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UBC Theses and Dissertations

Study of the action of two naturally-occuring tropolone derivatives on vascular smooth muscle. Leathem, Ann Marie 1970

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A S T U D Y O F T H E A C T I O N O F TWO N A T U R A L L Y - O C C U R I N G T R O P O L O N E D E R I V A T I V E S ON V A S C U L A R S M O O T H M U S C L E b y A N N E M A R I E L E A T H E M B . S . P . , 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 , 1 9 6 4 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E I N P H A R M A C Y i n t h e D i v i s i o n o f P h a r m a c o l o g y o f t h e F a c u l t y o f P h a r m a c e u t i c a l S c i e n c e s We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d 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 S e p t e m b e r , 1 9 7 0 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e fo r reference and study. I f u r t h e r agree t h a t permiss ion fo r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood that copying o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l ga in s h a l l not be a l lowed without my w r i t t e n p e r m i s s i o n . Department of The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada Date ABSTRACT Gamma-thujaplicin (GT) and beta-hydroxy t h u j a p l i c i n (BHT) are two of the isopropyl derivatives of tropolone found i n the heartwood of western red cedar (Thuja p l i c a t a D. Don). Several of the tropolones are e f f e c t i v e i n h i b i t o r s of the enzyme catechol-O-methy1transferase (COMT). In thi s work, the i s o l a t e d , s p i r a l l y - c u t rabbit thoracic aorta pre-paration has been used to study possible COMT i n h i b i t i o n as well as other pharmacological properties of the sodium s a l t s of GT and BHT. It was found that GT, BHT and py r o g a l l o l , a known COMT i n h i b i t o r , a l l potentiated the response of the aor t i c s t r i p to norepinephrine (NE) . However, since i t was shown that the chelating agent EDTA also potentiated the NE res-ponse, the potentiation by GT and BHT could not be attributed to COMT i n h i b i t i o n without further evidence. GT and BHT were found to have a stimulatory e f f e c t of their-own on a o r t i c smooth muscle tissue. BHT was a more potent agonist than GT. Phenoxybenzamine blocked the stimulatory e f f e c t s of BHT and GT. This suggested alpha-adrenergic receptor involvement although the halogenoalkyl-amine blocking agents are not absolutely s p e c i f i c i n th e i r action. Cocaine produces a small potentiation of the contrac-t i o n produced by GT and BHT. This potentiation suggests the involvement of endogenous NE. Cocaine does not cause relaxation of the GT and BHT responses which indicates that GT and BHT d i f f e r i n mode and perhaps s i t e of action from tyramine. BHT potentiates the tyramine response on the rabbit a o r t i c s t r i p . This may be due to COMT i n h i b i t i o n , increased NE release or merely additive e f f e c t s . GT was found to produce relaxation of a histamine-induced contraction. This relaxant e f f e c t was not prevented by beta-adrenergic blockade and i s l i k e l y due to a non-s p e c i f i c depressant e f f e c t by GT. Both GT and BHT have produced non-specific blocking e f f e c t s against NE and histamine as well as acetylcholine throughout this work. In reserpinized preparations, GT no longer produced a contraction of the a o r t i c s t r i p . Instead, a relaxation below normal tone was produced. I t would seem that the presence of endogenous NE i s required i n the tissue stores before GT can cause the s t r i p s to contract. GT