m e n t 2 ) . A f t e r one hour t h e s e m i x t u r e s were sampled and a s s a y e d . Maximum v a l u e s f o r k i n i n o g e n were d e t e r m i n e d i n a s e p a r a t e i n c u b a t i o n . R e s u l t s . Means f o r two e x p e r i m e n t s on the plasma o f d i f f e r e n t t u r t l e s a r e shown i n F i g u r e 14 b. Enough k a l l i k r e i n was p r e s e n t i n i t i a l l y t o degrade a l l i t s s u b s t r a t e . Decay was r a p i d i n t h e absence o f g l a s s ; a f t e r 15 m i n u t e s a t 20°C , t h e a c t i v i t y was so r e d u c e d t h a t no k i n i n was r e l e a s e d from the s u b s t r a t e . T h i s r a p i d i n h i b i t i o n i s o f the same o r d e r as t h a t d e s c r i b e d f o r human plasma ( M a r g o l i s , 1958). The p o t e n c y o f t h e s e i n a c t i v a t o r s i n t u r t l e plasma, com b i n e d w i t h the r a p i d l i b e r a t i o n o f plasma k a l l i k r e i n (compared i n F i g u r e s 14 a & b) s u g g e s t s t h a t the c o n v e r s i o n o f p r e  k a l l i k r e i n t o k a l l i k r e i n i s v e r y r a p i d i n d e e d , and t h a t amounts o f p r e k a l l i k r e i n may be even g r e a t e r t han the p r e v i o u s e x p e r i  ment seemed t o s u g g e s t . 76 D. E f f e c t o f Temperature on K i n i n P r o d u c t i o n Many p o i k i l o t h e r m enzymes have been f o u n d t o p o s s e s s , a t low temperatures,-- d i f f e r e n t k i n e t i c s t h a n t h e i r mammalian c o u n t e r p a r t s . I t seemed p o s s i b l e t h a t a t low t e m p e r a t u r e s k i n i n p r o d u c t i o n i n t h e t u r t l e , a p o i k i l o t h e r r t y m i g h t be more e f f i c i e n t t h a n i n t h e r a t , a homeotherm. K i n i n p r o d u c t i o n i n plasma from t h e s e a n i m a l s was t h e r e f o r e t e s t e d a t t h r e e d i f f e r e n t t e m p e r a t u r e s ; 37°C, a n o r m a l mammalian t e m p e r a t u r e ; o 20 C, a normal t e m p e r a t u r e f o r t h i s t u r t l e ( C a r r , 1952); and 5°C, a t e m p e r a t u r e a l s o e n c o u n t e r e d b y t h e t u r t l e . Method. 5 ml o f plasma from each a n i m a l was d i l u t e d . 1:2 w i t h s a l i n e (Appendix B) c o n t a i n i n g 1 mg 8-HQS0 4/ml u n d i l u t e d p l a s - .ma. T h i s m i x t u r e was p l a c e d i n 50 ml p o l y p r o p y l e n e t e s t t ubes i n a water b a t h p r e v i o u s l y e q u i l i b r a t e d a t 5, 20 o r 37°C. A t 2 time z e r o 6 g g l a s s beads (50 cm /ml) were added t o each tube and s u b j e c t e d t o c o n t i n u o u s s h a k i n g . A t t h e d e s i r e d i n t e r v a l s , samples were withdrawn, i n a c t i v a t e d by b o i l i n g , and s t o r e d f o r a s s a y . C o n t r o l i n c u b a t e s c o n t a i n i n g a) no g l a s s , and b) no i n  h i b i t o r , were i n c l u d e d f o r b o t h plasmas i n each e x p e r i m e n t . R e s u l t s . A b s o l u t e v a l u e s f o r k i n i n p r o d u c t i o n i n r a t and t u r  t l e plasma a r e g i v e n i n T a b l e 4. Rat..plasma u s u a l l y p r o d u c e d h i g h e r c o n c e n t r a t i o n s o f plasma k i n i n . T h e r e f o r e , t o a l l o w d i r e c t comparison o f t h e k i n e t i c s o f k i n i n r e l e a s e , d a t a a r e 77 T a b l e 4 . K i n i n p r o d u c t i o n i n r a t and t u r t l e p l a s m a a t d i f f e r  e n t t e m p e r a t u r e s , b y 50 cm 2  g l a s s / m l p l a s m a . A c t i v i t y (ug/ml p l a s m a , BK-eq) T e m p e r a t u r e S p e c i e s a t d i f f e r e n t s a m p l i n g t i m e s (min) _0 _1 2 5_ 10 30 _60 37°C R a t (2) * 0.01 0.7 1.5 1.7 1. 7 1.6 1. 6 T u r t l e (2) 0.01 0.5 1.2 1.3 1.3 1.2 1.2 o 20 C R a t (2) 0.01 0.3 0.8 1.2 1.5 1.5 T u r t l e (3) 0.01 0.05 0.1 0.3 0.7 1.0 1.0 5°C Rat (2) 0.01 0.01 0.01 0.07 0.4 1.2 1.3 T u r t l e (3) 0.01 0.01 0.01 0.06 0.4 1.7 1. 6 * v a l u e s i n p a r e n t h e s e s i n d i c a t e t h e number o f a n i m a l s u s e d i n d i f f e r e n t e x p e r i m e n t s t o o b t a i n t h e mean v a l u e s p r e s e n t e d i n t h i s t a b l e . 7 8 e x p r e s s e d i n F i g u r e 1 5 as p e r c e n t o f t h e maximum amount o f k i n i t h a t c o u l d be r e l e a s e d . Comparison o f t h e s e c u r v e s does n o t i n d i c a t e t h a t t h e r e was a d i f f e r e n c e i n t h e r e l a t i v e r a t e s a t which k i n i n was p r o d u c e d i n r a t and t u r t l e plasma, a t any o f the t e m p e r a t u r e s . Maximum v e l o c i t i e s o f k i n i n p r o d u c t i o n a t each tempera t u r e were c a l c u l a t e d f r o m F i g u r e 1 5 , u s i n g d a t a f r o m T a b l e 4. The Q-|_Q v a l u e s f o r r a t and t u r t l e k i n i n p r o d u c t i o n d i d n o t d i f f e r , b e i n g 1 . 9 o v e r t h e ra n g e 2 0 t o 3 7 ° C , and 1 . 3 from 5 t o 2 0 C. The form e r v a l u e i s i n good agreement w i t h Q]_Q v a l u e s c a l c u l a t e d b y M a r g o l i s ( 1 9 6 6 ) f o r human plasma o v e r the same range. I c o u l d f i n d no p u b l i s h e d v a l u e s f o r Q-,Q o f k i n i n p r o  d u c t i o n r a t e i n the 5 t o 2 0 ° C r a n g e . K i n i n p r o d u c t i o n i n b o t h plasmas had a d e f i n i t e l a g a t o 5 C; no k i n x n appeared u n t i l a t l e a s t 5 m i n u t e s o f i n c u b a t i o n , a f t e r which t h e r e was a r e l a t i v e l y r a p i d r e l e a s e o f k i n i n . T h i s was r e f l e c t e d i n t h e low Q-^Q f o r maximum r a t e o f k i n i n r e l e a s e a t low t e m p e r a t u r e s . T h i s l a g , o r t r a n s i e n t phase, i s a p r e d i c t a b l e c h a r a c t e r i s t i c o f a damped cascad e enzymic p r o  c e s s (Hemker and Hemker, 1 9 6 9 ) , and f u r t h e r s u p p o r t s the h y p o t h e s i s t h a t the k a l l i k r e i n - k i n i n s ystem o f b o t h t u r t l e and mammalian plasma may f i t t h i s model. E. K i n i n o g e n s i n T u r t l e Plasma I t has been mentioned (Chapter 1) t h a t t h e r e a r e a t l e a s t F i g u r e 15. E f f e c t o f t e m p e r a t u r e on g l a s s t r i g g e r e d k i n i n p r o d u c t i o n i n r a t and t u r t l e plasma. Both plasmas were t e s t e d a t 5, 20, and 37°C. K i n i n r e l e a s e d a t a p a r t i c u l a r s a m p l i n g time i s e x p r e s s e d as p e r c e n t o f t h e maximum p o s s i b l e , b a s e d on d a t a g i v e n i n T a b l e 4. V a l u e s a r e means o f 2 o r 3 e x p e r i m e n t s , as i n d i c a t e d i n T a b l e 4. F i g u r e 15 75t Turtle Plasma c 37°C 25h 10 30 Incubation Tim* - M i n t . 30 60 80 two k i n i n o g e n s i n mammalian p l a s m a , and t h a t t h e r e l a t i v e e f f i c i e n c y o f a w i d e number o f k a l l i k r e i n s and k i n i n o g e n a s e s i n r e l e a s i n g k i n i n s f r o m t h e s e p r e c u r s o r s i s a complex t o p i c , c u r r e n t l y a s u b j e c t f o r much r e s e a r c h (Hamberg, 1 9 6 9 ) . S i n c e i t was t h e aim o f t h i s s t u d y t o c h a r a c t e r i z e k i n i n r e l e a s e by t h e e n t i r e k a l l i k r e i n - k i n i n s y s t e m , an e x h a u s t i v e s t u d y o f t h e k i n i n o g e n complex p e r se i n t u r t l e p l a s m a was n o t c a r r i e d o u t . The e x p e r i m e n t s w i t h t r y p s i n and g l a s s s u r f a c e a c t i v a  t i o n d e s c r i b e d i n t h i s s e c t i o n were c o n d u c t e d t o d e t e r m i n e t h e p r o p o r t i o n o f t h e t o t a l k i n i n o g e n w h i c h i s a v a i l a b l e t o s u r f a c e a c t i v a t e d endogenous enzymes, and t o e s t a b l i s h more c l e a r l y w h e t h e r t h e d i f f e r e n t amounts o f k i n i n r e l e a s e d by t r y p s i n and g l a s s ( T a b l e 3) were due t o d i f f e r e n t k i n i n o g e n s . 1. E f f e c t o f t r y p s i n c o n c e n t r a t i o n on t u r t l e k i n i n r e l e a s e i n i n t a c t p l a s m a . Low c o n c e n t r a t i o n s o f t r y p s i n (0.1 mg/ml plasma) r e l e a s e k i n i n f r o m human p l a s m a i n an i n i t i a l r a p i d p h a s e , f o l l o w e d b y a p e r i o d o f s l o w e r p r o d u c t i o n . M a r g o l i s and B i s h o p (1963) have t a k e n t h i s phenomenon t o i n d i c a t e t h e p r e s e n c e o f two k i n i n o g e n m o i e t i e s , one a b e t t e r s u b s t r a t e f o r t r y p s i n t h a n t h e o t h e r . T h e r e f o r e , e x p e r i m e n t s s i m i l a r t o t h o s e o f M a r g o l i s and B i s h o p were c o n d u c t e d w i t h t u r t l e p l a s m a , and t h e r e s u l t s compared t o t h i s d a t a f o r human p l a s m a . Method. Plasma was i n c u b a t e d a t 37°C w i t h s a l i n e , 8-HQSO4 and 81 t r y p s i n , i n t h e f o l l o w i n g p r o p o r t i o n s : T r y p s i n P l a s m a S a l i n e (pH - 7 . 4 ) 8-HQS0 A 1. 2 mg 2 ml 4 ml 2 mg 2. 0.2 mg 2 ml 4 ml 2 mg A t a p p r o p r i a t e t i m e s s a m p l e s were removed f r o m e a c h t u b e , b o i l e d and s t o r e d f o r a s s a y ( C h a p t e r 2 ) . The e x p e r i m e n t was r e p e a t e d on p l a s m a f r o m t h r e e t u r t l e s , and c o n t r o l s c o n t a i n i n g a) no k i n i n a s e i n h i b i t o r , o r b) no p l a s m a , o r c) no t r y p s i n , b u t o t h e r w i s e s i m i l a r t o t h e t e s t i n c u b a t e s , were i n c l u d e d i n e a c h e x p e r i m e n t . R e s u l t s . None o f t h e c o n t r o l s c o n t a i n e d b i o l o g i c a l a c t i v i t y as measured on t h e i s o l a t e d r a t u t e r u s . F i g u r e 16 shows k i n i n p r o d u c t i o n i n t h e e x p e r i m e n t a l i n c u b a t e s , w i t h t h e two c o n  c e n t r a t i o n s o f t r y p s i n . Each p o i n t i s a mean, b a s e d on t h r e e v a l u e s . The r e s u l t s a r e s i m i l a r t o t h o s e o b t a i n e d f o r a c i d - t r e a t e d human p l a s m a by M a r g o l i s and B i s h o p ( 1 9 6 3 ) . 0.1 mg t r y p s i n r e l e a s e d k i n i n much more s l o w l y t h a n d i d 1.0 mg t r y p s i n / m l . The c u r v e f o r t u r t l e k i n i n p r o d u c t i o n b y 0.1 mg t r y p s i n / ml p l a s m a a p p e a r s t o be r e l a t i v e l y smooth, and does n o t show any s h a r p i n f l e c t i o n p o i n t , as f o u n d b y M a r g o l i s and B i s h o p . N o n e t h e l e s s , t h e i n i t i a l r a p i d r a t e o f p r o d u c t i o n f o l l o w e d by l a t e r s l o w e r k i n i n r e l e a s e ( a f t e r 10 m i n u t e s ) , m i g h t be i n t e r p r e t e d as s u p p o r t f o r t h e two k i n i n o g e n h y p o t h e s i s . F i g u r e 16. Comparison o f r a t e and y i e l d o f k i n i n p r o d u c t i o n i n unheated plasma by two c o n c e n t r a t i o n s o f t r y p s i n ; 1.0 and 0.1 mg/ml u n d i l u t e d plasma. V a l u e s a r e means f o r t h r e e e x p e r i m e n t s . 82 Figure 16 I n c u b a t i o n T i m e , M m 83 A c c o r d i n g t o F i g u r e 2, t r y p s i n a c t i v a t e s plasma k a l l i  k r e i n i n i n t a c t plasma (Webster, 1968), i n a d d i t i o n t o r e  l e a s i n g k i n i n d i r e c t l y f r o m k i n i n o g e n . I n h e a t e d plasma, on the o t h e r hand, a l l enzymic components have been i n a c t i v a t e d , and t r y p s i n a c t s o n l y on k i n i n o g e n . T h e r e f o r e , i f t u r t l e p l a s  ma i s s i m i l a r , e q u i v a l e n t c o n c e n t r a t i o n s o f t r y p s i n s h o u l d r e l e a s e k i n i n more r a p i d l y from i n t a c t t h a n from h e a t e d plasma. F i g u r e 17 compares r a t e s o f k i n i n r e l e a s e f r o m h e a t e d and un h e a t e d t u r t l e plasma b y 1.0 mg t r y p s i n / m l plasma. V a l u e s f o r h e a t e d plasma were t a k e n from F i g u r e 3 a, and t h o s e f o r un h e a t e d plasma from F i g u r e 16. In o r d e r t o a i d i n c o m p a r i s o n a l l d a t a a r e e x p r e s s e d as p e r c e n t o f t h e maximum k i n i n r e  l e a s e d . S i x d i f f e r e n t a n i m a l s were i n v o l v e d . I t i s c l e a r t h a t t u r t l e k i n i n was r e l e a s e d more r a p i d l y from i n t a c t plasma. The r e s u l t s i n t h i s F i g u r e t h e r e f o r e s u g g e s t t h a t t u r t l e plasma may be s i m i l a r t o mammalian plasma i n t h a t t r y p s i n c o n v e r t s p r e k a l l i k r e i n t o k a l l i k r e i n i n unheated plasma. 2• E f f e c t o f t r y p s i n on S u b s t r a t e 1 - d e p l e t e d plasma. A l t h o u g h p r e v i o u s e x p e r i m e n t s had e s t a b l i s h e d t h a t t r y p s i n always r e l e a s e d more t u r t l e k i n i n t h an d i d g l a s s s u r f a c e from a g i v e n sample of t u r t l e plasma, the two k i n i n o g e n h y p o t h e s i s c o u l d be t e s t e d more d i r e c t l y by d e t e r m i n i n g whether plasma d e p l e t e d o f s u b s t r a t e f o r g l a s s a c t i v a t e d k a l l i k r e i n s c o u l d s t i l l r e l e a s e t u r t l e k i n i n t o t r y p s i n . The e x p e r i m e n t d e s c r i b e d F i g u r e 1 7 . Comparison o f r a t e and y i e l d o f k i n i n p r o d u c t i o n i n u n h e a t e d and h e a t e d (to 65°C) t u r t l e plasma, by 1 . 0 mg t r y p s i n / m l u n d i l u t e d plasma. V a l u e s a r e means f o r t h r e e e x p e r i m e n t s . 84 Figure 17 I 85 h e r e was d e s i g n e d t o c a r r y o u t t h i s t e s t , and t o compared t h e r e s p o n s e o f t u r t l e plasma d i r e c t l y t o t h a t o f r a t plasma. Method. In each e x p e r i m e n t e v e r y sample o f plasma was t r e a t e d - i n 6 d i f f e r e n t ways: 1. Maximum k i n i n r e l e a s e d t o t r y p s i n . T h i s was d e t e r m i n e d by i n c u b a t i n g 2 ml plasma + 4 ml s a l i n e + 2 mg 8-HQS0 4 + 2 mg t r y p s i n i n s i l i c o n e d g l a s s t e s t t u b e s a t 37°C. A t 30 m i n u t e s , t h e s e were sampled and a s s a y e d i n t h e u s u a l manner. 2. Maximum k i n i n r e l e a s e d by g l a s s . 2 ml plasma were i n c u b a t e d w i t h 4 ml s a l i n e + 2 mg 8-HQSO4 and shaken a t 20°C w i t h 2.4 g g l a s s beads (50 cm /ml p l a s m a ) . A f t e r 50 m i n u t e s t h e s e tubes were sampled and a s s a y e d . 3. K i n i n r e l e a s e d by t r y p s i n f r o m g l a s s - d e p l e t e d plasma. a) 2 ml plasma was d i l u t e d w i t h 4 ml s a l i n e and shaken a t 20°C w i t h 2.4 g g l a s s beads f o r 50 m i n u t e s . No 8- HQS0 4 was added. b) 4 ml o f t h e m i x t u r e o b t a i n e d from #3 a was p l a c e d i n a s i l i c o n e d g l a s s tube and i n c u b a t e d w i t h 1.3 mg 8-HQSO^ and 1.3 mg t r y p s i n , a t 37°C. A f t e r 30 mi n u t e s t h i s i n c u b a t e was sampled, and a s s a y e d . 4. K i n i n r e l e a s e d by t r y p s i n f r o m t r y p s i n - d e p l e t e d plasma. a) 2 ml plasma was d i l u t e d w i t h 4 ml s a l i n e and r e a c t e d w i t h 2 mg t r y p s i n i n a s i l i c o n e d g l a s s tube a t 37°C. No 8-HQSO4 was added. 86 b) A f t e r 30 m i n u t e s , 4 ml o f t h e m i x t u r e f r o m #4 a was p l a c e d i n a f r e s h g l a s s tube, and r e - i n c u b a t e d a t 37°C w i t h 1.3 mg 8-HQSO4 and 1.3 mg t r y p s i n . A f t e r 30 m i n u t e s t h i s •tube was sampled and a s s a y e d . 5. K i n i n r e l e a s e d by g l a s s f r o m t r y p s i n - d e p l e t e d plasma. 4 ml o f t r y p s i n - d e p l e t e d plasma p r e p a r a e d as i n #4 a was p l a c e d i n a 50 ml p o l y p r o p y l e n e tube w i t h 1.3 mg 8-HQSO^ and 1.6 g g l a s s beads and shaken a t 20°C f o r 50 m i n u t e s , sampled and a s s a y e d . 6. K i n i n r e l e a s e d , by g l a s s f r o m g l a s s - d e p l e t e d plasma. 4 ml o f g l a s s - d e p l e t e d plasma, p r e p a r e d as i n #3 a was i n  cubated, w i t h 1.3 mg 8-HQSO^ and 1.6 g g l a s s , as d e s c r i b e d i n #5 b. R e s u l t s . Means f o r 7 e x p e r i m e n t s on t u r t l e plasma and 5 e x p e r i  ments on r a t plasma a r e g i v e n i n F i g u r e 18. The r e s u l t s a r e e x p r e s s e d as p e r c e n t o f maximum k i n i n r e l e a s e d , b a s e d on t h e v a l u e o f t u r t l e k i n i n d e v e l o p e d by t r y p s i n ( i n c u b a t e #1, above) from any plasma. In b o t h plasmas k i n i n r e l e a s e d by g l a s s (G-l) formed a r e l a t i v e l y c o n s t a n t p r o p o r t i o n o f t h e t o t a l r e l e a s e d by t r y p s i n ; about 75% i n the r a t , 65% i n the t u r t l e . Plasma d e p l e t  ed o f s u b s t r a t e f o r g l a s s - a c t i v a t e d enzymes always r e l e a s e d a d d i t i o n a l k i n i n when r e - i n c u b a t e d "with t r y p s i n . T h i s amount o f k i n i n , a bout 2 9% i n the r a t , and 40% i n the t u r t l e , was a p p r o x i m a t e l y e q u a l t o the d i f f e r e n c e between r e l a t i v e amounts F i g u r e 18. R e l a t i v e e f f e c t s of t r y p s i n and g l a s s i n r e l e a s i n g k i n i n from i n t a c t t u r t l e and r a t plasmas. C o n d i t i o n s of i n c u b a t i o n are d e s c r i b e d i n the t e x t , p 85-86. Values f o r t u r t l e and r a t plasma are based on 7 and 5 experiments r e s p e c t i v e l y . T - l t r y p s i n G-l g l a s s s u r f a c e T-2 g l a s s s u r f a c e f o l l o w e d by t r y p s i n T-3 t r y p s i n f o l l o w e d by t r y p s i n G-2 t r y p s i n f o l l o w e d by g l a s s s u r f a c e G-3 g l a s s s u r f a c e f o l l o w e d by g l a s s s u r f a c e 87 F i g u r e 18 88 o f t r y p s i n and g l a s s r e l e a s e d k i n i n (column T - l minus column G - l ) . A l t h o u g h t h e r e were no s i g n i f i c a n t d i f f e r e n c e s i n t h e s e p r o p o r t i o n s between r a t ' and t u r t l e plasma, i t i s p o s s i b l e t h a t a l a r g e r sample s i z e m i g h t i n d i c a t e t h a t t u r t l e plasma r e l e a s e s r e l a t i v e l y l e s s k i n i n t o g l a s s t h a n r a t plasma. In b o t h plasmas the r e s i d u a l k i n i n o g e n (column T -2) was s i g n i f i c a n t l y l e s s t h a n the amount d e g r a d e d by g l a s s a c t i v a t e d enzymes (column G-l) . On the o t h e r hand, g l a s s d e p l e t e d plasma developed, an i n s i g n i f i c a n t amount o f e x t r a k i n i n (column G-2) when r e - i n c u b a t e d w i t h f r e s h g l a s s . A l s o , t r y p s i n depleted plasma d i d n o t y i e l d s i g n i f i c a n t k i n i n a c t i v i t y when r e - i n c u b a t e d w i t h g l a s s (column G-3) o r w i t h f r e s h t r y p s i n (column T - 3 ) . These f a c t s s u g g e s t t h a t i n b o t h plasmas some k i n i n may e x i s t i n a p r e c u r s o r l i n k a g e w h i c h i s n o t a v a i l a b l e t o g l a s s a c t i v a t e d enzymes; t h i s a p p e a r s t o s u p p o r t the h y p o t h e s i s t h a t t u r t l e plasma, l i k e mammalian plasma, c o n t a i n s a t l e a s t two m o l e c u l a r s p e c i e s o f k i n i n o g e n . However, i t i s n o t p o s s i b l e on t h e b a s i s o f t h e s e ex p e r i m e n t s a l o n e t o e l i m i n a t e an a l t e r n a t i v e h y p o t h e s i s t o e x p l a i n the p a r t i a l e x h a u s t i o n o f k i n i n o g e n by g l a s s ; i . e . , t h a t some f a c t o r e s s e n t i a l t o the r e a c t i o n may e x i s t i n a l i m i t e d s u p p l y , and d i s a p p e a r r a p i d l y on a c t i v a t i o n w i t h g l a s s . However, t h i s i s n o t the c a s e f o r mammalian plasma 89 ( M a r g o l i s and B i s h o p , 1 9 6 3 ) . F. D i s c u s s i o n and C o n c l u s i o n s The s i g n i f i c a n c e o f e a c h o f t h e e x p e r i m e n t s d e s c r i b e d i n t h i s c h a p t e r h a s b e e n d i s c u s s e d i n d i v i d u a l l y , and t h e g e n e r a l c o n c l u s i o n s w i l l be s u m m a r i z e d i n C h a p t e r 7. B r i e f l y , t u r t l e p l a s m a was t e s t e d q u a l i t a t i v e l y a g a i n s t a m o del o f t h e mammal i a n k a l l i k r e i n - k i n i n s y s t e m as shown i n F i g u r e 2. None o f t h e s e d i r e c t o r i n d i r e c t t e s t s s u g g e s t e d t h a t t h e m o del was n o t e q u a l l y a p p l i c a b l e t o t u r t l e p l a s m a . C e r t a i n o t h e r a s p e c t s o f t h e s y s t e m , s u c h as t h e amount o f p r e k a l l i k r e i n , t h e k i n e t i c s o f t h e i n a c t i v a t i o n o f k a l l i k r e i n , t h e r e s p o n s e o f r e a c t i o n r a t e t o t e m p e r a t u r e , and t h e n a t u r e o f t h e k i n i n o g e n complex were compared d i r e c t l y t o mammalian p l a s m a , a n d no q u a l i t a t i v e d i f f e r e n c e s were n o t e d . I t may be c o n c l u d e d t h a t t u r t l e p l a s m a , i n c o n t r a s t t o snake and b i r d p l a s m a , h a s a f u l l y d e v e l o p e d k a l l i k r e i n - k i n i n s y s t e m c a p a b l e o f p r o d u c i n g up t o 3.0 ug/ml p l a s m a , b r a d y k i n i n - e q u i v a l e n t , a l e v e l c o m p a r a b l e t o some mammals, a n d w h i c h h a s t h e p o t e n t i a l f o r endogenous a c t i v a t i o n . I n f u r t h e r work, more d e t a i l e d s t u d i e s o f t h e v a r i o u s r e a c t i o n s i n v e s t i g a t e d , w i t h t h e u s e o f p u r i f i e d enzymes and s u b s t r a t e s , w o u l d be t h e most f r u i t f u l a p p r o a c h . N o n e t h e l e s s , t h e b a s i c work p r e s e n t e d h e r e must be e x t e n d e d t o a w i d e v a r i e t y o f o t h e r s p e c i e s b e f o r e any g e n e r a l i z a t i o n s can be made. 90 CHAPTER 5. CHEMICAL AND PHARMACOLOGICAL TESTS ON PURIFIED TURTLE K I N I N A. P u r i f i c a t i o n o f G l a s s R e l e a s e d T u r t l e K i n i n The e x p e r i m e n t s d e s c r i b e d i n C h a p t e r 4 i n d i c a t e d t h a t t h e endogenous e n z y m i c mechanisms f o r r e l e a s e o f t u r t l e k i n i n were c l o s e l y s i m i l a r t o t h o s e o f mammals. However, t h i s d i d n o t i m p l y t h a t t h e k i n i n s p r o d u c e d were i d e n t i c a l , a n d t h e r e  f o r e p u r i f i c a t i o n o f t h e g l a s s r e l e a s e d f a c t o r was c a r r i e d o u t , w i t h a v i e w t o c o n d u c t i n g p h a r m a c o l o g i c a l and c h e m i c a l t e s t s on t h e p e p t i d e . T h i s method o f r e l e a s e was c h o s e n s i n c e t h e k i n i n i s p r o d u c e d b y endogenous enzymes, r a t h e r t h a n b y a r t i  f i c i a l l y added 1 enzymes. A w i d e v a r i e t y o f methods h a v e been e m p l o y e d t o r e l e a s e , e x t r a c t and p u r i f y mammalian p l a s m a k i n i n s . These were summar i z e d by E r d o s (1966) and by S c h a c h t e r ( 1 9 6 9 ) . Many a n a l y s e s o f p u r i f i e d k i n i n s h ave b e e n made, b u t t o d a t e t h e r e h a s been no amino a c i d s e quence o r a n a l y s i s o f g l a s s r e l e a s e d k i n i n ( S c h a c h t e r , 1 9 6 9 ) . T h i s seems t o be a s t r a n g e o m m i s s i o n , i n v i e w o f t h e g e n e r a l l y a c c e p t e d o p i n i o n t h a t t h i s method o f r e  l e a s e may p a r a l l e l p h y s i o l o g i c a l r e l e a s e o f t h e s e h y p o t e n s i v e p e p t i d e s . 1. Method. I n 1967 B r o c k l e h u r s t and Z e i t l i n p u b l i s h e d a method f o r t h e d e t e r m i n a t i o n o f l o w l e v e l s o f f r e e k i n i n i n 91 s m a l l b l o o d samples. R e c o v e r y o f a r t i f i c a l l y added b r a d y k i n i n was c l o s e t o 80% i n t r i a l e x p e r i m e n t s . The p r o c e d u r e a l s o had t h e advantage o f e x c l u d i n g a wide v a r i e t y o f i n t e r f e r i n g sub s t a n c e s such as a d r e n a l i n e , h i s t a m i n e , o x y t o c i n , and 5-hydroxy- t r y p t a m i n e . T h i s method i s p r e s e n t e d i n i t s o r i g i n a l f o r m i n Appendix C. A number o f p r e l i m i n a r y p u r i f i c a t i o n s o f t u r t l e k i n i n were c a r r i e d o u t , a c c o r d i n g t o t h i s method, u s i n g s m a l l amounts o f plasma ( l e s s t h a n 200 m l ) . These r e s u l t e d i n the a d o p t i o n o f a number o f m o d i f i c a t i o n s o f t h e b u t a n o l e x t r a c t i o n t e c h n i q u e , m a i n l y t o accomodate the l a r g e r amounts o f plasma r e q u i r e d . The e x a c t p r o c e d u r e f o l l o w e d d u r i n g t h e f i n a l p u r  i f i c a t i o n i s p r e s e n t e d below; i t combines f e a t u r e s o f the methods d e s c r i b e d by B r o c k l e h u r s t and Z e i t l i n (1967) and Z a c e s t and M a s h f o r d (1967), and i s s i m i l a r t o t h a t o f Habermann (1966) . Through a l l e x t r a c t i o n and p u r i f i c a t i o n p r o c e d u r e s , k i n i n a c t i v i t y was f o l l o w e d b y means o f the i s o l a t e d r a t u t e r u s b i o a s s a y . a. R e l e a s e o f k i n i n . 900 ml o f plasma was o b t a i n e d from 30 t u r t l e s by the s t a n d a r d method (Chapter 2) and s t o r e d not more than 18 h o u r s a t 0°C i n s i l i c o n e d 2 L b e a k e r s c o v e r e d w i t h p a r a f i l m . P r i o r t o a c t i v a t i o n , " t h e plasma was d i l u t e d w i t h 900 ml t u r t l e s a l i n e (Appendix B) which c o n t a i n e d 900 mg 8 - H Q S O 4 (1 mg/ml u n d i l u t e d p l a s m a ) . 450 ml o f t h i s f l u i d were p l a c e d i n each o f 4 s i l i c o n e d 1 L E r l e n m e y e r f l a s k s and warmed 92 t o 25°C on a m a g n e t i c s t i r r e r ( F i s h e r F l e x a - M i x ) . 135 g o f a c i d washed g l a s s b a l l o t i n i (25 cm /ml u n d i l u t e d plasma) were added t o each f l a s k , and the e n t i r e m i x t u r e was s t i r r e d a t a moderate speed, w i t h a m a g n e t i c s t i r r e r . b. E x t r a c t i o n p r o c e d u r e . A f t e r 50 m i n u t e s o f i n c u b a  t i o n t h e s u p e r n a t a n t from each f l a s k d e s c r i b e d above was p o u r e d i n t o 900 ml o f b o i l i n g 95% e t h y l a l c o h o l , i n s i l i c o n e d 2 L b e a k e r s . The g l a s s beads r e m a i n i n g i n the f l a s k were t w i c e washed w i t h 100 ml o f 80% e t h y l a l c o h o l , and t h e washings were added t o the o r i g i n a l s u p e r n a t a n t . The e t h a n o l e x t r a c t was b o i l e d f o r 10 m i n u t e s , c o o l e d , and f i l t e r e d t h r o u g h Whatman #1 f i l t e r p aper on a Buchner f u n n e l , under water a s p i r a t i o n . The p r e c i p i t a t e and f i l t e r paper were p e r i o d i c a l l y washed w i t h 100 ml 80% e t h a n o l and d i s c a r d e d , and the washings were added t o t h e f i l t r a t e . The p o o l e d , o p a l e s c e n t g r e e n f i l t r a t e , now about 2.5 l i t e r s , was r e d u c e d t o a b o u t 50 ml i n a r o t a r y f l a s h e v a p o r a t o r , a t 40°C, under a r e d u c e d p r e s s u r e o f 0.2 - 0.5 mm Hg. The c o n c e n t r a t e d s o l u t i o n was a c i d i f i e d w i t h 1.5 ml g l a c i a l a c e t i c a c i d and t w i c e e x t r a c t e d w i t h 2 t o 3 volumes o f d i - e t h y l e t h e r . T h i s e t h e r e x t r a c t was r e d u c e d t o d r y n e s s i n a s i l i c o n e d f l a s k and s t o r e d a t - 20°C f o r l a t e r e s t i m a t i o n o f k i n i n c o n  t e n t . P r e v i o u s t r i a l e x p e r i m e n t s had i n d i c a t e d t h a t under the above c o n d i t i o n s l i t t l e k i n i n i s e x t r a c t e d by e t h e r , though 93 many i m p u r i t i e s a r e removed. These e x p e r i m e n t s , and t h e r a t i o n  a l e f o r i n c l u d i n g t h i s s t e p i n t h e method, a r e d e s c r i b e d i n d e t a i l i n A p p e n d i x E. A f t e r e t h e r e x t r a c t i o n , t h e aqueous p h a s e w h i c h c o n t a i n e d t h e t u r t l e k i n i n was r e d u c e d t o d r y n e s s u n d e r vacuum, a t 40°C. The d r i e d s a mple was t h e n t a k e n up i n 25 ml o f warm (60°C) s a t u r a t e d N a C l s o l u t i o n , a c i d i f i e d t o pH 1.5 w i t h H C l . T h i s s o l u t i o n was e x t r a c t e d a t 60°C w i t h 50, 25, and 25 ml o f r e  d i s t i l l e d 1-N b u t a n o l i n a 125 ml s e p a r a t i n g f u n n e l . The p o o l  ed b u t a n o l e x t r a c t was r a p i d l y r e d u c e d t o d r y n e s s i n a f l a s h e v a p o r a t o r , i n i t i a l l y a t 0.2 mm Hg and 40°C, and i n t h e f i n a l s t a g e s , a t 0.1 mm Hg and 80°C, i n o r d e r t o remove t h e l a s t t r a c e s o f b u t a n o l . The r e s i d u e , w h i c h c o n t a i n e d c o n s i d e r a b l e N a C l , was s t o r e d d r y a t - 20°C u n t i l a s s a y , and f u r t h e r p u r i  f i c a t i o n . The p r e s e n c e o f N a C l was a n e c e s s a r y c o n d i t i o n , s i n c e t h e e f f i c i e n c y o f t h e e x t r a c t i o n i n t h e a b s e n c e o f N a C l was v e r y l o w (see A p p e n d i x F ) . c. Column c h r o m a t o g r a p h y . i . G e l f i l t r a t i o n . The d r y powder f r o m t h e b u t a n o l e x t r a c t was t a k e n u p i n 16 ml o f 0.02 M ammonium a c e t a t e b u f f e r , pH = 5.0 (Ap p e n d i x B ) . 0.3 ml was removed f o r b i o a s s a y , and f o r e s t i m a t i o n o f t h e t o t a l p e p t i d e c o n t e n t b y t h e method o f Lowry e t a l ( 1 9 5 1 ) . The r e m a i n d e r was s u b j e c t e d t o g e l f i l  t r a t i o n on a 90 x 2.5 cm Sephadex G-25 column b u i l t as d e s c r i b e d i n C h a p t e r 2. The b u f f e r i n t h e column above t h e g e l was 94 a l l o w e d t o s i n k t o t h e l e v e l o f t h e f i l t e r p a p e r and t h e 16 ml o f e x t r a c t were a p p l i e d t o t h e column i n a 20 ml p l a s t i c s y r i n g e t h r o u g h a t t a c h e d p o l y e t h y l e n e t u b i n g ( I n t r a m e d i c , 1*90) . T h i s f l u i d was a l s o a l l o w e d t o s i n k t o t h e l e v e l o f t h e f i l t e r p a p e r . The column was t h e n s h u t o f f and f r e s h b u f f e r was added s l o w l y t o t h e t o p o f t h e c o l u m n , w h i c h was s u b s e q u e n t l y c o n n e c t e d b y p l a s t i c t u b i n g t o a 1 L r e s e r v o i r f l a s k c o n t a i n i n g 0.02 M b u f f e r . F l o w t h r o u g h t h e column was a d j u s t e d t o 12- 18 m l / h o u r , and t h e e l u a t e was f e d t o an LKB f r a c t i o n c o l l e c t o r s e t a t 5 m l / t u b e . F r a c t i o n s e l u t e d . a f t e r t h e v o i d v o lume were t e s t e d f o r b i o l o g i c a l a c t i v i t y on t h e r a t u t e r u s , f o r "Lowry" p e p t i d e , and f o r i o n i c s t r e n g t h b y c o n d u c t i v i t y measurements. The a c t i v e f r a c t i o n s i n t h e s i n g l e peak o b t a i n e d f r o m t h i s c olumn were p o o l e d , and a s a mple was removed f r o m t h i s p o o l f o r l a t e r a s s a y . i i . I o n e x c hange. The combined a c t i v i t y , now i n 70 m l , was l o a d e d a t 6 m l / h o u r on t o a 20 x 0.9 cm CM-Sephadex column ( C h a p t e r 2) i n 0.02 M ammonium a c e t a t e b u f f e r . An a d d i t i o n a l 50 m l o f b u f f e r were washed t h r o u g h t h e column, and c o l l e c t e d , t o be t e s t e d f o r p o s s i b l e b i o l o g i c a l a c t i v i t y . No k i n i n was d e t e c t e d , t h e r e b y e n s u r i n g t h a t t h e t u r t l e k i n i n h a d a t t a c h e d t o t h e r e s i n , w h i l e o t h e r i m p u r i t i e s w o u l d have washed t h r o u g h . An i o n i c g r a d i e n t t o 1.0 M o f t h e same b u f f e r 95 was t h e n i n i t i a t e d , and t h e e l u a t e f r o m t h e column was c o l l e c t  ed i n 5 ml f r a c t i o n s . B i o l o g i c a l a c t i v i t y was e s t i m a t e d on t h e r a t u t e r u s b i o a s s a y a f t e r n e u t r i l i z a t i o n o f t h e s a m p l e s w i t h s m a l l amounts o f NaHCO^. The i o n i c s t r e n g t h o f t h e f r a c t i o n s was m e a s u r e d , and "Lowry" p e p t i d e was d e t e r m i n e d on t h a t f r a c  t i o n w h i c h c o n t a i n e d peak a c t i v i t y . 