causes relaxation of tyramine-induced contractions i n reserpinized s t r i p s . The i s o l a t e d rabbit thoracic aorta was found not to be a suitable preparation for the pharmacological i n v e s t i -gation of COMT i n h i b i t i o n by GT and BHT due to t h e i r own i v agonistic e f f e c t s on thi s tissue as well as th e i r non-s p e c i f i c chelating properties. However, th i s tissue was useful i n providing information on other pharmacological actions of these compounds. Signatures of Examiners V TABLE OF CONTENTS PAGE ABSTRACT i i LIST OF TABLES v i i LIST OF FIGURES. . . . . . ix LIST OF TABLES (APPENDIX) x INTRODUCTION . 1 The Thujaplicins 1 Adrenergic Neurons . 5 Reserpine and Catecholamine Storage •. . . 7 Inactivation of Transmitter . . . . . . . 9 Tyramine . . . . 11 Cocaine 13 Beta-adrenergic Blocking Agents 14 Alpha-adrenergic Blocking Agents. . . . . . . . . . 20 Catechol-O-Methyltransferase. 26 Catechol-O-Methyltransferase I n h i b i t i o n by Tropolones 30 METHODS AND MATERIALS 36 Isolated Rabbit A o r t i c Strips . . . . 36 RESULTS The E f f e c t of Beta-hydroxy T h u j a p l i c i n , Gamma-Thujaplici n , Ethylenediamine Tetra-acetic acid and Pyrogallol on the Response of the Isolated v i P A G E A o r t a t o N o r e p i n e p h r i n e . 4 1 B e t a - H y d r o x y T h u j a p l i c i n 4 7 B e t a - h y d r o x y t h u j a p l i c i n a s a n a g o n i s t . . . . . 4 7 E f f e c t o f E D T A o n t h e b e t a - h y d r o x y t h u j a p l i c i n r e s p o n s e . 4 8 T h e e f f e c t o f p h e n o x y b e n z a m i n e o n t h e b e t a -h y d r o x y t h u j a p l i c i n r e s p o n s e . . . . . . . . . . 5 0 T h e e f f e c t o f p r o n e t h a l o l o n t h e b e t a - h y d r o x y t h u j a p l i c i n r e s p o n s e . . . . . . . . 5 1 T h e e f f e c t o f b e t a - h y d r o x y t h u j a p l i c i n o n t h e t y r a m i n e r e s p o n s e . . . . 5 3 T h e e f f e c t o f b e t a - h y d r o x y t h u j a p l i c i n o n t h e t y r a m i n e r e s p o n s e a f t e r p r e t r e a t m e n t w i t h i p r o n i a z i d . . . . . . . . . . * . . . . . . . . . 5 7 T h e e f f e c t o f i p r o n i a z i d o n t h e b e t a - h y d r o x y t h u j a p l i c i n r e s p o n s e . . . . . . 6 1 T h e e f f e c t o f c o c a i n e o n t h e b e t a - h y d r o x y t h u j a p l i c i n r e s p o n s e 6 1 G a m m a - T h u j a p l i c i n . . . . . . . 6 4 G a m m a - t h u j a p l i c i n a s a n a g o n i s t 6 4 T h e e f f e c t o f c o c a i n e o n t h e g a m m a - t h u j a p l i c i n r e s p o n s e 6 6 T h e b e t a - a d r e n e r g i c e f f e c t s o f g a m m a - t h u j a p l i c i n 6 6 T h e e f f e c t o f p r o p r a n o l o l o n t h e g a m m a -t h u j a p l i c i n r e s p o n s e 6 9 T h e e f f e c t o f r e s e r p i n i z a t i o n o n t h e g a m m a -t h u j a p l i c i n r e s p o n s e 7 0 D I S C U S S I O N 7 4 S U M M A R Y A N D C O N C L U S I O N S 8 1 B I B L I O G R A P H Y . 8 4 A P P E N D I X . . . . . . . . . . . 9 0 v i i L I S T O F T A B L E S T A B L E P A G E I . P e r c e n t i n c r e a s e i n t h e d u r a t i o n - 5 0 o f t h e r e s p o n s e t o n o r e p i n e p h r i n e 0 . 8 x l O - ^ M i n t h e p r e s e n c e o f a p o t e n t i a t o r . . . . . . . . . 4 3 I I . P e r c e n t i n c r e a s e i n t h e h e i g h t o f c o n t r a c -t i o n o f t h e n o r e p i n e p h r i n e 0 . 8 x 1 0 ~ 7 M r e s p o n s e i n t h e p r e s e n c e o f a p o t e n t i a t o r . . . 4 4 I I I . B e t a - h y d r o x y t h u j a p l i c i n a s a n a g o n i s t . . . . 