2. R e s u l t s . A summary o f t h e p u r i f i c a t i o n i s g i v e n i n T a b l e 5. The i n c r e a s e i n p u r i f i c a t i o n a t e a c h s t e p was c a l  c u l a t e d f r o m t h e s p e c i f i c a c t i v i t y , w h i c h was b a s e d on t h e t o t a l ug b r a d y k i n i n - e q u i v a l e n t / t o t a l mg "Lowry" p e p t i d e . On t h i s c r i t e r i o n , o v e r a l l p u r i f i c a t i o n was 1475 f o l d o v e r t h e o r i g i n a l p l a s m a c o n c e n t r a t i o n . The e l u t i o n p a t t e r n o f t u r t l e k i n i n on Sephadex G-25 i s p r e s e n t e d i n F i g u r e 19. The a c t i v e m a t e r i a l was c l e a r l y s e p  a r a t e d f r o m a m a j o r p r o t e i n peak, and. was e l u t e d j u s t b e f o r e t h e s a l t . A l l b u t a n o l e x t r a c t s had c o n s i d e r a b l e N a C l , s i n c e c o n d i t i o n s o f e x t r a c t i o n r e q u i r e d a s a t u r a t e d N a C l s o l u t i o n . T h i s was n o t a d i s a d v a n t a g e , s i n c e t h e s a l t peak s e r v e d as a c o n v e n i e n t m a r k e r t o h e l p l o c a t e k i n i n a c t i v i t y , and t h e G-25 column was an e f f e c t i v e d e s a l t i n g mechanism. T u r t l e k i n i n was a l s o e l u t e d f r o m CM-Sephadex i n a s i n g l e peak, a l t h o u g h one f r a c t i o n c o n t a i n e d a r e l a t i v e l y h i g h l e v e l o f k i n i n ( F i g u r e 2 0 ) , c a u s i n g a " s h o u l d e r " b e f o r e t h e main T a b l e 5. The p u r i f i c a t i o n o f g l a s s r e l e a s e d t u r t l e k i n i n . S t a g e o f p u r i - T o t a l volume, T o t a l p e p t i d e , T o t a l r a t u t e r u s R e c o v e r y , % . S p e c i f i c F a c t o r f i c a t i o n ml. mg "Lowry" pep- o x y t o c i c a c t i v i t y , t i d e . ug BK-eq.' b a c t i v i t y , o f pur- ug BK-eq/ i f i c a - c mg "Lowry" t i o n . O r i g i n a l plasma 900 468.0 2490- 250 - 0. 53 1 B u t a n o l e x t r a c t 16 60.0 + 531- 18 21.3 8.9 16. 8 Sephadex G-25 p o o l e d e l u a t e s 75 1. 95 370± 40 69. 7 189 357 £r, ! — CM-Sephadex p o o l e d e l u a t e s 25 0.45 352± 26 94.5 782 1475 a. . . P e p t i d e d e t e r m i n e d b y t h e method o f Lowry, e t a l (1951). b. . . O x y t o c i c a c t i v i t y d e t e r m i n e d b y t h e 4 - p o i n t method o f H o l t o n (1948), w i t h e r r o r s , e x p r e s s e d as c o n f i d e n c e l i m i t s , p = 0.05. c. ..The f a c t o r o f p u r i f i c a t i o n = s p e c i f i c a c t i v i t y o f t h e p r e p a r a t i o n / s p e c i f i c a c t i v i t y o f t h e o r i g i n a l plasma. F i g u r e 19. P u r i f i c a t i o n o f g l a s s - r e l e a s e d t u r t l e k i n i n on a 90 x 2.5 cm Sephadex G-25 g e l f i l t r a t i o n column, b u i l t i n 0.02 M ammonium a c e t a t e b u f f e r , pH = 5.0. 97 Figure 19 Elution Volume (Mis) F i g u r e 20. P u r i f i c a t i o n o f g l a s s r e l e a s e d t u r t l e k i n i n on a 20 x 0.9 cm CM-Sephadex i o n exchange column. The column was b u i l t i n 0.02 M ammonium a c e t a t e b u f f e r , pH = 5.0, and d e v e l o p e d w i t h a c o n c e n t r a t i o n g r a d  i e n t t o 1.0 M ammonium a c e t a t e , pH = 5.0. 98 F i g u r e 20 99 peak. The t u r t l e k i n i n i n t h i s f r a c t i o n was l a t e r t e s t e d f o r amino a c i d c o n t e n t ( t h i s c h a p t e r , p 125) and i t d i d n o t d i f f e r f r o m t h e k i n i n i n t h e r e m a i n d e r o f t h e peak. 3. D i s c u s s i o n and C o n c l u s i o n s . I f t u r t l e k i n i n i s as a c t i v e on t h e r a t u t e r u s as b r a d y k i n i n , and i f "Lowry" p e p t i d e i s an e x a c t measurement o f p e p t i d e c o n t e n t , t h e n a s p e c i f i c a c t i v i t y ( r a t u t e r u s a c t i v i t y / m g "Lowry" p e p t i d e ) o f 1000 w o u l d i n d i c a t e a p u r e p r e p a r a t i o n . W h i l e t h e r e i s no b a s i s f o r a s s u m i n g t h a t e i t h e r o f t h e s e c o n d i t i o n s i s s t r i c t l y t r u e , t h e e x p e r i m e n t a l v a l u e f o r s p e c i f i c a c t i v i t y o f t h e CM-Sephadex e l u a t e , w h i c h was 782, s u g g e s t s t h a t t h e p r e p a r a t i o n was a p p r o a c h i n g p u r i t y . T h i s o b s e r v a t i o n was l a t e r c o r r o b o r a t e d by amino a c i d a n a l y s i s ( p l 2 7 ) . The b e h a v i o u r o f t h e g l a s s - r e l e a s e d t u r t l e k i n i n i n column c h r o m a t o g r a p h y was v e r y s i m i l a r t o t h a t o b s e r v e d b y Habermann (1966) f o r t h e g l a s s r e l e a s e d " c o n t a c t " k i n i n o f b e e f serum. H i s d a t a gave no i n d i c a t i o n t h a t more t h a n one a c t i v e p e p t i d e was p r e s e n t i n t h e e x t r a c t . As Habermann p o i n t s o u t , t h i s i s n o t c o n c l u s i v e p r o o f t h a t o n l y one p e p t i d e i s r e l e a s e d f r o m k i n i n o g e n by p l a s m a k a l l i k r e i n s , s i n c e t h e r e i s c o n s i d e r a b l e e v i d e n c e t h a t b o t h l y s - b r a d y k i n i n and m e t - l y s - b r a d y k i n i n a r e r a p i d l y c o n v e r t e d t o b r a d y k i n i n i n i n t a c t p l a s m a ( F i g u r e 1 ) , and t h a t serum k a l l i k r e i n i t s e l f s p l i t s m e t - l y s - b r a d y k i n i n i n t o t h e d i p e p t i d e m e t - l y s , and b r a d y k i n i n . 100 In any c a s e , t h e method d e s c r i b e d i n t h i s s e c t i o n s u c c e e d  ed i n p r o v i d i n g about 350 ug b r a d y k i n i n - e q u i v a l e n t o f p u r i f i e d t u r t l e k i n i n . T h i s was ample f o r amino a c i d a n a l y s i s and o t h e r c h e m i c a l and p h a r m a c o l o g i c a l t e s t s , and was d e r i v e d f r o m an amount o f t u r t l e plasma (900 ml) which was s m a l l compared w i t h t h e volumes u s e d by o t h e r w o r k e r s i n s t u d i e s o f mammalian plasma k i n i n s (e.g., E l l i o t t e t a l , 1961; P i e r c e and Webster, 1961) . B. P h a r m a c o l o g i c a l S t u d i e s The mammalian plasma k i n i n s b r a d y k i n i n , l y s - b r a d y k i n i n , and m e t - l y s - b r a d y k i n i n have q u a l i t a t i v e l y t h e same a c t i o n i n a wide v a r i e t y o f b i o a s s a y s (Erdos, 1966). However, some o f t h e s e a s s a y s w i l l d i s t i n g u i s h between t h e s e r e l a t e d p e p t i d e s on a q u a n t i t a t i v e b a s i s . T u r t l e k i n i n f r o m c r u d e e x t r a c t s and p u r i f i e d e l u a t e s was t h e r e f o r e a s s a y e d a g a i n s t s y n t h e t i c b r a d y  k i n i n i n s e v e r a l b i o a s s a y s which show q u a l i t a t i v e d i f f e r e n c e s between b r a d y k i n i n and l y s - b r a d y k i n i n . These were the f o l l o w i n g ; t h e c o n t r a c t i o n o f the g u i n e a p i g i l e u m , t h e r e l a x a t i o n o f t h e r a t duodenum, and the d e p r e s s i o n o f r a t and r a b b i t b l o o d p r e s s u r e (Sturmer and Berde, 1963). E d e r y and G r u n f e l d (1969) have shown t h a t t r e a t m e n t o f the r a t u t e r u s w i t h c h y m o t r y p s i n s p e c i f i c a l l y s e n s i t i z e d the p r e p a r a t i o n t o b r a d y k i n i n , and t o a few c l o s e l y r e l a t e d pep t i d e s ; t h e r e f o r e t h i s b i o a s s a y was a l s o used as a p h a r m a c o l o g i c a l t e s t . 101 1. Methods. a. G e n e r a l . I n a l l a s s a y s t h e b r a d y k i n i n - e q u i v a l e n t o f an a c t i v e e l u a t e was e s t i m a t e d b y t h e s t a t i s t i c a l method o f H o l t o n (1949). The r e s p o n s e s o f the t e s t p r e p a r a t i o n t o h i g h and low doses o f t h e s t a n d a r d and unknown were matched i n f o u r - p o i n t g r o u p s . In each a s s a y , 3 t o 5 o f t h e s e groups were o b t a i n e d , and a p p l i c a t i o n o f H o l t o n ' s s t a t i s t i c a l method a l l o w e d 95% c o n f i d e n c e l i m i t s t o be a s s i g n e d t o t h e c a l c u l a t e d v a l u e f o r p o t e n c y . Where t h e amounts o f unknown were n o t l i m i t i n g ( i . e . , f o r t h e more s e n s i t i v e b i o a s s a y s ) , 5 groups were always used, t o i n c r e a s e a c c u r a c y . C o n c e n t r a t e d t u r t l e k i n i n a t v a r i o u s d e g r e e s o f p u r i  f i c a t i o n was a s s a y e d . The b u t a n o l e x t r a c t s (17 f o l d p u r i  f i c a t i o n ) and Sephadex G-25 e l u a t e s (357 f o l d p u r i f i c a t i o n ) were from p i l o t p u r i f i c a t i o n a t t e m p t s , i n v o l v i n g r e l a t i v e l y s m a l l amounts o f k i n i n . CM-Sephadex e l u a t e s (1475 f o l d p u r i  f i c a t i o n ) were from t h e f i n a l p u r i f i c a t i o n d e s c r i b e d i n t h i s c h a p t e r . A l l s o l u t i o n s t o be t e s t e d were a d j u s t e d t o pH 7.0 - 7.5 w i t h s m a l l amounts o f s a t u r a t e d NaOH. The c o n c e n t r a t i o n o f t h e b u f f e r s containing the t u r t l e k i n i n d i d n o t i n t e r f e r e w i t h the r a t u t e r u s , g u i n e a p i g i l e u m , o r r a t duodenum b i o a s s a y s , s i n c e t h e s e were a l l s u f f i c i e n t l y s e n s i t i v e t o a l l o w s i g n i f i c a n t 102 d i l u t i o n o f t h e b u f f e r . However, t h e ammonium a c e t a t e b u f f e r c o n t a i n i n g t h e t u r t l e k i n i n f r o m CM-Sephadex columns was a b o u t 0.8 M, and f r e q u e n t l y gave s m a l l p r e s s o r r e s p o n s e s i n b l o o d p r e s s u r e b i o a s s a y s . T h i s was overcome e i t h e r b y l y o p h i l y z i n g t h e s a m p l e s , o r b y p l a c i n g t h e s t a n d a r d i n an e q u i m o l a r s o l u  t i o n o f t h e b u f f e r , b. B i o a s s a y s . The r a t u t e r u s b i o a s s a y was c a r r i e d o u t as d e s c r i b e d i n C h a p t e r 2. O t h e r a s s a y s a r e b r i e f l y d e s c r i b e d b e l o w . i . R a t u t e r u s s e n s i t i z e d w i t h c h y m o t r y p s i n . I n t h i s a s s a y a v a l u e was o b t a i n e d f o r t h e p o t e n c y o f t h e unknown e x a c t l y as d e s c r i b e d f o r t h e r a t u t e r u s b i o a s s a y . The same u t e r  us was t h e n t r e a t e d w i t h 500 ug c h y m o t r y p s i n f o r 60 s e c o n d s ( E d e r y and G r u n f e l d , 1 9 6 9 ) . The a s s a y was t h e n r e p e a t e d on t h i s s e n s i t i z e d p r e p a r a t i o n . i i . R e l a x a t i o n o f t h e r a t duodenum. T h i s a s s a y was b a s e d on a d e s c r i p t i o n b y A n t o n i o ( 1 9 6 8 ) . R a t s ( W i s t a r s t r a i n ) o f e i t h e r s e x , w e i g h i n g 200-250 g were s a c r i f i c e d as d e s c r i b e d f o r t h e r a t u t e r u s b i o a s s a y . The abdomen was opened and t h e duodenum was removed, f l u s h e d w i t h T y r o d e 1 s s o l u t i o n and p l a c e d i n a 5 ml o r g a n b a t h connected, t o a 2 l i t e r r e s e r v o i r i n a t h e r m a l l y r e g u l a t e d w a t e r b a t h . The t e m p e r a t u r e f o r most a s s a y s was 3 7°C. The s u p p o r t i n g f l u i d t h r o u g h o u t was T y r o d e ' s s o l u t i o n as u s e d by A n t o n i o , w h i c h was composed as f o l l o w s : 103 C h e m i c a l Amount (g/L) C h e m i c a l Amount (g/L) N a C l 8.00 NaHC0 3 1.00 KC1 0.20 N aH 2P04'H 20 0.055 C a C l 2 * 0.22 G l u c o s e 1.00 ( D e x t r o s e ) M g C l 2 * 6 H 2 0 0.20 * . . . A n t o n i o showed t h a t t h e c o n c e n t r a t i o n o f C a + + a f f e c t e d t h e s e n s i t i v i t y o f t h e p r e p a r a t i o n , and t h a t 1 mM C a + + , o r 0.22 g C a C l 2 / L a l l o w e d t h e g r e a t e s t r e l a x a t i o n r e s p o n s e t o b r a d y k i n i n . B o t h t h e o r g a n b a t h and r e s e r v o i r were b u b b l e d w i t h a i r . R e s p o n s e s were r e c o r d e d e x a c t l y as d e s c r i b e d f o r t h e r a t u t e r u s a s s a y , e x c e p t t h a t measurements were t a k e n t o t h e n e a r e s t 0.1 mm. i i i . C o n t r a c t i o n o f t h e g u i n e a p i g i l e u m . A d u l t male g u i n e a p i g s (500 g) were k i l l e d b y a b l o w on t h e h e a d , b l e d , and t h e i l e u m was removed t h r o u g h an a b d o m i n a l i n c i s i o n . The t i s s u e was f l u s h e d w i t h T y r o d e ' s s o l u t i o n , and a p i e c e (5 cm i n l e n g t h ) was suspended i n t h e 5 ml o r g a n b a t h . The r e  m a i n d e r o f t h e a s s a y was c a r r i e d o u t a s d e s c r i b e d f o r t h e r e l a x a t i o n o f t h e r a t duodenum, e x c e p t t h a t t h e t e m p e r a t u r e o f t h e b a t h was r e g u l a t e d b e t ween 30 and 35°C, d e p e n d i n g on t h e s p o n t a n e i t y o f t h e p r e p a r a t i o n . L o w e r i n g t h e a s s a y t e m p e r a t u r e u s u a l l y d e c r e a s e d s p o n t a n e o u s r e s p o n s e s . i v . D e p r e s s i o n o f r a t b l o o d p r e s s u r e . The t e c h n i q u e .used i n t h i s a s s a y was b a s e d on a method d e s c r i b e d b y v a n Dyke e t a l ( 1 9 5 5 ) . M a l e W i s t a r r a t s (220-260 g) were a n e s t h e t i z e d 104 I n 45 t o 60 m i n u t e s b y s u b c u t a n e o u s i n j e c t i o n o f u r e t h a n e (175 mg/100 g b o d y w e i g h t ) - A s m a l l s a g i t t a l i n c i s i o n was made i n t h e v e n t r a l s u r f a c e o f t h e n e c k . U s i n g b l u n t d i s s e c t i o n t e c h n i q u e s , t h e j u g u l a r v e i n was l o c a t e d and f r e e d o f c o n n e c  t i v e t i s s u e . Two l i g a t u r e s were p l a c e d a r o u n d t h e v e i n , a n d t h e one d i s t a l t o t h e h e a r t t i e d . The v e s s e l was p u n c t u r e d w i t h a 22 gauge n e e d l e and c a n n u l a t e d w i t h PE 10 t u b i n g ( I n t r a - m e d i c ) , w h i c h was t i e d i n w i t h t h e two l i g a t u r e s . I n t h e same manner, t h e c a r o t i d a r t e r y was d i s s e c t e d f r e e o f s u r r o u n d i n g t i s s u e and c a n n u l a t e d w i t h PE 50 t u b i n g . 1 ml o f 50' mg% H e p a r i n s o l u t i o n ( i n 0.9% N a C l ) p e r 100 g b o dy w e i g h t was i m m e d i a t e l y g i v e n i n t r a v e n o u s l y , t o p r e v e n t t h e f o r m a t i o n o f b l o o d c l o t s d u r i n g t h e a s s a y . I n j e c t i o n s o f t h e a c t i v e a g e n t s were g i v e n i n t o t h e j u g  u l a r v e i n b y means o f s i l i c o n e d g l a s s s y r i n g e s , and washed t h r o u g h w i t h 0.9% N a C l . The t o t a l v o lume o f t h e i n j e c t i o n n e v e r e x c e e d e d 0.2 m l . The d e p r e s s o r r e s p o n s e s were m e a s u r e d i n mm Hg f r o m t h e c a r o t i d a r t e r y , w h i c h was c o n n e c t e d t o a S t a t h a m P 2 3AA p r e s s u r e t r a n s d u c e r c o u p l e d t o a Beckman dyno- g r a p h r e c o r d e r . P o t e n c y o f t h e unknown sample was e s t i m a t e d b y t h e u s u a l f o u r - p o i n t a s s a y . I n e v e r y a s s a y c o n t r o l i n j e c t i o n s o f 0.2 ml o f t h e b u f f e r ( u s u a l l y ammonium a c e t a t e a d j u s t e d t o pH 7.0 w i t h NaOH) s u p p o r t i n g t h e unknown were g i v e n . S e n s i t i v i t y t o 105 t h i s v e h i c l e v a r i e d between a n i m a l s . Responses t o k i n i n s were r e g a r d e d as v a l i d when t h e s e c o n t r o l i n j e c t i o n s e l i c i t e d no r e s p o n s e i n t h e same p r e p a r a t i o n s , o r when the k i n i n s were p l a c e d i n e q u i m o l a r s o l u t i o n s o f b u f f e r . I t i s s t a n d a r d p r o c e d u r e i n t h i s t y p e o f b i o a s s a y t o t r e a t t h e a n i m a l w i t h an a d r e n e r g i c b l o c k i n g a g e n t such as d i b e n z y l e n e (van Dyke, e t a l , 1955) t o s t a b l i l i z e b l o o d p r e s s u r e , and t o p r e v e n t i n t e r f e r e n c e by changes i n c a t e c h o l a m i n e l e v e l s . I t has a l s o been r e p o r t e d t h a t t h i s t r e a t m e n t p o t e n t i a t e s some b l o o d p r e s s u r e r e s p o n s e s t o b r a d y k i n i n ( A l - K a t i b , e t a l , 19 69). However, I f o u n d t h a t t h e s e p r e p a r a t i o n s became v e r y i n s e n s i t i v e t o b r a d y k i n i n a f t e r a d m i n i s t r a t i o n o f 0.5 mg d.ibenzylene/100 g body w e i g h t ( F i g u r e 2 1 ) . T h e r e f o r e no b l o c k i n g a g e n t was used i n t h e s e b i o a s s a y s . v. D e p r e s s i o n o f r a b b i t b l o o d p r e s s u r e . Male r a b b i t s (1.5-2.0 kg) were i n j e c t e d i n t r a p e r i t o n e a l l y w i t h 155 mg u r e - thane/100 g body w e i g h t , i n 0.9% s a l i n e , and i n 2 h o u r s an e s t h e s i a was deep enough t o a l l o w s u r g e r y . The j u g u l a r v e i n and c a r o t i d a r t e r y were exposed and c a n n u l a t e d as d e s c r i b e d f o r the r a t b l o o d p r e s s u r e b i o a s s a y . I n j e c t i o n s o f b r a d y k i n i n and t u r t l e k i n i n were g i v e n by the i n t r a v e n o u s r o u t e , i n a t o t a l volume n o t e x c e e d i n g 0.4 ml. Responses t o a g o n i s t s and c o n t r o l s were r e c o r d e d as d e s c r i b e d i n t h e p r e v i o u s a s s a y , and p o t e n c y o f the unknown was c a l c u l a t e d i n the u s u a l manner, F i g u r e 21. Responses t y p i c a l o f s y n t h e t i c b r a d y k i n i n (B) and p u r i f i e d t u r t l e k i n i n (T) i n s i x b i o a s s a y s . A s i n g l e f o u r - p o i n t g r o u p i s g i v e n f o r each a s s a y , e x c e p t f o r t h e r a t b l o o d p r e s s u r e b i o a s s a y , where the two groups show t h e r e s p o n s e o f one r a t t o b r a d y k i n i n and t u r t l e k i n i n b e f o r e (#1) and a f t e r (#2) a d m i n i s t r a t i o n o f 0.5 mg d i b e n z y l e n e / 1 0 0 g body w e i g h t . Arrows w i t h a "C" i n d i c a t e c o n t r a c t i o n , t h o s e w i t h an "R" i n d i c a t e r e l a x a t i o n . Drum speed f o r a l l smooth muscle b i o a s s a y s was 2 mm/min, and f o r b l o o d p r e s s u r e a s s a y s was 24 mm/min. 106 F i g u r e 21 Sensitized Rat Uterus ro O I N t o o t— cn h- cn cn _ CN 10 o o _- Guinea Pig Ileum H CDCD CN O O — CN Rat Duodenum CXL ft ^ «o — o I20h 80 Rat Blood Pressure - 1 .5 8 .015T .25B .03T I •03 T .06 T , 6B i.o B CO o X E E 1201 80 Rabbit Blood Pressure 2 T 2B 4T l B 107 u s i n g t h r e e f o u r - p o i n t g r o u p s . 2. R e s u l t s and D i s c u s s i o n In a l l b i o a s s a y s t h e p u r i f i e d t u r t l e k i n i n gave r e s p o n s e s . t y p i c a l o f mammalian plasma k i n i n s , as shown i n F i g u r e 21. I t c o n t r a c t e d t h e r a t u t e r u s and g u i n e a p i g i l e u m a f t e r a t y p i c a l l y l o n g l a t e n t p e r i o d , i t r e l a x e d , t h e r a t duodenum, and i t l o w e r e d the b l o o d p r e s s u r e o f r a t and r a b b i t . C h y m o t r y p s i n s e n s i t i z e d t h e r a t u t e r u s 2 t o 5 t i m e s t o b o t h b r a d y k i n i n and t u r t l e k i n i n , and t h i s s u g g e s t e d a marked s i m i l a r i t y between the two a c t i v e a g e n t s . A summary o f t h e q u a n t i t a t i v e r e s u l t s o b t a i n e d i n each a s s a y i s g i v e n i n T a b l e 6. V a l u e s i n t h e f i n a l column a r e r a t i o s o f t h e r a t u t e r u s a c t i v i t y / t e s t b i o a s s a y a c t i v i t y , f o r each o f t h e b i o a s s a y s l i s t e d i n t h e f i r s t column. S y n t h e t i c b r a d y k i n i n was used as s t a n d a r d i n a l l b i o a s s a y s . T h e r e f o r e , i f t u r t l e k i n i n were c h e m i c a l l y i d e n t i c a l t o b r a d y  k i n i n , none o f t h e s e r a t i o s would be s i g n i f i c a n t l y d i f f e r e n t from one, p r o v i d i n g t h e same t u r t l e k i n i n p r e p a r a t i o n was used f o r b o t h a s s a y s i n v o l v e d i n any r a t i o . T h i s was t r u e f o r the r a t u t e r u s / s e n s i t i z e d r a t u t e r u s r a t i o , and f o r t h e r a t u t e r u s / r a t duodenum r a t i o s . A l s o , the r a t i o o f r a t u t e r u s / r a t b l o o d p r e s s u r e , a t 1.3 t 0.2. was o n l y s l i g h t l y g r e a t e r than one, and c o u l d n o t be t a k e n t o i n d i c a t e any d i f f e r e n c e . However, the r a t i o s o f r a t u t e r u s / g u i n e a p i g i l e u m a c t i v i t y , and r a t u t e r u s / T a b l e 6. P h a r m a c o l o g i c a l s t u d i e s ; the r a t i o s o f d i f f e r e n t b i o l o g i c a l a c t i v i t i e s o f t u r t l e k i n i n , a s s a y e d a g a i n s t s y n t h e t i c b r a d y k i n i n . Method o f A s s a y T u r t l e k i n i n A s s a y v a l u e ; Rat u t e r u s R a t i o ; r a t u t e r u s / p r e p a r a t i o n BK-eq, ug/ml. a c t i v i t y ; b i o l o g i c a l a c t i v i t y a s s a y e d 3 . BK-eq, ug/ml. a s s a y e d Rat u t e r u s , s e n s i  t i z e d w i t h chymo- t r y p s i n B u t a n o l e x t r a c t 2.5 ± 0.1 b 2.3 - 0.2 0.9 + 0. 1 Rat duodenum Sephadex 0.16 ± 0.03 c 0.17 - 0.01 1.0 + 0. 1 G-25 e l u a t e 0.19 ± 0.03 c 0.19 ± 0.01 1.0 + 0. 1 Rat b l o o d p r e s s u r e CM-Sephadex e l u a t e 10.1 - 1.5 13.2 - 1.0 1.3 + 0. 2 G u i n e a p i g i l e u m Sephadex G-25 0.10 - 0.04 0.19 - 0.01 2.0 + 0. 8 e l u a t e CM-Sephadex e l u a t e 5.2 - 0.7 13.2 - 1.0 2.5 + 0. 4 R a b b i t b l o o d p r e s s u r e CM-Sephadex e l u a t e 0.95 0.15 6.7 - 0.8 7.0 + 1. 8 a...The s t a g e s o f p u r i f i c a t i o n , and de g r e e s o f p u r i t y a r e i n i d c a t e d i n T a b l e 5. b...The 4 - p o i n t method o f H o l t o n (1948) was used i n a l l b i o a s s a y s ; e r r o r s a r e e x p r e s s e d as c o n f i d e n c e l i m i t s , p = 0.05. c . . . V a l u e s o b t a i n e d d u r i n g two s e p a r a t e runs t h r o u g h Sephadex G-25. 109 r a b b i t b l o o d p r e s s u r e a c t i v i t y were m a r k e d l y d i f f e r e n t f r o m one. Compared t o v a l u e s o b t a i n e d i n r a t u t e r u s a s s a y s , b r a d y  k i n i n was 2.5 t i m e s more a c t i v e t h a n t u r t l e k i n i n on t h e g u i n e a p i g i l e u m , and 7.0 t i m e s more a c t i v e t h a n t u r t l e k i n i n on t h e r a b b i t b l o o d p r e s s u r e . These d a t a s t r o n g l y i n d i c a t e d t h a t t u r t l e k i n i n and b r a d y k i n i n were c h e m i c a l l y d i f f e r e n t . T hese r e s u l t s may be compared t o t h o s e o f E r d o s , Miwa and Graham (1967) who r e p o r t e d t h a t a l l i g a t o r and t u r t l e plasma a c t i v a t e d w i t h g l a s s beads r e l e a s e d a s u b s t a n c e o x y t o c i c t o t h e r a t u t e r u s , and c o n c l u d e d t h a t t h e a c t i v e a gent was a p e p t i d e p h a r m a c o l o g i c a l l y i n d i s t i n g u i s h a b l e f r o m mammalian plasma k i n i n s , p o s s i b l y a m i x t u r e o f b r a d y k i n i n and l y s - b r a d y k i n i n . C l o s e r e x a m i n a t i o n o f t h e s e f i n d i n g s r e v e a l s t h a t t h e y a r e n o t i n c o m p a t i b l e w i t h r e s u l t s g i v e n i n t h i s t h e s i s . F i r s t l y , t h e s e w o r k e r s d i d p h a r m a c o l o g i c a l s t u d i e s o n l y on a l l i g a t o r plasma, showing t h a t the a c t i v e p r i n c i p l e c o n t r a c t e d t h e i s o l a t e d g u i n e a p i g i l e u m , caused v a s o d i l a  t i o n i n the dog h i n d l i m b , and caused a d e p r e s s i o n o f r a b b i t b l o o d p r e s s u r e . S e c o n d l y , r a t i o s o f a c t i v i t i e s f o r the a l l i  g a t o r k i n i n were c a l c u l a t e d o n l y f o r r a t u t e r u s / g u i n e a p i g i l e u m and r a t u t e r u s / d o g h i n d limb b l o o d f l o w . F u r t h e r m o r e , no d a t a were g i v e n f o r any o f t h e s e , a s s a y s . I f a l l i g a t o r k i n i n i s i d e n t i c a l t o t u r t l e k i n i n , t h e r a t u t e r u s / g u i n e a p i g i l e u m a c t i v i t y s h o u l d have been d i f f e r e n t from one, 110 b u t E r d o s e t a l o n l y remark t h a t t h e r a t i o s were s i m i l a r t o b r a d y k i n i n . C. C h e m i c a l S t u d i e s T u r t l e k i n i n was compared t o b r a d y k i n i n i n a wide v a r i e t y o f s p e c i f i c and i n d i r e c t t e s t s . 1. M i s c e l l a n e o u s T e s t s . D u r i n g t h e p r o c e s s e s o f r e l e a s e , e x t r a c t i o n and p u r i  f i c a t i o n , t u r t l e k i n i n was s u b j e c t e d t o a number o f t r e a t  ments which would i n a c t i v a t e o r e x c l u d e o t h e r s u b s t a n c e s known t o o c c u r i n plasma, and which a r e o x y t o c i c t o t h e r a t u t e r u s . a * B o i l i n g . B r a d y k i n i n i s n o t i n a c t i v a t e d b y b o i l i n g a t n e u t r a l o r a c i d pH (Rocha e S i l v a , 1949). The g l a s s - r e l e a s e d t u r t l e k i n i n s t u d i e d h e r e was b o i l e d f o r a t l e a s t 10 m i n u t e s a t n e u t r a l pH i n a l l e x p e r i m e n t s ( C h a p t e r s 4 and 5) and m a i n t a i n e d f u l l a c t i v i t y (e.g., F i g u r e 1 0 ) . O x y t o c i n and i t s a n a l o g u e s a r e i n a c t i v a t e d by t h i s p r o c e s s . k* T r y p s i n . S y n t h e t i c b r a d y k i n i n i s o n l y v e r y s l o w l y i n a c t i v a t e d by i n c u b a t i o n w i t h t r y p s i n a t 37°C (Oates and Melmon, 1966). Many e x p e r i m e n t s w i t h t u r t l e plasma i n d i c a t e d t h a t the a c t i v i t y r e l e a s e d by a r e l a t i v e l y h i g h c o n c e n t r a t i o n o f t r y p s i n showed no d e t e c t a b l e l o s s o f a c t i v i t y a f t e r 30 m inutes of i n c u b a t i o n a t 37°C (e.g., F i g u r e 17). In con t r a s t , S ubstance P i s c o m p l e t e l y i n a c t i v a t e d under t h e s e I l l c o n d i t i o n s (Pernow, 1 9 5 5 ) . c. B u t a n o l e x t r a c t i o n . The b u t a n o l e x t r a c t i o n a t l o w pH d e s c r i b e d i n C h a p t e r 5 h a s b e e n shown b y B r o c k l e h u r s t and Z e i t l i n (1967) t o e x t r a c t b r a d y k i n i n f r o m t h e aqueous l a y e r , w h i l e l a r g e l y e x c l u d i n g h i s t a m i n e , a d r e n a l i n e and 5 - h y d r o x y t r y p t a m i n e . These w o r k e r s f o u n d t h a t o n l y 0.4% o f t h e h i s t a m i n e , and 3.2% o f t h e 5 - h y d r o x y t r y p t a m i n e were t a k e n up i n t h e b u t a n o l . The p r e s e n t work w i t h t u r t l e k i n i n i n d i c a t e d t h a t t h i s a c t i v e p e p t i d e was e x t r a c t e d b y t h e same p r o c e d u r e , and s u b s e q u e n t column c h r o m a t o g r a p h y ( F i g  u r e s 19 and 20) s u g g e s t e d t h a t o n l y one a c t i v e f a c t o r h a d b e en e x t r a c t e d . I n a l l o f t h e i n d i r e c t t e s t s d e s c r i b e d above, t u r t l e k i n i n was i n d i s t i n g u i s h a b l e f r o m mammalian p l a s m a k i n i n s , b u t d i f f e r e d f r o m many o t h e r a c t i v e p r i n c i p l e s c o n t a i n e d i n v e r t e b r a t e p l a s m a . 2. P a p e r C h r o m a t o g r a p h y . T u r t l e k i n i n o b t a i n e d f r o m Sephadex G-25 columns was compared t o s y n t h e t i c b r a d y k i n i n (Sandoz) and l y s - b r a d y k i n i n (Schwarz) i n t h r e e p a p e r chromatograms, u s i n g a b u t a n o l - a c e t i c a c i d - w a t e r s o l v e n t s y s t e m . Method. The t e c h n i q u e u s e d has b een d e s c r i b e d b y P e r k s (1966) and by V i z s o l y i ( 1 9 6 7 ) , and i s g i v e n i n d e t a i l i n A p p e n d i x D. T h r e e e x p e r i m e n t s were c a r r i e d o u t , one a 12 112 h o u r r u n , t h e o t h e r s 24 h o u r s . I n e a c h e x p e r i m e n t t h e t u r t l e k i n i n sample was r u n b e s i d e b o t h b r a d y k i n i n and l y s - b r a d y k i n i n . B i o l o g i c a l a c t i v i t y i n e a c h d i v i s i o n o r i n a r b i t r a r y d i v  i s i o n s ( A p p e n d i x D) was e s t i m a t e d b y a s s a y a g a i n s t s y n t h e t i c b r a d y k i n i n . R e s u l t s . F i g u r e 22 i s a r e p r e s e n t a t i o n o f r e s u l t s f r o m t h e 12 h o u r e x p e r i m e n t . M ost b r a d y k i n i n and t u r t l e k i n i n r a n a t Rf 0.3 - 0.4. T h i s i s i n a g r e e m e n t w i t h p u b l i s h e d v a l u e s f o r Rf o f b r a d y k i n i n i n t h i s s o l v e n t s y s t e m (Oates and Melmon, 1 9 6 6 ) . M ost l y s - b r a d y k i n i n r a n a t Rf 0.2 - 0.3, and was t h e r e f o r e p a r t i a l l y s e p a r a t e d f r o m b r a d y k i n i n and. t u r t l e k i n i n . I t was t h e p u r p o s e o f t h e two 24 h o u r chromatograms ( F i g u r e 23) t o i n c r e a s e t h e p o s s i b l e r e s o l u t i o n o f t h e k i n i n s b y i n c r e a s i n g t h e l e n g t h o f t h e r u n . A l t h o u g h t h e s o l v e n t f r o n t r a n c o m p l e t e l y o f f t h e p a p e r d u r i n g t h i s t i m e , a l l b i o l o g i c a l a c t i v i t y was s u f f i c i e n t l y r e t a r d e d t o r e m a i n on t h e p a p e r . The a r b i t r a r y u n i t s were o b t a i n e d b y d i v i d i n g t h e d i s t a n c e f r o m t h e o r i g i n t o t h e b o t t o m o f t h e p a p e r i n t o 10 s e c t i o n s , and e l u t i n g e a c h s e c t i o n s e p a r a t e l y . These u n i t s a r e t h e r e f o r e n o t c o m p a r a b l e t o R^ v a l u e s , b u t a r e a good measure o f t h e d i s t a n c e moved f r o m t h e o r i g i n . The r e s u l t s i n F i g u r e 2 3 show t h a t l y s - b r a d y k i n i n was s h a r p l y s e p a r a t e d (by one c o m p l e t e u n i t ) f r o m b o t h b r a d y k i n i n F i g u r e 22. Paper chromatography o f l y s - b r a d y k i n i n , t u r t l e k i n i n , and b r a d y k i n i n i n b u t a n o l - a c e t i c a c i d - water (4:1:5) on Whatman 3MM chromatography paper a 12 hour r u n . 113 F i g u r e 22 12 Hr Chroma to gram F i g u r e 23. Paper chromatography o f l y s - b r a d y k i n i n , t u r t l e k i n i n , and b r a d y k i n i n i n b u t a n o l - a c e t i c a c i d - w a ter (4:1:5) on Whatman 3MM chromatography p a p e r ; 2 4 h o u r r u n s . S i n c e the s o l v e n t f r o n t r a n o f f the pa p e r d u r i n g t h i s time, r e s u l t s a r e g i v e n i n a r b i t r a r y u n i t s (see p 112) . F i g u r e 23 115 and t u r t l e k i n i n a c t i v i t y . T h e r e was no d i f f e r e n c e i n t h e b e h a v i o u r o f b r a d y k i n i n and t u r t l e k i n i n i n t h i s sytem. 3. G e l F i l t r a t i o n on Sephadex G-25 The e l u t i o n p a t t e r n o f t u r t l e k i n i n from Sephadex G-25 ( F i g u r e 19) was compared t o t h a t o f b r a d y k i n i n e l u t e d under s i m i l a r c o n d i t i o n s . Method. S y n t h e t i c b r a d y k i n i n was mixed w i t h b l u e d e x t r a n and N a C l , and a p p l i e d t o a 95 x 2.5 cm Sephadex.G-25 column, which was b u i l t i n 0.02 M ammonium a c e t a t e b u f f e r , and e l u t e d as d e s c r i b e d i n C h a p t e r 2. The e x p e r i m e n t w i t h t u r t l e k i n i n was d e s c r i b e d i n t h i s c h a p t e r , p 93 - 94. R e s u l t s . The e l u t i o n p a t t e r n o f t u r t l e k i n i n g i v e n i n F i g u r e 19 may be compared t o t h a t o f s y n t h e t i c b r a d y k i n i n , shown i n F i g u r e 24. A l t h o u g h t h e columns d i f f e r e d s l i g h t l y i n s i z e and e l u t i o n volume, and i n t h e amount o f b r a d y k i n i n - e q u i v a l - e n t i n t h e e l u a t e , p a t t e r n s o f e l u t i o n were s i m i l a r , b o t h a c t i v e peaks coming j u s t b e f o r e t h e NaCl peak. 4. Ion Exchange Chromatography on CM-Sephadex The i o n exchange p r o p e r t i e s o f t u r t l e k i n i n , b r a d y k i n i n , and l y s - b r a d y k i n i n were compared i n a number o f ways: a) b y e l u t i o n o f b r a d y k i n i n and t u r t l e k i n i n f r o m columns d e v e l o p e d w i t h 0.5 M ammonium a c e t a t e b u f f e r ; b) b y s e p a r a t i o n o f b r a d y k i n i n ahd l y s - b r a d y k i n i n on a column d e v e l o p e d w i t h 1.0 M b u f f e r , and c o m p a r i s o n o f the F i g u r e 24. E l u t i o n p a t t e r n o f s y n t h e t i c b r a d y k i n i n on a 95 x 2.5 cm Sephadex G-25 g e l f i l t r a t i o n column, i n 0.02 M ammonium a c e t a t e b u f f e r , pH = 5.0. 116 Figure 24 117 e l u t i o n o f t u r t l e k i n i n i n t h e same system; c) b y a d i r e c t c o m parison, i n which e q u a l p o r t i o n s o f b r a d y k i n i n and t u r t l e k i n i n were mixed and e l u t e d f r o m a CM-Sephadex column d e v e l o p e d w i t h 0.6 M b u f f e r . Methods. a) A p p r o x i m a t e l y 10 ug b r a d y k i n i n - e q u i v a l e n t o f g l a s s r e l e a s e d t u r t l e k i n i n p u r i f i e d on Sephadex G-25 i n a p i l o t e x p e r i m e n t were l o a d e d i n 5 ml on t o a s m a l l (17 x 0.9 cm) CM-Sephadex column b u i l t i n 0.02 M ammonium a c e t a t e b u f f e r as d e s c r i b e d i n C h a p t e r 2. An i o n i c s t r e n g t h g r a d i e n t t o 0.5 M b u f f e r was a p p l i e d , and the a c t i v i t y c o l l e c t e d i n d i f f e r e n t f r a c t i o n s was e s t i m a t e d on t h e r a t u t e r u s , a f t e r n e u t r i l i z a - t i o n w i t h NaHCC>3. Two o t h e r columns o f the same s i z e , one l o a d e d w i t h 10 ug s y n t h e t i c b r a d y k i n i n , and t h e o t h e r w i t h 10 ug b r a d y k i n i n - e q u i v a l e n t o f l y s - b r a d y k i n i n were d e v e l o p e d i n e x a c t l y t h e same manner. b) A m i x t u r e of 10 ug b r a d y k i n i n and 10 - 1 ug l y s - b r a d y k i n i n were l o a d e d i n 2 ml on t o a CM-Sephadex column b u i l t i n 0.02 M ammonium a c e t a t e b u f f e r . The column was d e v e l  oped u s i n g a g r a d i e n t t o 1.0 M b u f f e r , e x a c t l y as d e s c r i b e d i n C h a p t e r 5 f o r the p u r i f i c a t i o n o f t u r t l e k i n i n . A c t i v i t y o f the 6.2 ml f r a c t i o n s was a s s a y e d , and the e l u t i o n p a t t e r n was compared t o t h a t o f t u r t l e k i n i n i n t h e same system. c) 6.7 ug b r a d y k i n i n was mixed w i t h 6.6.- 0.45 ug o f 118 t u r t l e k i n i n , b r a d y k i n i n - e q u i v a l e n t (a s ample f r o m t h e CM- Sephadex e l u a t e l i s t e d i n T a b l e 5 ) , - and a d j u s t e d t o 3 ml 0.3 M ammonium a c e t a t e b u f f e r , pH = 5.0. T h i s m i x t u r e was l o a d e d on a CM-Sephadex column a l s o b u i l t i n 0.3 M b u f f e r . The column was t h e n washed w i t h 50 m l o f t h e b u f f e r , and t h e e l u a t e was c h e c k e d f o r b i o l o g i c a l a c t i v i t y . A c o n c e n t r a t i o n g r a d i e n t t o 0.6 M b u f f e r , pH = 5.0, was t h e n a p p l i e d t o t h e column i n t h e u s u a l manner, and 5 ml f r a c t i o n s were c o l l e c t e d and a s s a y e d . R e s u l t s . a) F i g u r e 25 compares t h e e l u t i o n o f t u r t l e k i n i n and b r a d y k i n i n on s e p a r a t e c o l u m n s , b y 0.5 M b u f f e r . The p a t t e r n s a r e i d e n t i c a l . No b i o l o g i c a l a c t i v i t y was d e t e c t e d i n t h e e l u a t e f r o m t h e column l o a d e d w i t h l y s - b r a d y k i n i n , and i t was c o n c l u d e d t h a t a more c o n c e n t r a t e d b u f f e r w o u l d be r e  q u i r e d t o d e t a c h t h i s p e p t i d e f r o m t h e r e s i n . b) F i g u r e 26 a i l l u s t r a t e s t h e s e p a r a t i o n o f b r a d y k i n i n and l y s - b r a d y k i n i n b y a g r a d i e n t t o 1.0 M b u f f e r . The r e s o l u  t i o n o f t h e s e p e p t i d e s i s v e r y c l e a r , t h e two peaks b e i n g s e p a r a t e d by 57 ml o f e l u a t e . The e l u t i o n p a t t e r n o f t u r t l e k i n i n i n t h e same s y s t e m i s a l s o i n c l u d e d i n t h i s F i g u r e . S i n c e p r e v i o u s r e s u l t s e s t a b l i s h e d t h a t b r a d y k i n i n i s e l u t e d a t t h e same i o n i c s t r e n g t h as t u r t l e k i n i n ( F i g u r e 2 5 ) , t h e f i r s t peak i n F i g u r e 26 a may be i d e n t i f i e d as b r a d y k i n i n , F i g u r e 25. E l u t i o n o f s y n t h e t i c b r a d y k i n i n and t u r t l e k i n i n f r o m s e p a r a t e CM-Sephadex columns b u i l t i n 0.02 M ammonium a c e t a t e b u f f e r , pH =• 5.0, and d e v e l o p e d w i t h a c o n c e n t r a t i o n g r a d i e n t t o 0.5 M ammonium a c e t a t e b u f f e r , pH = 5.0. 119 F i g u r e 25 Elut ion Volume, Ml 120 F i g u r e 2 6 a. S e p a r a t i o n o f s y n t h e t i c b r a d y k i n i n from s y n t h e t i c l y s - b r a d y k i n i n on a CM-Sephadex column (20 x 0.9 cm) d e v e l o p e d w i t h a c o n c e n t r a t i o n g r a d i e n t from 0.02 M t o 1.0 ammonium a c e t a t e b u f f e r , pH = 5.0. b. The e l u t i o n p a t t e r n o f t u r t l e k i n i n i n t h e same system (from F i g u r e 20), i n c l u d e d f o r compa r i s o n . 120 F i g u r e 2 6 E lu t ion Vo l ume , M l 121 and t h e s e c o n d as l y s - b r a d y k i n i n . S i n c e m e t - l y s - b r a d y k i n i n r u n s c l o s e t o l y s - b r a d y k i n i n i n t h i s sytem (Miwa, E r d o s , and S e k i , 1969) i t can be c o n c l u d  ed t h a t t u r t l e k i n i n i s a l s o d i f f e r e n t f r o m m e t - l y s - b r a d y k i n i n . c) The h y p o t h e s i s t h a t b r a d y k i n i n i s i n s e p a r a b l e f r o m t u r t l e k i n i n i n i o n exchange chromatography on CM-Sephadex was t e s t e d more r i g o r o u s l y by t h e chromatography o f a mix t u r e o f t h e two, u s i n g a more s h a l l o w g r a d i e n t t o i n c r e a s e r e s o l u t i o n . R e s u l t s o f t h e t e s t ( F i g u r e 27) i n d i c a t e t h a t a s i n g l e peak o f a c t i v i t y was e l u t e d . P o o l e d a c t i v i t y f r o m e l u t i o n volume 90 - 140 ml was e s t i m a t e d a t 10.9 - 1.5 ug b r a d y k i n i n - e q u i v a l e n t . T h i s t o t a l i s s i g n i f i c a n t l y g r e a t e r (p<.01) t h a n e i t h e r the b r a d y k i n i n (6.7 ug) o r the t u r t l e k i n i n (6.6 * 0.45) i n i t i a l l y l o a d e d on the column. The peak must t h e r e f o r e c o n t a i n b o t h a c t i v e p r i n c i p l e s . Assuming t h i s t o be t r u e , t h e t o t a l r e c o v e r y o f b i o l o g i c a l a c t i v i t y f r o m the column would be 10.9/13.3 = 74.4%, a normal r e c o v e r y f o r CM-Sephadex. On the b a s i s o f the e x p e r i m e n t s d e s c r i b e d above i t was c o n c l u d e d t h a t t h e g l a s s r e l e a s e d t u r t l e k i n i n i s d i f f e r e n t from l y s - b r a d y k i n i n and from m e t - l y s - b r a d y k i n i n , b u t i s v e r y s i m i l a r t o b r a d y k i n i n . 5. Amino A c i d A n a l y s i s Samples o f p u r i f i e d t u r t l e k i n i n and s y n t h e t i c b r a d y k i n i n F i g u r e 27. E l u t i o n p a t t e r n o f a m i x t u r e o f s y n t h e t i c b r a d y k i n i r and p u r i f i e d t u r t l e k i n i n f r o m a CM-Sephadex column (15 x 0.9 cm) d e v e l o p e d w i t h a s h a l l o w c o n c e n t r a  t i o n g r a d i e n t , from 0.3 M t o 0.6 M ammonium a c e t a t e , pH = 5.0. 122 I j I i F i g u r e 27 123 w e r e h y d r o l y z e d , and s u b j e c t e d t o amino a c i d a n a l y s i s . M ethod. T h r e e s a m p l e s o f p u r i f i e d t u r t l e k i n i n were h y d r o l y z e d s e p a r a t e l y . Two s a m p l e s (100 - 10 and 145 - 10 ug BK-eq) 'were t a k e n f r o m t h e m a i n peak i n t h e CM-Sephadex e l u a t e ( F i g u r e 2 0 ) , and one f r o m t h e " s h o u l d e r " , a t e l u t i o n volume 85 ml (62 - 4 ug B K - e q ) . One s a mple o f s y n t h e t i c b r a d y k i n i n ( S c h w a r z , 100 ug) was a l s o h y d r o l y z e d s e p a r a t e l y . The s a m p l e s o f t u r t l e k i n i n i n 0.8 M ammonium a c e t a t e , and s a m p l e s o f s y n t h e t i c b r a d y k i n i n i n 0.2 M a c e t i c a c i d w ere l y o p h i l i z e d i n 50 ml vacuum f l a s k s . I n e a ch c a s e t h e r e s i d u e was d i s s o l v e d i n g l a s s d i s t i l l e d w a t e r and r e - l y o - p h i l i z e d a t l e a s t 3 t i m e s t o r e d u c e l e v e l s o f ammonium a c e t a t e and a c e t i c a c i d . The r e s i d u e was t h e n t a k e n up i n 4 m l o f 6 N t r i p l e d i s  t i l l e d H C l , and t r a n s f e r r e d t o an a c i d washed 6 m l h y d r o l y s i s b u l b . U s i n g PE 10 t u b i n g ( I n t r a m e d i c ) , N 2 gas was b u b b l e d t h r o u g h t h e l i q u i d f o r 15 m i n u t e s t o d i s p l a c e d i s s o l v e d 0 2 . The f l u i d was t h e n f r o z e n b y i m m e r s i n g t h e h y d r o l y s i s b u l b i n l i q u i d N 2 , and s u b s e q u e n t l y e v a c u a t e d f o r 30 m i n u t e s a t 0.01 mm Hg p r e s s u r e . U s i n g a n a t u r a l gas f l a m e , t h e neck o f t h e b u l b was s e a l e d u n d e r t h e same vacuum, and t h e b u l b was p l a c e d i n an oven ( F i s h e r Isotemp) a t 107°C, f o r 24 o r 36 h o u r s , a c c o r d  i n g t o t h e s a mple. A f t e r t h i s t i m e , t h e s e a l e d h y d r o l y s i s b u l b s were removed 124 from the oven, c o o l e d , c a r e f u l l y opened, and t h e c o n t e n t s were t r a n s f e r r e d t o a c i d washed 50 ml round bottom f l a s k s . These f l a s k s were c o o l e d i n i c e f o r 15 m i n u t e s , t h e n a t t a c h e d 'to a f l a s h e v a p o r a t o r and r o t a t e d under vacuum i n a w a ter b a t h a t 37°C. A f t e r 30 m i n u t e s , the d r i e d r e s i d u e s were r e d i s s o l v e d i n 2 ml i c e d d i s t i l l e d w a t e r and t h e sample r e - d r i e d . T h i s w a s h i n g was r e p e a t e d 3 t i m e s t o remove the l a s t t r a c e s o f HC1. ' o Samples were s t o r e d d r y a t - 20 C u n t i l a n a l y s i s . Amino a c i d a n a l y s i s was p e r f o r m e d on a B i o c a l 200 a n a l y z e r The d r i e d h y d r o l y s a t e s were d i s s o l v e d i n 1 ml o f sodium c i t  r a t e b u f f e r , pH - 2.2. A l i q u o t s c o n t a i n i n g t h e d e s i r e d amount o f m a t e r i a l were removed w i t h a c i d washed p i p e t t e s and p l a c  ed on t h e column i n a volume o f 1 ml. A f t e r c o m p l e t i o n o f t h e 5 h o u r a n a l y s i s , t h e s e p a r a t e d peaks o b t a i n e d on t h e r e c o r d c o u l d be q u a n t i f i e d by c o m p arison o f the a r e a s w i t h t h o s e o f s t a n d a r d amino a c i d s . In c a l c u l a t i o n o f m o l a r r a t i o s , a r g - i n i n e was u s e d as r e f e r e n c e (Arg = 2.00) s i n c e i t i s r e l a  t i v e l y s t a b l e under the c o n d i t i o n s o f a c i d h y d r o l y s i s . R e s u l t s . T a b l e 7 summarizes the r e s u l t s o f the a n a l y s e s . S y n t h e t i c b r a d y k i n i n h y d r o l y z e d f o r 24 h o u r s gave mola r r a t i o s o f amino a c i d s e x a c t l y as would be p r e d i c t e d from a con s i d e r a t i o n o f i t s m o l e c u l a r s t r u c t u r e . However, t u r t l e k i n i n h y d r o l y z e d f o r the same p e r i o d gave d i f f e r e n t molar r a t i o s . 125 T a b l e 7. M o l a r r a t i o s o f amino a c i d s i n p u r i f i e d t u r t l e k i n i n and i n s y n t h e t i c b r a d y k i n i n h y d r o l y z e d f o r 24 and 36 h o u r s . E x p e r i m e n t a l R a t i o s Deduced R a t i o s Amino a c i d . 24 hour h y d r o l y s i s 36 ho u r h y d r o l y s i s BK BK T K a T K b T K a BK TK A r g 2.00 2.00 2.00 2.00 2.00 2 2 Pro 2.93 2.60 6.80 3.00 3.16 3 3 -Gly 1.06 1.28 2.00 1.16 1.15 1 1 Phe 2.01 1.63 0.28 1.68 1.82 2 2 Ser 1.07 1.21 0.05 0.15 0.14 1 0 Thr 0.01 0.12 0.12 0.99 0.99 0 1 Amino a c i d s p r e s e n t i n t r a c e amounts o n l y were: A l a , V a l , L y s , Asp, G l u , Met, l i e , Leu, T y r , and H i s . A l l v a l u e s were l e s s t h a n 0.25, most l e s s t h a n 0.10. a . . . t u r t l e k i n i n from main peak, e l u t i o n volume 120 ( F i g u r e 20) b . . . t u r t l e k i n i n f r o m " s h o u l d e r " , e l u t i o n volume 85 ( F i g u r e 20) 12 6 Compared t o b r a d y k i n i n , t h i s t u r t l e k i n i n h y d r o l y z e d f o r 24 h o u r s had l a r g e amounts o f p r o l i n e (M.R. = 6.8), and g l y c i n e (M.R. = 2.0), low l e v e l s o f p h e n y l a l a n i n e (M.R. = 0.28), 'and o n l y t r a c e s o f o t h e r amino a c i d s . The h i g h p r o l i n e c o n  t e n t , and l a c k o f most o t h e r amino a c i d s , s u g g e s t e d t h a t t h e t u r t l e k i n i n was e i t h e r i n c o m p l e t e l y h y d r o l y z e d , o r m a r k e d l y d i f f e r e n t f r o m b r a d y k i n i n . Samples o f p u r i f i e d t u r t l e k i n i n were t h e r e f o r e h y d r o  l y z e d f o r 36 h o u r s , and a n a l y z e d as b e f o r e . R e s u l t s i n T a b l e 7 show t h a t t h e m o l a r r a t i o s o f A r g ( 2 ) , Pro (3), G l y (1), and Phe (2) were now e x a c t l y as would be p r e d i c t e d f o r b r a d y  k i n i n . However, the t u r t l e k i n i n d i f f e r e d i n t h e a bsence o f one m o l a r r a t i o o f s e r i n e , and i n the p r e s e n c e o f one m o l a r r a t i o o f t h r e o n i n e . A p a r t from t h i s d i s c r e p a n c y , the m o l a r r a t i o s f o r a l l amino a c i d s were e i t h e r v e r y c l o s e t o t h e whole numbers e x p e c t e d f o r b r a d y k i n i n , o r were p r e s e n t i n t r a c e amounts o n l y ; t h i s s u g g e s t e d t h a t t h e t u r t l e k i n i n i n the CM-Sephadex e l u a t e was h i g h l y p u r i f i e d . The t u r t l e k i n i n samples from th e " s h o u l d e r " i n the CM-Sephadex e l u a t e had t h e same a n a l y s i s as the k i n i n from the main peak ( T a b l e 7 ) . T a b l e 8 compares the b i o l o g i c a l a c t i v i t y o f the b r a d y k i n i n and t u r t l e k i n i n p r e p a r a t i o n s t o t h e amount o f p e p t i d e measur ed, f o r each a n a l y s i s . The r a t i o s f o r b r a d y k i n i n measured/ 12 7 T a b l e 8. R e l a t i o n o f b i o l o g i c a l a c t i v i t y o f b r a d y k i n i n and t u r t l e k i n i n t o nM o f k i n i n measured by amino a c i d a n a l y s i s . B i o l o g i c a l a c t i v i t y Amount o f k i n i n nM measured/ o f k i n i n a p p l i e d t o measured, by ana- P e p t i d e a n a l y z e r (nM BK-eq) l y z e r (nM Arg/2) nM a p p l i e d B r a d y k i n i n 37.7 30.4 0.806 18.9 14.7 0.788 14.2 12.4 0.873 T u r t l e k i n i n 58.5 - 6.5* 45.0 0. 769 68.8 ± 2.0 66.6 0.968 27.4 - 2.0 26.1 0.952 * . . . t h e s e v a l u e s were d e t e r m i n e d by r a t u t e r u s a s s a y , u s i n g s y n t h e t i c b r a d y k i n i n , 0.1 mg/ml, as s t a n d a r d . 128 b r a d y k i n i n a p p l i e d i n d i c a t e d t h a t t h e a n a l y z e r measured 80 - 8 5 % o f t h e p e p t i d e a p p l i e d . S i n c e t u r t l e k i n i n was a s s a y e d f o r b i o l o g i c a l a c t i v i t y a g a i n s t s y n t h e t i c b r a d y k i n i n , t h e s i m i l a r r a t i o s o b t a i n e d f o r t u r t l e k i n i n p r o v i d e good e v i  dence t h a t t h i s k i n i n i s f u l l y as a c t i v e as b r a d y k i n i n on t h e r a t u t e r u s . S e r i n e and t h r e o n i n e r u n c l o s e t o one a n o t h e r d u r i n g amino a c i d a n a l y s i s , and t h e r e f o r e a s e r i e s o f t e s t s were c a r r i e d o u t i n o r d e r t o e l i m i n a t e any p o s s i b l e e r r o r o r a m b i g u i t y i n t h e i m p o r t a n t c o n c l u s i o n t h a t t u r t l e k i n i n c o n t a i n e d t h r e o n i n e , b u t no s e r i n e . The i d e n t i t y and r e s o l u  t i o n o f t h e s e two p e a k s was t e s t e d b y a n a l y z i n g t h e f o l l o w i n g m i x t u r e s ; a) an a l i q u o t o f b r a d y k i n i n h y d r o l y s a t e + 40 nM o f L- t h r e o n i n e , and b) an a l i q u o t o f t u r t l e k i n i n h y d r o l y s a t e + 40 nM o f L- s e r i n e . R e s u l t s shown i n F i g u r e 28 i n d i c a t e c l e a r l y t h a t e a c h o f t h e s e t e s t m i x t u r e s d i f f e r e d f r o m i t s s i m p l e h y d r o l y s a t e c o n t r o l o n l y i n t h e a p p e a r a n c e o f a p p r o x i m a t e l y 40 nM o f t h e e x t r a amino a c i d . T h i s r e s u l t e x c l u d e d any p o s s i b i l i t y o f a c o n f u s i o n b e t ween t h e two c l o s e l y r e l a t e d amino a c i d s . D. D i s c u s s i o n and C o n c l u s i o n s Amino a c i d a n a l y s i s o f t u r t l e k i n i n r e v e a l e d m o l a r r a t i o s i d e n t i c a l t o b r a d y k i n i n , e x c e p t f o r t h e a p p a r e n t s u b s t i t u t i o n o f t h r e o n i n e f o r s e r i n e . S i n c e i n c o m p l e t e l y h y d r o l y z e d t u r t l e F i g u r e 28. C o p i e s o f t r a c e s from amino a c i d a n a l y s i s o f h y d r o - l y s a t e s o f s y n t h e t i c b r a d y k i n i n and p u r i f i e d t u r t l e k i n i n showing a s e r i n e peak f o r s y n t h e t i c b r a d y  k i n i n (BK), a t h r e o n i n e peak f o r p u r i f i e d t u r t l e k i n i n (TK), and b o t h t h r e o n i n e and s e r i n e peaks f o r b r a d y k i n i n + s y n t h e t i c t h r e o n i n e (BK + Thr) and f o r t u r t l e k i n i n + s y n t h e t i c s e r i n e (TK + S e r ) . 12 9 F i g u r e 28 I BK TK a < 0) to O *» _c a, — < r - O O O BK-Thr TK + Ser < r — C O -2 O a < cO O 130 k i n i n c o n t a i n e d o n l y t r a c e s o f t h r e o n i n e ( T a b l e 7 ) , t h e r e i s l i t t l e d o u b t t h a t t h e t h r e o n i n e i s an i n t e g r a l p a r t o f t h e m o l e c u l e . The s i m p l e s t c o n c l u s i o n c o n s i s t e n t w i t h a l l a v a i l a b l e e v i d e n c e i s t h a t t u r t l e k i n i n may be an a n a l o g u e o f b r a d y k i n i n i n w h i c h t h r e o n i n e r e p l a c e s s e r i n e , and i t i s t h e r e f o r e 6- t h r - b r a d y k i n i n : 1 2 3 4 5 6 7 8 9 N H 2 - A r g - P r o - P r o - G l y - P h e - S e r - P r o - P h e - A r g - C O O H B r a d y k i n i n N H 2 - A r g - P r o - P r o - G l y - P h e - T h r - P r o - P h e - A r g - C O O H 6 - t h r - b r a d y k i n i n , t h e p r o p o s e d t u r t l e k i n i n S u p p o r t f o r t h i s h y p o t h e s i s comes f r o m t h e p h a r m a c o l o g i  c a l p r o p e r t i e s o f t u r t l e k i n i n . 6 - t h r - b r a d y k i n i n was s y n t h e  s i z e d and s t u d i e d b y S c h r o d e r and Hempel ( 1 9 6 4 ) , and a l t h o u g h i t was as a c t i v e as b r a d y k i n i n on t h e r a t u t e r u s , t h e r a t i o o f r a t u t e r u s / g u i n e a p i g i l e u m a c t i v i t y was 3, and t h e r a t i o o f r a t u t e r u s / r a b b i t b l o o d p r e s s u r e a c t i v i t y was 10. These d a t a were o b t a i n e d u s i n g s i m p l e c o m p a r i s o n s o f t h e t h r e s h o l d dose f o r r a t u t e r u s and g u i n e a p i g i l e u m a c t i v i t i e s , and s i n g l e r e a d i n g s f r o m l o g d o s e - r e s p o n s e c u r v e s f o r r a b b i t b l o o d p r e s s u r e . No e s t i m a t e s o f e r r o r s o f t h e s e a s s a y s a r e p o s s i b l e . T h e r e f o r e , t h e r a t i o s p r e s e n t e d b y t h e s e w o r k e r s must be r e g a r d e d as a p p r o x i m a t e , and c o u l d e a s i l y have 9 5 % c o n f i d e n c e l i m i t s o f - 100%. N o n e t h e l e s s , t h e v a l u e s g i v e n c o n t r a s t 131 w i t h t h e wide range o f r a t i o s o b t a i n e d f o r many o t h e r s y n t h e t i c b r a d y k i n i n a n a l o g u e s ( S c h r 6 d e r and Ltibke, 1966), and i n t h e s e c i r c u m s t a n c e s t h e y may be r e g a r d e d as b e i n g i n good a g r e e  ment w i t h v a l u e s o b t a i n e d f o r t u r t l e k i n i n ( T a b l e 6 ) . T u r t l e k i n i n i s a l s o s i m i l a r t o s y n t h e t i c 6 - t h r - b r a d y k i n i n i n t h a t b o t h a r e f u l l y as a c t i v e as b r a d y k i n i n on t h e r a t u t e r u s ( T a b l e 8; S c h r o d e r and Hempel, 1964). 132 CHAPTER 6. PRELIMINARY STUDIES OF THE EFFECT OF BRADYKININ ON BLOOD PRESSURE I N THE TURTLE Once i t h a s been e s t a b l i s h e d t h a t t u r t l e k i n i n d i f f e r s i n s t r u c t u r e f r o m b r a d y k i n i n ( C h a p t e r 5 ) , i t becomes e s s e n t i a l t o examine t h o r o u g h l y t h e e f f e c t s o f t h i s new k i n i n on s u c h a s p e c t s o f t u r t l e p h y s i o l o g y as t h e c i r c u l a t i o n . Due t o a l a c k o f p u r i f i e d t u r t l e k i n i n , i t has n o t b e e n p o s s i b l e t o c a r r y o u t more t h a n p r e l i m i n a r y s t u d i e s w i t h t h e u s e o f s y n  t h e t i c b r a d y k i n i n . T h e r e i s no p u b l i s h e d r e c o r d o f a t t e m p t s t o examine t h e e f f e c t s o f mammalian p l a s m a k i n i n s on b l o o d p r e s s u r e o f any r e p t i l e , a l t h o u g h i t i s known t h a t h i g h c o n c e n t r a t i o n s o f b r a d y k i n i n and l y s - b r a d y k i n i n c a u s e l i t t l e change i n b l o o d p r e s s u r e o f b i r d s ( E r d o s , 1966) and f i s h ( V o g e l e t a l , 1 9 6 9 ) . These r e s u l t s a r e n o t s u r p r i s i n g , s i n c e n e i t h e r o f t h e s e g r o u p s a p p e a r t o have a c o m p l e t e p l a s m a k a l l i k r e i n - k i n i n s y s t e m ( C h a p t e r 3 ) . By c o n t r a s t , t u r t l e p l a s m a does have a f u l l y d e v e l o p e d k i n i n p r o d u c i n g enzyme s y s t e m ( C h a p t e r 4) and i n  v e s t i g a t i o n o f t h e e f f e c t s o f b r a d y k i n i n on t h e b l o o d p r e s s u r e o f t h e s e a n i m a l s m i g h t be a more p r o m i s i n g l i n e o f e n q u i r y , and w o u l d a t l e a s t s e r v e as a u s e f u l b a s e f o r c o m p a r i s o n i n f u t u r e e x p e r i m e n t s w i t h 6 - t h r - b r a d y k i n i n , t h e p r o p o s e d t u r t l e . k i n i n . 133 Many w o r k e r s have f o u n d t h a t p r e - t r e a t m e n t o f a mammal w i t h an a l p h a - a d r e n e r g i c b l o c k e r (e.g., phenoxybenzamine) does n o t r e d u c e t h e v a s o d i l a t o r and h y p o t e n s i v e e f f e c t o f b r a d y k i n i n and i n most c a s e s a c t u a l l y i n c r e a s e s t h i s r e s p o n s e ( H a r r i s o n e t a l , 1968; Lang and P e a r s o n , 1968; L l o y d , 1962; Nakano, 1965; Rocha e S i l v a e t a l , 1960). The same i s t r u e o f t h e v e n o c o n s t r i c t o r a c t i o n o f b r a d y k i n i n i n r a b b i t s (Bobbin and Guth, 1968) and o f b r o n c h o c o n s t r i c t i o n i n t h e g u i n e a p i g ( C o l l i e r , e t a l , 1965). T h e r e a r e many r e p o r t s o f a b i p h a s i c r e s p o n s e t o i n t r a - a r t e r i a l i n j e c t i o n s o f k i n i n s (Lang and Pe a r s o n , 1968; A l - K a t i b and Baba, 1969); t h e r e i s an i n i t i a l h y p o t e n s i o n , f o l l o w e d by a p r e s s o r r e s p o n s e . The l a t t e r phase • i s i n p a r t due t o t h e r e l e a s e o f c a t e c h o l a m i n e s f r o m t h e a d r e n a l m e d u l l a ( F e l d b e r g and L e w i s , 1964) and can be a b o l i s h  ed by b l o c k a d e o f t h e autonomic n e r v o u s system. A l l w o r k e r s s t u d y i n g _in v i v o r e s p o n s e s have found, t h a t i n t r a - a r t e r i a l , r a t h e r t h a n i n t r a v e n o u s i n j e c t i o n s , have the l o w e s t t h r e s h o l d . I t was the o b j e c t o f t h e s e p r e l i m i n a r y s t u d i e s t o c h a r a c t  e r i z e t h e r e s p o n s e o f t u r t l e b l o o d p r e s s u r e t o i n t r a - a r t e r i a l i n j e c t i o n o f b r a d y k i n i n , and t o d e t e r m i n e whether a d r e n e r g i c b l o c k a d e would m o d i f y t h i s r e s p o n s e . A. Method T u r t l e s (P_. s c r i p t a e l e g a n s ) 1.0 - 2.0 k g i n w e i g h t were a n e s t h e t i z e d by subcutaneous i n j e c t i o n o f sodium p e n t o b a r b i t a l 134 (60 mg/kg). The a n i m a l was t a p e d u p s i d e down t o a p o l y s t y r e n e f o r m , and a s q u a r e a b o u t 5 x 5 cm was c u t i n t h e p l a s t r o n w i t h a bone saw ( D e s o u t t e r C o . ) . Removal o f t h i s p o r t i o n o f s h e l l , and c a r e f u l d i s s e c t i o n o f u n d e r l y i n g m u s c l e r e v e a l e d t h e h e a r t , and m a j o r b l o o d v e s s e l s l e a d i n g f r o m i t . U s i n g w a t c h m a k e r ' s f o r c e p s and f i n e s c i s s o r s , t h e a r t e r i e s were f r e e d o f mesen t e r i e s and c o n n e c t i v e t i s s u e . C a n n u l a t i o n s were a c c o m p l i s h e d b y p l a c i n g a c l a m p on t h e v e s s e l , m a k i n g a s m a l l V - s h a p e d i n c i s i o n i n i t s v e n t r a l s u r f a c e d i s t a l t o t h e c l a m p , and i n s e r t  i n g t h e b e v e l e d end o f t h e p o l y e t h y l e n e c a n n u l a (PE 60, 190 o r 200, I n t r a m e d i c ) t h r o u g h t h e i n c i s i o n and t o w a r d s t h e h e a r t . The c a n n u l a was s e c u r e d b y a l i g a t u r e p r e v i o u s l y p l a c e d a r o u n d t h e a r t e r y . Where i n j e c t i o n s were t o be g i v e n i n t o t h e same v e s s e l , a s i z e #2 7 n e e d l e c o n n e c t e d t o PE 10 t u b i n g was i n s e r t e d i n t o t h e c a n n u l a a b o u t V f r o m t h e b e v e l e d end, p r i o r t o i t s p l a c e m e n t i n t h e v e s s e l . I n e a ch e x p e r i m e n t two o f t h e f o l l o w i n g a r t e r i e s were c a n n u l a t e d ; l e f t a o r t a , r i g h t s u b c l a v i a n , l e f t s u b c l a v i a n , and l e f t p u l m o n a r y a r t e r i e s (see W h i t e and R o s s , 1966, f o r a d i a  gram o f t h e h e a r t and m a j o r b l o o d v e s s e l s o f t h i s t u r t l e ) . B l o o d p r e s s u r e was r e c o r d e d i n mm Hg b y means o f a S t a t h a m P 23BB t r a n s d u c e r c o u p l e d t o a 6 c h a n n e l Beckman d y n o g r a p h . B l o o d f l o w was a l s o r e c o r d e d f r o m one o f t h e s e v e s s e l s , u s i n g a D o p p l e r u l t r a s o n i c f l o w m e t e r c o u p l e d t o t h e Beckman r e c o r d e r . 135 The c o l l a r bore) was t i e d around t h e i n t a c t a r t e r y , and any e x t r a s p a ce f i l l e d i n w i t h A q u a s o n i c g e l . The f o l l o w i n g a r b i t r a r y measure o f mean b l o o d f l o w was used; h e i g h t o f p u l s e peak (cm) x h e a r t r a t e ( b e a t s / m i n ) . These v a l u e s s h o u l d be d i r e c t l y p r o p o r t i o n a l t o mean b l o o d f l o w . I n j e c t i o n s o f a l l a g e n t s were g i v e n i n t r a - a r t e r i a l l y . A c t i v e a g e n t s were d i s s o l v e d i n b u f f e r e d t u r t l e s a l i n e (Appen d i x B) and washed i n w i t h s a l i n e so t h a t t h e t o t a l volume o f the i n j e c t i o n n e v e r exceeded 0.3 ml. Drugs u s e d were b r a d y k i n i n , L - e p i n e p h r i n e , a c e t y l c h o l i n e HC1, phenoxybenzamine, p r o p a n o l o l , a t r o p i n e , and sodium p e n t o b a r b i t a l (Appendix A ) . B. R e s u l t s A s e r i o u s f l a w i n most e x p e r i m e n t s was the s t e a d y d e c r e a s e i n b l o o d p r e s s u r e , though b l o o d l o s s was i n s i g n i f i c a n t . V a l u e s i n d i c a t i n g the magnitude o f t h i s change i n 6 e x p e r i m e n t s a r e g i v e n i n T a b l e 9. A s s o c i a t e d w i t h t h e l o s s o f b l o o d p r e s s u r e was a g e n e r a l d e c r e a s e i n s e n s i t i v i t y o f t h e p r e p a r a t i o n t o any d r u g . N o n e t h e l e s s , a l l a n i m a l s gave p r e s s o r r e s p o n s e s t o b o t h a d r e n a l i n e and b r a d y k i n i n i n r e l a t i v e l y low c o n c e n t r a t i o n s . On a p e r w e i g h t b a s i s a d r e n a l i n e was always more p o t e n t t h a n b r a d y k i n i n . T h r e s h o l d doses f o r b r a d y k i n i n were as low as 0.5 ug/kg body w e i g h t , b u t were as h i g h as 10 ug/kg i n some p r e  p a r a t i o n s . T y p i c a l r e s p o n s e s t o s y n t h e t i c b r a d y k i n i n and a d r e n a l i n e 136 T a b l e 9. D e c r e a s e i n b l o o d p r e s s u r e o f 6 t u r t l e s o v e r the 5 10 h o u r e x p e r i m e n t a l p e r i o d . E x p e r i m e n t * A r t e r i a l B l o o d P r e s s u r e (mm Hg) I n i t i a l F i n a l 1 3 6/29 14/11 2 2 8/10 5/1 3 2 6/21 10/5 4 18/9 9/3 5 16/13 11/5 6 2 9/25 14/7 measured i n r i g h t s u b c l a v i a n a r t e r y . 137 are g i v e n i n F i g u r e 29 a. The reduced responses of the same animal to h i g h e r doses of the same agents a f t e r a p p l i c a t i o n of 1.2 mg phenoxybenzamine/kg and 5.4 ug p r o p a n o l o l / k g are shown i n F i g u r e 29 b. F i g u r e 30 i s a summary of the e f f e c t s of phenoxybenzamine ' on b l o o d p r e s s u r e responses to b r a d y k i n i n and a d r e n a l i n e , i n t h r e e d i f f e r e n t a n i m a l s . I n j e c t i o n s of p r o p a n o l o l t o 10 ug/kg had no e f f e c t on these responses, a l t h o u g h the drug seemed to s t a b i l i z e h e a r t r a t e . A l l of these p r e s s o r responses were accom panied by a decrease i n b l o o d f l o w (e.g., F i g u r e 29 a ) . However, minor changes i n the p o s i t i o n of the c o l l a r - s h a p e d probe around the v e s s e l d i r e c t l y a f f e c t e d the f l o w i t s e l f ; hence "base l i n e " a c t i v i t y was c o n s t a n t l y changing, making these r e s u l t s u n s a t i s f a c t o r y . In any case, a dose of phenoxybenzamine s u f f i c  i e n t to reduce the e f f e c t of a d r e n a l i n e always s i g n i f i c a n t l y lowered the a c t i v i t y of b r a d y k i n i n . Treatment w i t h 1.2 mg phe noxybenzamine/kg, or any l a r g e r amount, always r e s u l t e d i n a t l e a s t a 10 f o l d r e d u c t i o n i n responses to both compounds. C. D i s c u s s i o n and C o n c l u s i o n s White and Ross (1966) found t h a t normal a o r t i c b l o o d p r e s s u r e i n j?. s c r i p t a elegans was about 30 mm Hg a t s y s t o l e , a v a l u e s i m i l a r to those o b t a i n e d f o r i n i t i a l p r e s s u r e s i n the p r e s e n t study (Table 9). However, the c o n t i n u a l l o s s of p r e s s u r e i n a l l p r e p a r a t i o n s suggested t h a t the a n e s t h e t i c and/or s u r g i c a l F i g u r e 29 a. T y p i c a l r e s p o n s e s o f t u r t l e b l o o d p r e s s u r e and f l o w t o i n t r a - a r t e r i a l i n j e c t i o n s o f s y n t h e t i c b r a d y k i n i n (B) and a d r e n a l i n e (Ad). b._ Reduced r e s p o n s e s o f t h e same a n i m a l t o h i g h e r doses o f b r a d y k i n i n and a d r e n a l i n e , a f t e r a d m i n i s  t r a t i o n o f 1.2 mg phenoxybenzamine/kg and 5.4 ug p r o p a n o l o l / k g . 138 Figure 29 a Z I e 5ug B 20 • 1 « • mm• 6 )0 . 2 ug Ad b Time, sec '' ' Blood Flow ''M^M-M^M.n^uM.MMHHuuM.uMHn,,,.^, Systemic Blood Pressure Pulmonary Blood Pressure • 1 Time, sec "<'"" "''"''/''I'jI'lJ— Blood Flow wwwmmmmmmmmmwmm—Systemic Blood Pressure lOug B l O u g Ad :;mmmh- PuI monory Blood Pressure F i g u r e 30. E f f e c t o f a d r e n e r g i c b l o c k a d e on t h e p r e s s o r r e s p o n s e t o b r a d y k i n i n i n 3 d i f f e r e n t t u r t l e s . S o l i d l i n e s a r e a p p r o x i m a t e l o g d o s e - r e s p o n s e c u r v e s f o r s y n t h e t i c b r a d y k i n i n (BK) and a d r e n a l i n e (Ad) p r i o r t o b l o c k  ade. Dashed l i n e s a r e a p p r o x i m a t e l o g d o s e - r e s p o n s e c u r v e s a f t e r a p p l i c a t i o n o f 1.2 mg phenoxybenzamine/ k g . 139 I | I i Figure 30 140 t r e a t m e n t s i g n i f i c a n t l y a l t e r e d t h e hemodynamics o f t h e s e a n i m a l The e x t r e m e l y low p r e s s u r e s e n c o u n t e r e d n e a r t h e end o f some e x p e r i m e n t s (e.g., #2, T a b l e 9) p r o b a b l y i n d i c a t e t h a t t h e a n i m a l s were n e a r d e a t h ; r e s p o n s e s o b t a i n e d f r o m such p r e p  a r a t i o n s a r e o f d u b i o u s v a l u e . Lang and P e a r s o n (1968), P e a r s o n and Lang (1969), and A l - K a t i b and Baba (1969) f o u n d t h a t t h e s i z e o f t h e p r e s s o r r e  sponses t o i n t r a - c a r o t i d i n j e c t i o n o f k i n i n s was i n v e r s e l y r e l a t e d t o a r t e r i a l p r e s s u r e s i n dogs and r a b b i t s . A t low p r e s s u r e s t h e r e was sometimes no h y p o t e n s i v e component t o the r e s p o n s e . S i n c e the p r e s s o r r e s p o n s e , w h i c h p e r s i s t e d i n a d r e n a l e c t o m i z e d a n i m a l s (Lang and Pearson, 1968) c o u l d be r e d u c e d b y a d r e n e r g i c b l o c k a d e o r d e c e r e b r a t i o n s , i t was s u g g e s t e d t h a t p a r t o f t h i s r e s p o n s e c o u l d be c e n t r a l l y m e d i a t e d . However, o t h e r w orkers (Lecomte, e t a l , 1964) have found t h a t p r e s s o r e f f e c t s can be c o m p l e t e l y a b o l i s h e d by a d r e n a l e c t o m y i n the r a b b i t . The h y p e r t e n s i v e e f f e c t o f b r a d y k i n i n i n the t u r t l e , w h i c h i s a l s o a b o l i s h e d by a d r e n e r g i c b l o c k a d e , t h e r e f o r e has c e r t a i n s i m i l a r i t i e s w i t h t h e o b s e r v a t i o n s by the a u t h o r s mentioned above. However, the t u r t l e d i f f e r e d from a l l mammals s t u d i e d i n t h a t no h y p o t e n s i v e r e s p o n s e was e v e r evoked by any dose o f b r a d y k i n i n , u p t o 50 ug/kg body w e i g h t . I t i s t h e r e f o r e i m p o s s i b l e t o c o n c l u d e t h a t t h e s e p r e l i m i n a r y s t u d i e s p r o v i d e 141 e v i d e n c e t h a t b r a d y k i n i n has an e f f e c t on t u r t l e b l o o d p r e s s u r e a n a l a g o u s t o i t s e f f e c t i n mammals. 142 CHAPTER 7. SUMMARY AND GENERAL DISCUSSION Whole b l o o d and plasma o f v a r i o u s f i s h , a m p hibians, r e p t i l e s and mammals was compared w i t h r e s p e c t t o t h e t y p e o f k a l l i k r e i n - k i n i n s y s t e m each c o n t a i n e d ( Chapter 3 ) . The f o l l o w i n g components o f t h e s y s t e m ( o u t l i n e d i n F i g u r e 2) were t e s t e d as f o l l o w s : Component K i n i n o g e n , i n whole b l o o d K i n i n o g e n , i n plasma K i n i n a s e , i n u n d e n a t u r e d plasma A c t i v a t a b l e plasma k a l l i  k r e i n s i n u n d e n a t u r e d plasma K i n i n o g e n , i n u n d e n a t u r e d plasma T e s t t r y p s i n on d e n a t u r e d b l o o d t r y p s i n and hog p a n c r e a s k a l l i k r e i n on d e n a t u r e d plasma a. r a t e o f i n a c t i v a t i o n o f s y n t h e t i c b r a d y k i n i n i n plasma b. e f f e c t o f 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 k i n i n a s e i n  h i b i t o r on k i n i n a s e a c t i v i t y e f f e c t i v e n e s s o f g l a s s s u r  f a c e i n a c t i v a t i n g k a l l i k r e i n i n t h e p r e s e n c e o f k i n i n a s e i n h i b i t o r e f f e c t i v e n e s s o f g l a s s s u r  f a c e , t r y p s i n , and hog pan c r e a s k a l l i k r e i n i n r e l e a s  i n g k i n i n i n t h e p r e s e n c e o f k i n i n a s e i n h i b i t o r T e s t e d plasmas were c l e a r l y s e p a r a t e d i n t o two g r o u p s : Group 1. Mammalian and t u r t l e plasmas. H e a t e d plasma r e  l e a s e d r e l a t i v e l y l a r g e amounts o f k i n i n when i n c u b a t e d w i t h 143 t r y p s i n o r hog p a n c r e a s k a l l i k r e i n , a l w a y s l e s s w i t h h o g p a n  c r e a s k a l l i k r e i n . K i n i n a s e a c t i v i t y i n i n t a c t p l a s m a s was t o t a l l y i n h i b i t e d b y l o w d o s e s o f 8-HQSO^, and i n t h e p r e  s e n c e o f t h i s i n h i b i t o r , g l a s s b e a d s and t r y p s i n c a u s e d t h e r e l e a s e o f endogenous k i n i n . G r o up 2. T r o u t , f r o g and Amphiuma p l a s m a s . These p l a s m a s were s i m i l a r t o t h o s e i n g r o u p 1 i n o n l y two r e s p e c t s ; a) t h e p o s s e s s i o n o f k i n i n a s e a c t i v i t y , and b) t h e r e l e a s e o f b i o  l o g i c a l a c t i v i t y f r o m h e a t e d p l a s m a , b y hog p a n c r e a s k a l l i  k r e i n . T r y p s i n r e l e a s e d o n l y t r a c e s o f a k i n i n - l i k e f a c t o r f r o m h e a t e d p l a s m a s , w h i l e h o g p a n c r e a s k a l l i k r e i n l i b e r a t e d f a r l a r g e r amounts o f a c t i v i t y . K i n i n a s e i n i n t a c t p l a s m a s was o n l y p a r t i a l l y i n h i b i t e d b y h i g h d o s e s o f 8-HQSO4, and no t r e a t m e n t ( g l a s s , t r y p s i n , o r hog p a n c r e a s k a l l i k r e i n ) c a u s e d t h e r e l e a s e o f a k i n i n - l i k e f a c t o r i n u n d e n a t u r e d p l a s m a . The s u g g e s t i o n t h a t t u r t l e p l a s m a c o n t a i n e d a k a l l i k r e i n - k i n i n s y s t e m s i m i l a r t o t h a t o f mammals was t e s t e d more r i g o r  o u s l y b y c o m p a r i n g t h e two s y s t e m s i n a v a r i e t y o f e x p e r i m e n t s ( C h a p t e r 4 ) . T u r t l e p l a s m a was f o u n d t o be s i m i l a r t o mammalian p l a s m a i n t h e f o l l o w i n g c h a r a c t e r i s t i c s : 1. G l a s s a c t i v a t i o n i n t h e p r e s e n c e o f 8-HQSO4 p r o d u c e d k i n i n , t h e r a t e b e i n g d i r e c t l y p r o p o r t i o n a l t o t h e g l a s s s u r f a c e a r e a . 2. R a t e s o f a c t i v a t i o n and de c a y o f p l a s m a k a l l i k r e i n were 144 s i m i l a r . 3. Plasma k a l l i k r e i n may be a c t i v a t e d b y t r y p s i n , as i t i s i n mammals. 4. P r e k a l l i k r e i n was p r e s e n t i n amounts f a r l a r g e r t h a n r e q u i r e d t o deg r a d e a l l k i n i n o g e n . 5. A t l e a s t two t y p e s o f k i n i n o g e n were p r e s e n t i n t u r t l e p lasma. 6. The e f f e c t o f t e m p e r a t u r e on the r a t e o f k i n i n r e l e a s e b y plasma k a l l i k r e i n s was s i m i l a r i n t u r t l e and r a t plasma. Mammalian plasma u s u a l l y r e l e a s e d more k i n i n / m l t h a n d i d t u r t l e plasma, b u t no q u a l i t a t i v e d i f f e r e n c e s i n t h e mechanisms o f r e l e a s e were n o t e d . A l t h o u g h no e v i d e n c e had been o b t a i n e d t o s u g g e s t t h a t t h e t u r t l e plasma k a l l i k r e i n - k i n i n s ystem d i f f e r e d f r o m t h a t o f mammals, the p o s s i b i l i t y r e m a i n e d t h a t the a c t i v e p e p t i d e s r e l e a s e d by the a c t i o n o f t h e enzyme systems might be d i f f e r  e n t . G l a s s r e l e a s e d t u r t l e k i n i n was t h e r e f o r e s u b j e c t e d t o a v a r i e t y o f p h a r m a c o l o g i c a l and c h e m i c a l t e s t s . Two p h a r m a c o l o g i c a l r a t i o s , namely g u i n e a p i g i l e u m / r a t u t e r u s a c t i v i t y , and r a b b i t b l o o d p r e s s u r e / r a t u t e r u s a c t i v i t y , were s i g n i f i c a n t l y g r e a t e r than one, b e i n g 2.5 and 7.0 r e s p e c t i v e l y . S i n c e the a s s a y s a l l u s e d s y n t h e t i c b r a d y k i n i n as s t a n d a r d , t h e s e v a l u e s s t r o n g l y s u g g e s t e d t h a t t u r t l e k i n i n was c h e m i c a l l y d i f f e r e n t from t h e mammalian k i n i n . 145 P u r i f i e d t u r t l e k i n i n was compared to synthetic bradykinin i n gel f i l t r a t i o n experiments (Sephadex G-25), i n ion exchange chromatography (CM-Sephadex), and i n paper chromatography i n b'utanol-acetic acid-water. While these t e s t s c l e a r l y d i s  tinguished between bradykinin and l y s - b r a d y k i n i n , t u r t l e k i n i n and bradykinin were not separated. However, amino acid a n a l y s i s of the purest preparation of t u r t l e k i n i n established that i t was chemically d i f f e r e n t . Repeated analyses indicated that molar r a t i o s of p r o l i n e (3), arginine (2), phenylalanine (2), and g l y c i n e (1) were i d e n t i c a l to those of bradykinin, but that t u r t l e k i n i n d i f f e r e d from the mammalian k i n i n i n the presence of one molar r a t i o of threonine, and i n the absence of one molar r a t i o of serine. This evidence l e d to the hypothesis that t u r t l e k i n i n was 6-thr-bradykinin, as f o l l o w s : NH2-Arq-Pro-Pro-Phe-Gly-Thr-Pro-Phe-Arg-C00H Proposed t u r t l e k i n i n Marked s i m i l a r i t i e s i n the pharmacological a c t i v i t y of synthetic 6-thr-bradykinin (Schrtider and Hempel, 1964) and t u r t l e k i n i n i n c e r t a i n bioassays offered support f o r t h i s hypothesis. Although f i n a l conclusions must await sequence analysis of the a c t i v e peptide, the evidence presented i n t h i s thesis suggests that t u r t l e plasma contains a k a l l i k r e i n - k i n i n system 146 v e r y s i m i l a r t o t h a t o f mammalian plasma, b u t b a s e d i n s t e a d on t h r e o n i n e c o n t a i n i n g p e p t i d e s , and c u l m i n a t i n g i n the p r o  d u c t i o n o f 6 - t h r - b r a d y k i n i n as the n a t u r a l plasma k i n i n o f t h e t u r t l e J?. s c r i p t a e l e g a n s . I t seems p o s s i b l e t h a t t r e a t  ment o f t u r t l e plasma w i t h a c i d o r g l a n d u l a r k a l l i k r e i n c o u l d r e s u l t i n the p r o d u c t i o n o f m e t - l y s - 6 - t h r - b r a d y k i n i n o r l y s - 6 - t h r - b r a d y k i n i n , i n t h e same way t h a t mammalian plasma g i v e s r i s e t o m e t - l y s - b r a d y k i n i n and l y s - b r a d y k i n i n (see P i e r c e , 1968) . Non-mammalian k i n i n s c l o s e l y r e l a t e d t o b r a d y k i n i n have a l r e a d y been i d e n t i f i e d ( P i s a n o , 1968), b u t t o d a t e a l l mod i f i c a t i o n s a r e by a d d i t i o n o f C o r N t e r m i n a l groups t o the b r a d y k i n i n m o i e t y ( C o l l i e r , 1968; S c h a c h t e r , 1969; P i s a n o , 1968) . T h i s r e p o r t i s the f i r s t e v i d e n c e t o s u g g e s t t h a t 6- t h r - b r a d y k i n i n o c c u r s i n n a t u r e , and i t i s the f i r s t example o f a n a t u r a l l y o c c u r r i n g a n a l o g u e o f b r a d y k i n i n i n which t h e r e has been a r e p l a c e m e n t w i t h i n the f u n d a m e n t a l n o n a p e p t i d e c h a i n . As a r e s u l t o f an e x h a u s t i v e s t u d y o f s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s i n s y n t h e t i c a n a l o g u e s o f b r a d y k i n i n , Schrtider and Hempel (1964) t e n t a t i v e l y c l a s s i f i e d i n d i v i d u a l amino a c i d p o s i t i o n s o f t h e b r a d y k i n i n m o l e c u l e a c c o r d i n g to t h e i r r e  l a t i v e i n f l u e n c e on b i o l o g i c a l a c t i v i t y ; the s e r i e s , i n o r d e r o f d e s c e n d i n g importance, i s as f o l l o w s : 147 7,8> 1> 9> 5> 4> 7> 2) 3> 6 Changes i n the residue at p o s i t i o n 6 have the l e a s t e f f e c t on b i o l o g i c a l a c t i v i t y . Therefore, a mutation which could cause the interchange of serine and threonine at t h i s p o s i t i o n i s a reasonable p o s s i b i l i t y , since i t might w e l l leave the molecule, and therefore the animal, p h y s i o l o g i c a l l y f u n c t i o n a l . In a d d i t i o n , the interchange of these two residues requires but a s i n g l e base change i n the genetic code (Vleigenthart and Versteeg, 1967). This r a i s e s the p o s s i b i l i t y that other mutations could, have produced yet d i f f e r e n t analogues i n other vertebrate plasmas, and i t i s possible that further work could reveal a pattern of molecular evolution throughout the v e r t e  brate tree. The d i s t r i b u t i o n of plasma k i n i n s i n major vertebrate groups i s summarized, i n the f o l l o w i n g diagram: Birds Mammals b r a d y k i n i n - l i k e peptide (Erdos, 1967) \ o r n i t h o k i n i n ? (Werle, 1966) bradykinin ( E l l i o t t et a l 1961) Reptiles ( t u r t l e s , a l l i g a t o r s ? ) I 6-thr-bradykinin (Dunn and Perks, 1970) F i s h , Amphibians, Reptiles (snakes) no evidence f o r a bradykinin analogue 148 O b s e r v a t i o n s i n t h e plasmas o f b i r d s , r e p t i l e s and f i s h a r e p i t i f u l l y s c a r c e . T h e r e f o r e , c o n n e c t i n g l i n e s i n t h i s d i a  gram do n o t i m p l y e v o l u t i o n a r y pathways f o r plasma k i n i n s . A d i s c u s s i o n o f the e v o l u t i o n o f plasma k i n i n s and t h e enzyme systems which r e l e a s e them i s d i f f i c u l t a t t h i s t i m e, s i n c e so few s p e c i e s have been examined. However, the f a c t t h a t some r e p t i l e s ( t u r t l e s and a l l i g a t o r s ) can p r o d u c e an a c t i v e p lasma k i n i n , w h i l e o t h e r r e p t i l e s (snakes) c a n n o t do so, e n c o u r a g e s s p e c u l a t i o n t h a t the plasma k a l l i k r e i n - k i n i n system may have a r i s e n w i t h i n t h i s group. However, i n v e s t i g a t i o n s t h r o u g h o u t t h i s d i v e r s e c l a s s o f v e r t e b r a t e s , and a more comp l e t e s u r v e y o f a m p h i b i a n and f i s h plasmas must p r e c e d e 'any m e a n i n g f u l d i s c u s s i o n o f the o r i g i n o f t h i s enzyme system, and t h e a c t i v e p o l y p e p t i d e s p r o d u c e d by i t . F u r t h e r s t u d i e s o f b i r d plasma a r e r e q u i r e d t o e l u c i d a t e the n a t u r e o f i t s k i n i n s . Is the b r a d y k i n i n - l i k e f a c t o r d e s c r i b e d b y E r d o s e t a l (1967) b r a d y k i n i n , 6 - t h r - b r a d y k i n i n , o r a n o t h e r c l o s e l y r e l a t e d p e p t i d e ? The answer t o t h i s ques t i o n must be known i n o r d e r t o p o s i t i o n the a v i a n plasma k a l l i k r e i n - k i n i n system r e l a t i v e to t h a t o f r e p t i l e s and mammals. In b i r d s , the n a t u r e o f o r n i t h o k i n i n i s a s p e c i a l problem, s i n c e i t s s t r u c t u r e and pharmacology a r e e n t i r e l y d i f f e r e n t from b r a d y k i n i n . I t i s t h e r e f o r e n o t a t r u e k i n i n (see D e f i n i  t i o n s and A b b r e v i a t i o n s , p xvi) . T h i s type o f system c o u l d 149 e x i s t i n many v e r t e b r a t e plasmas, and be u n d e t e c t e d a t t h i s t i m e , s i n c e o r n i t h o k i n i n i s i n a c t i v e on mammalian t i s s u e s (Werle, e t a l , 1966) and would n o t be n o t e d i n s t a n d a r d b i o - as*say p r o c e d u r e s . I t s h o u l d be p o i n t e d o u t t h a t t h e b i o a s s a y s u s e d t o c h a r a c t e r i z e k i n i n s f r o m a l l s o u r c e s ( e x c e p t o r n i t h o k i n i n ) have i n v o l v e d mammalian t i s s u e s . A s s a y s u s i n g t i s s u e s f r o m the a n i m a l w h i c h p r o d u c e d th e b r a d y k i n i n a n a l o g u e might r e s u l t i n a r a n k i n g o f amino a c i d p o s i t i o n s q u i t e d i f f e r e n t f r o m t h a t p r e s e n t e d on page 147 f o r b r a d y k i n i n . T h e r e f o r e , t r e a t  ment o f p o i k i l o t h e r m plasmas w i t h t r y p s i n and g l a s s (and other, k i n i n - p r o d u c i n g agents) might appear t o r e l e a s e no k i n i n s i m p l y b e c a u s e the f a c t o r s l i b e r a t e d were n o t a c t i v e on the i s o l a t e d r a t u t e r u s . S i m i l a r problems were i n v o l v e d i n methods u s e d i n p r e  l i m i n a r y s t u d i e s o f the p h y s i o l o g y o f k i n i n s i n t u r t l e s (Chap t e r 6 ) . The b r i e f e x p e r i m e n t s on the e f f e c t o f b r a d y k i n i n on t u r t l e b l o o d p r e s s u r e do n o t b e g i n t o s u g g e s t a p h y s i o l o g i c a l r o l e f o r b r a d y k i n i n , i f o n l y b e c a u s e b r a d y k i n i n i s not the n a t u r a l plasma k i n i n o f t h i s t u r t l e . A l t h o u g h r e s u l t s were e n c o u r a g i n g i n t h a t low doses o f b r a d y k i n i n u s u a l l y c a u s e d b l o o d p r e s s u r e changes, f u t u r e e x p e r i m e n t s s h o u l d i n v e s t i g a t e t h e e f f e c t s o f 6 - t h r - b r a d y k i n i n on the t u r t l e i t s e l f . Responses might be enhanced by the use o f i n v i v o k i n i n a s e i n h i b i t o r s 150 (Erdos and Yang, 1966). The use o f p e r f u s e d o r g a n s , such as t h e b l o o d p e r f u s e d l u n g ( a f t e r Hauge, e t a l , 1966) m i g h t a l s o be a f r u i t f u l a p p r o a c h . In t h e end, i t i s e n t i r e l y p o s s i b l e t h a t k i n i n s have l i t t l e e f f e c t on t h e v a s c u l a t u r e o f t h e t u r t l e , and o f o t h e r p o i k i l o t h e r m s , and t h a t t h e i r r e a l p h y s i o l o g i c a l f u n c t i o n l i e s i n a h i t h e r t o u n s u s p e c t e d r o l e . T h i s p r o b l e m i s a l s o o f g r e a t c o n c e r n i n t h e numerous s t u d i e s o f mammalian plasma k i n i n s , s i n c e many wo r k e r s f e e l t h a t t h e most i m p o r t a n t e f f e c t s o f t h e p e p t i d e s may s t i l l be unknown. In any c a s e , the d a t a p r e s e n t e d i n t h i s t h e s i s shows t h a t the p h y s i o l o g i c a l f u n c t i o n i n t h e t u r t l e w ould depend on t h e a c t i o n s o f a new plasma . k i n i n , 6 - t h r - b r a d y k i n i n . 151 LITERATURE CITED A l - K a t i b , H.A., and W.I. Baba. 1969. The a c t i o n o f c e r t a i n h a l o a l k y l a m i n e s on some b i o l o g i c a l a c t i v i t i e s o f b r a d y  k i n i n . B r . J . P h a r m a c o l . 36:635. A n a s t a s i , A., V. E r s p a m e r , and G. B e r t a c c i n i . 1965. O c c u r r e n c e o f b r a d y k i n i n i n t h e s k i n o f Rana t e m p o r a r i a . Comp. Bio c h e m . P h y s i o l . 14:43. 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S c i . 45:89. 159 APPENDIX A Chemicals Used Bradykinin (Sandoz Pharmaceuticals, BRS 640, 0.1 mg/ml) Bradykinin (Schwarz Bioresearch, l o t #6901) Lys-bradykinin (Schwarz Bioresearch, l o t #6902) Trypsin ( N u t r i t i o n a l Biochemicals Corp., 2X) K a l l i k r e i n ( N u t r i t i o n a l Biochemicals Corp., 10 K.U./mg) Alpha-chymotrypsin ( N u t r i t i o n a l Biochemicals Corp.) 8-hydroxyquinoline s u l f a t e (Matheson, Coleman and B e l l ) 3-rmercaptoproprionic acid (Sigma Chemical Co.) 2-mercaptoethanol (Sigma Chemical Co.) Ethylenediaztv t e t r a a c e t i c acid, sodium s a l t (Matheson, Coleman and B e l l ) L-epinephrine (Sigma Chemical Co.) Phenoxybenzamine hydrochloride (Smith, K l i n e , and French) Atropine s u l f a t e (Matheson, Coleman and B e l l ) A c e t y l c h o l i n e chloride (Sigma Chemical Co.) Propanolol (Inderal, I.C.I. 45,520, Ayerst Laboratories, Mtl) Bromolysergic acid diethylamide (BOL #148, Sandoz) Heparin ( N u t r i t i o n a l Biochemical Corp.) Sodium pentobarbital (Nembutal, Abbott, Mtl) Urethane (Ethyl Carbamate, Matheson, Coleman and B e l l ) .MS-222 (Tricane methanesolfonate, Kent, Chemicals Ltd) D i e t h y l e t h e r ( F i s h e r ) E t h y l a l c o h o l (95%, R e l i a n c e C h e m i c a l Co.) 1 - N - b u t a n o l ( F i s h e r ) A c e t i c a c i d ( F i s h e r ) H y d r o c h l o r i c a c i d ( F i s h e r ) Ammonium h y d r o x i d e ( F i s h e r ) Sodium H y d r o x i d e ( F i s h e r ) N a C l ( F i s h e r ) KC1 ( F i s h e r ) Na 2HP04 ( F i s h e r ) N a H 2 P 0 4 ( F i s h e r ) C a C l 2 ( F i s h e r ) - M g C l 2 - 6 H 2 0 ( F i s h e r ) D e x t r o s e ( F i s h e r ) Sodium p h e n o l s u l f o n p h t h a l e i n ( J . T . Baker) L - s e r i n e (Sigma Co.) L - t h r e o n i n e (Sigma Co.) N . B . . . . a l l c h e m i c a l s u s e d were o f Reagent g r a d e 160 APPENDIX B C o m p o s i t i o n o f P h y s i o l o g i c a l S o l u t i o n s , ( g / l i t e r ) N a C l KC1 C a C l 2 M g C l 2 NaHC0 3 N a 2 H P 0 4 NaH 2 P 0 4 Mammals 9.0 0.42 0.24 - 0.20 t r a c e t r a c e T u r t l e 8.0 0.20 0.20 0.05 1.00 t r a c e 0.04 A m p h i b i a n s 6. 5 0.14 0.12 - 0.20 t r a c e t r a c e F i s h 7.4 0.36 0.17 - 0. 31 1.60 0.04 Ammonium A c e t a t e B u f f e r , 0.02 M. pH = 5.0 1 M a c e t i c a c i d = 60.05 g / l i t e r , and 0.02 M a c e t i c a c i d = 1.201 g . l i t e r . S i n c e S g a c e t i c a c i d = 1.049, 0.02 M a c e t i c a c i d = 1.145 m l / l i t e r . T h e r e f o r e 1.145 ml a c e t i c a c i d were added t o 990 ml d i s t i l l e d w a t e r , and t h e pH a d j u s t e d t o 5.0 w i t h NH 4OH, and t h e t o t a l v o l ume made up t o 1000 m l . P h o s p h a t e B u f f e r f o r T r y p s i n I n c u b a t i o n s , C h a p t e r 3 Two s o l u t i o n s were p r e p a r e d as f o l l o w s : a) 4.543 g o f Na 2HP04 were added t o 1000 ml h o t d i s t i l l e d w a t e r . b) 1.270 g N a H 2 P 0 4 were added t o 1000 ml o f h o t d i s t i l l e d w a t e r . S o l u t i o n s a) and b) were combined so t h a t t h e pH was 7.35; t h e f i n a l b u f f e r was 0.02 M f o r a n i o n s . 161 APPENDIX C D e t e r m i n a t i o n o f Plasma K i n i n and K i n i n o g e n L e v e l s The procedure d e s c r i b e d below was taken from a method d e s c r i b e d by B r o c k l e h u r s t and Z e i t l i n (1967). A. C o l l e c t i o n o f Sample 5-1 ml of whole bl o o d was taken q u i c k l y i n t o a c h i l l e d p o l y s t y r e n e s y r i n g e through a s i l i c o n e d needle. 0.1 ml was used f o r m i c r o h e m a t o c r i t d e t e r m i n a t i o n , the remainder b e i n g immed i a t e l y i n a c t i v a t e d by f o r c i b l y e j e c t i n g i t through the needle i n t o 15 ml of c h i l l e d e t h a n o l i n a 50 ml Nalgene c e n t r i f u g e tube. The sample c o u l d be covered w i t h p a r a f i l m and stored, on i c e f o r a t l e a s t two days. B. P r e p a r a t i o n and Assay The coagulated p r o t e i n was scraped from the s i d e s of the c e n t r i f u g e tube, and the sample was c e n t r i f u g e d a t 2000 g f o r a t l e a s t 30 minutes. The supernatant should c o n t a i n the f r e e k i n i n w h i l e the p r e c i p i t a t e c o n t a i n s the p r e c u r s o r . The p r e  c i p i t a t e was washed w i t h 10 ml 80% e t h a n o l and r e - c e n t r i f u g e d . The washings were added to the f i r s t supernatant and k e p t on i c e . 1- K i n i n o g e n . The washed p r e c i p i t a t e was suspended i n 10 ml 80% e t h a n o l and p l a c e d i n b o i l i n g water f o r 10 minutes to g i v e .complete d e n a t u r a t i o n . The suspension was c e n t r i f u g e d a t 4,000 g and the supernatant d i s c a r d e d . The p r e c i p i t a t e was washed 162 twice with 10 ml d i s t i l l e d water, then suspended i n 15 ml of 2.5 M NaCl s o l u t i o n . This suspension was homogenized i n a ground glass blender, and 0.2 ml ali q u o t s of the homogenate were incubated at 37°C f o r 30 minutes with 1 mg of pure t r y p s i n , i n 5 ml 0.02 M sodium phosphate b u f f e r , pH = 7.35. The t r y p s i n was then i n a c t i v a t e d by heating i n a b o i l i n g water bath f o r 10 minutes. These solutions were assayed i n t h i s s t a t e , or stored at - 20°C u n t i l assay, which involved comparison with synthetic bradykinin on the i s o l a t e d r a t uterus i n the presence of at ropine (1 ug/ml) . 2. Free K i n i n . The k i n i n - c o n t a i n i n g ethanol extract was placed i n a b o i l i n g water bath f o r 10 minutes to destroy l a t e n t -enzyme a c t i v i t y , then evaporated to dryness under reduced press ure. The sample was reconstituted i n 11 ml warm d i s t i l l e d water (60°C) saturated with NaCl and a c i d i f i e d to pH 1.5 with HCl. This s o l u t i o n was extracted i n a separating funnel with 10, then, 5 ml of 1-N-butanol, which was then pooled and evaporated to dryness, beginning at 15°C, and r i s i n g to 50°C, under reduced pressure. The pressure was f i n a l l y reduced to 0.1 mm Hg and the temperature r a i s e d to 80°C f o r 5 minutes to remove the l a s t traces of butanol. These dry samples were stored at - 20°C u n t i l estimation of b i o l o g i c a l a c t i v i t y on the r a t uterus, i n the presence of atropine (1 ug/ml) and a 5-HT antagonist (bromo- l y s e r g i c acid diethylamide, 0.5 ug/ml). 163 APPENDIX D P a p e r C h r o m a t o g r a p h y , i n B u t a n o l - A c e t i c A c i d - W a t e r 1-N b u t a n o l , a c e t i c a c i d , and w a t e r i n a 4:1:5 r a t i o , and i n a t o t a l volume o f 200 ml were p e r i o d i c a l l y s h a k e n i n a 250 ml s e p a r a t o r y f u n n e l , o v e r a 2 h o u r p e r i o d . T h i s s o l v e n t s y s t e m was t h e n a l l o w e d t o s e p a r a t e i n t o 2 l a y e r s . The aqueous l a y e r was d i v i d e d among f o u r P e t r i d i s h e s and p l a c e d a t t h e b o t t o m o f a 12" x 12" x 24" g l a s s c h r o m a t o g r a p h y t a n k . A g l a s s l i d was s e a l e d on w i t h L u b r i s e a l , and t h e s y s t e m a l l o w e d t o e q u i l i  b r a t e a t room t e m p e r a t u r e f o r a t l e a s t 1 h o u r . S t a n d a r d s ( s y n t h e t i c b r a d y k i n i n and l y s - b r a d y k i n i n ) and unknowns ( t u r t l e k i n i n ) were a p p l i e d i n 10 u l H a m i l t o n s y r i n g e s t o a s h e e t o f Whatman 3 MM c h r o m a t o g r a p h y p a p e r c u t t o 20 V x 8 5/8". A s t r e a m o f a i r s u p p l i e d b y a p o r t a b l e h a i r d r y e r was u s e d t o d r y i n t h e samples a t t h e o r i g i n . The chromatogram was t h e n p l a c e d i n t h e e q u i l i b r a t e d c h r o m a t o g r a p h i c t a n k , and t h e s y s t e m a l l o w e d t o e q u i l i b r a t e f o r a n o t h e r h o u r . The b u t a n o l l a y e r o f t h e s o l v e n t was t h e n added t o t h e b a t h a t t h e t o p o f t h e t a n k t h r o u g h an o p e n i n g i n t h e g l a s s l i d w h i c h was i m m e d i a t e l y s t o p p e r e d . T h i s s y s t e m was a l l o w e d t o r u n f o r 12 o r 24 h o u r s . The chromatogram was t h e n removed, t h e s o l v e n t f r o n t marked, and t h e r e m a i n i n g s o l v e n t d r i v e n o f f by a f l o w o f a i r f r o m a fume hood f a n . L o n g i t u d i n a l s t r i p s o f t h e d r y chromatograms were c u t 164 a t t h e w i d t h o f t h e o r i g i n + 0.5 cm on e i t h e r s i d e , t o a l l o w f o r d i f f u s i o n . These s t r i p s were i n t u r n c u t i n t o 10 e q u a l p a r t s b e t w e e n t h e o r i g i n and t h e s o l v e n t f r o n t . I n t h e c a s e d f t h e 24 h o u r chromatograms, where t h e s o l v e n t f r o n t r a n o f f t h e p a p e r , t h e d i s t a n c e b e t w e e n t h e o r i g i n and t h e b o t t o m o f t h e p a p e r was d i v i d e d i n t o 10 e q u a l p a r t s . T h e r e f o r e , t h e s e d i v i s i o n s were a r b i t r a r y u n i t s , w h i l e t h o s e o b t a i n e d f o r t h e 12 h o u r chromatogram r e p r e s e n t e d i n d i v i d u a l R^'s. The r e c t  a n g l e s o b t a i n e d i n e i t h e r c a s e were f o l d e d and p l a c e d i n  d i v i d u a l l y i n s i l i c o n e d 5 ml b e a k e r s c o n t a i n i n g 1 ml 0.02 M ammonium a c e t a t e , pH = 5.0. A f t e r 6 h o u r s a t 4°C, t h e e l u a t e was r e c o v e r e d f r o m t h e p a p e r b y w r a p p i n g each i n p a r a f i l m and s q u e e z i n g m a n u a l l y . E l u a t e s were s t o r e d i n t h e s i l i c o n e d b e a k e r s a t 4°C u n t i l a s s a y on t h e i s o l a t e d r a t u t e r u s b i o  a s s a y . 165 APPENDIX E A method f o r k i n i n e x t r a c t i o n d e s c r i b e d b y Z a c e s t and M a s h f o r d (1967) i n c l u d e d an e t h e r e x t r a c t i o n o f t h e a l c o h o l c o n c e n t r a t e (see C h a p t e r 5 ) . I t was t h e o b j e c t o f t h i s e x p e r i  ment t o a s s e s s t h e e f f i c i e n c y o f t h i s added s t e p i n r e m o v i n g k i n i n f r o m t h e a l c o h o l e x t r a c t . M ethod. The f o l l o w i n g s o l u t i o n was p r e p a r e d : 50 ug s y n t h e t i c b r a d y k i n i n + 5 ml 9 5 % e t h y l a l c o h o l + 1 ml g l a c i a l a c e t i c a c i d + 44 ml i n a c t i v e p r o t e i n s o l u t i o n f r o m t u r t l e p l a s m a . T h i s m i x t u r e was t w i c e e x t r a c t e d w i t h 2 v o l u m e s o f d i  e t h y l e t h e r . The e x t r a c t was p o o l e d , and e v a p o r a t e d t o d r y n e s s u n d e r r e d u c e d p r e s s u r e . The t h i c k g r e e n p r e c i p i t a t e was t a k e n up i n 10 ml L o c k e ' s s o l u t i o n s , t h e pH a d j u s t e d t o 5.0 w i t h NaHC03, and t h e b r a d y k i n i n c o n t e n t e s t i m a t e d on t h e i s o l a t e d r a t u t e r u s . The b i o l o g i c a l a c t i v i t y r e m a i n i n g i n t h e aqueous l a y e r was a l s o measured a t t h i s t i m e . R e s u l t s . As f o l l o w s : S o l u t i o n T o t a l B r a d y k i n i n (ug) P e r c e n t E t h e r e x t r a c t 0.5 ug 1.0 Aqueous l a y e r 4 4 - 7 . 4 88 - 9.3 D i s c u s s i o n . I t was c o n c l u d e d t h a t t h e e t h e r e x t r a c t i o n removed an i n s i g n i f i c a n t p r o p o r t i o n o f t h e k i n i n f r o m t h e e t h a n o l e x t r a c t , w h i l e t h e t r e a t m e n t d i d e x t r a c t c o n s i d e r a b l e amounts o f i m p u r i t i e s ( l i p i d s , e t c ) . 166 APPENDIX F E f f i c i e n c y o f B u t a n o l i n E x t r a c t i n g B r a d y k i n i n The o b j e c t o f t h i s e x p e r i m e n t was t o d e t e r m i n e w h e t h e r t h e u s e o f a s a t u r a t e d s o d i u m c h l o r i d e s o l u t i o n was an a b s o  l u t e r e q u i r e m e n t f o r t h e e x t r a c t i o n o f k i n i n s b y b u t a n o l , s i n c e I h a d o r i g i n a l l y c o n s i d e r e d i t t o be i n c o n v e n i e n t t o remove t h e e x t r a N a C l f r o m t h e e x t r a c t . M e thod. Two s o l u t i o n s c o n t a i n i n g s y n t h e t i c b r a d y k i n i n were p r e  p a r e d : a) 15 ug BK + 25 ml s a t u r a t e d N a C l (60°C, pH = 1.5) b) 15 ug BK + 25 m l d i s t i l l e d w a t e r (60°C, pH = 1.5) B o t h o f t h e s e s o l u t i o n s were e x t r a c t e d w i t h 50, t h e n 25 ml o f 1-N b u t a n o l . The e x t r a c t s were p o o l e d i n each c a s e , and r e d u c e d t o d r y n e s s u n d e r l o w p r e s s u r e . The d r i e d s a m p l e s were r e c o n s t i t u t e d i n L o c k e ' s s o l u t i o n , and t h e b r a d y k i n i n c o n t e n t d e t e r m i n e d b y a s s a y on t h e i s o l a t e d r a t u t e r u s . R e s u l t s . As f o l l o w s : T e s t S o l u t i o n B r a d y k i n i n Added B r a d y k i n i n R e c o v e r e d S a t u r a t e d N a C l 15 ug 17 ± 4.2 ug No N a C l 15 ug 0.33 - 0.1 ug D i s c u s s i o n . I t was c o n c l u d e d t h a t ; a) t h e e x t r a c t i o n i s e x t r e m e l y e f f i c i e n t i n t h e a b s e n c e o f p l a s m a , b e i n g c l o s e t o 100%, and b) s a t u r a t i o n o f t h e aqueous l a y e r w i t h N a C l i s an a b s o - l u t e d r e q u i r e m e n t f o r e f f i c i e n t e x t r a c t i o n o f b r a d y k i n i n b y b u t a n o l . 

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