4 9 I V . B e t a - h y d r o x y t h u j a p l i c i n a s a n a g o n i s t i n t h e p r e s e n c e o f e t h y l e n e d i a m i n e t e t r a - a c e t i c a c i d 5 0 V . T h e b e t a - h y d r o x y t h u j a p l i c i n r e s p o n s e a f t e r a l p h a - a d r e n e r g i c b l o c k a d e 5 1 V I . T h e e f f e c t o f p r o n e t h a l o l o n t h e b e t a - h y d r o x y t h u j a p l i c i n r e s p o n s e . . . . . . 5 2 V I I . T h e e f f e c t o f b e t a - h y d r o x y t h u j a p l i c i n o n t h e t y r a m i n e r e s p o n s e 5 5 V I I I . T h e e f f e c t o f b e t a - h y d r o x y t h u j a p l i c i n o n t h e t y r a m i n e r e s p o n s e e x p r e s s e d r e l a t i v e t o n o r e -p i n e p h r i n e c o n t r o l . . . . . . 5 6 I X . C o m p a r a t i v e D u r a t i o n - 5 0 o f t h e r e s p o n s e s t o t y r a m i n e a n d t o t y r a m i n e p l u s b e t a - h y d r o x y t h u j a p l i c i n . . . . . . . . . . . . . . . . . . 5 6 X . T h e e f f e c t o f b e t a - h y d r o x y t h u j a p l i c i n o n t h e t y r a m i n e r e s p o n s e a f t e r p r e t r e a t m e n t w i t h i p r o n i a z i d 5 8 X I . T h e e f f e c t o f i p r o n i a z i d o n t h e b e t a - h y d r o x y t h u j a p l i c i n r e s p o n s e . . . . . . . . . . . . . 6 2 X I I . T h e e f f e c t o f c o c a i n e o n t h e b e t a - h y d r o x y t h u j a p l i c i n r e s p o n s e 6 4 X I I I . G a m m a - t h u j a p l i c i n a s a n a g o n i s t 6 5 v i i i TABLE PAGE XIV. The e f f e c t o f c o c a i n e on t h e g a m m a - t h u j a p l i c i n response 67 XV. The r e l a x a n t e f f e c t o f g a m m a - t h u j a p l i c i n on the h i s t a m i n e c o n t r a c t i o n . . . . 6 8 XVI. - The e f f e c t o f p r o p r a n o l o l on t h e gamma-t h u j a p l i c i n - i n d u c e d r e l a x a t i o n o f t h e h i s t a -mine c o n t r a c t i o n . . . . . . . . 70 X V I I . R e l a x a t i o n produced by g a m m a - t h u j a p l i c i n i n r e s e r p i n i z e d r a b b i t a o r t i c s t r i p s 71 i x L I S T O F F I G U R E S F I G U R E P A G E 1 . S t r u c t u r e s o f t h e t h u j a p l i c i n s . . . . . . . . . 2 2 . B i o s y n t h e s i s o f n o r e p i n e p h r i n e . 1 0 3 . B e t a - a d r e n e r g i c b l o c k i n g a g e n t s 1 5 4 . A l p h a - a d r e n e r g i c b l o c k i n g a g e n t s 2 1 5 . P a t h w a y s o f m e t a b o l i s m o f n o r e p i n e p h r i n e . . . . 2 9 6 . T h e p o s t u l a t e d c o m p l e x f o r m e d b e t w e e n t h e t r o p o l o n e r i n g , a d i v a l e n t c a t i o n , S -a d e n o s y l m e t h i o n i n e a n d c a t e c h o l - O - m e t h y l -t r a n s f e r a s e . . , 3 2 7 . T h e p e r c e n t i n c r e a s e i n d u r a t i o n - 5 0 o f a n o r e p i n e p h r i n e 0 . 8 x 1 0 ~ 7 M r e s p o n s e i n t h e p r e s e n c e o f a p o t e n t i a t o r 4 5 8 . T h e p e r c e n t i n c r e a s e i n t h e h e i g h t o f a n o r e p i n e p h r i n e 0 . 8 x 1 0 - 7 M r e s p o n s e i n t h e p r e s e n c e o f a p o t e n t i a t o r . . . . . . . . . . . . 4 6 9 . A b e t a - h y d r o x y t h u j a p l i c i n c o n t r a c t i o n . . . . . 4 7 1 0 . B e t a - h y d r o x y t h u j a p l i c i n p l u s t y r a m i n e 5 3 1 1 . T h e e f f e c t o f b e t a - h y d r o X y t h u j a p l i c i n o n t h e t y r a m i n e r e s p o n s e a f t e r p r e t r e a t m e n t w i t h i p r o n i a z i d 5 9 1 2 . T h e e f f e c t s o f c o c a i n e o n t h e r e s p o n s e t o t y r a m i n e a n d b e t a - h y d r o x y t h u j a p l i c i n . . . . . . 6 3 1 3 . G a m m a - t h u j a p l i c i n a s a n a g o n i s t . . . . . . . . . 6 6 1 4 . T h e r e l a x a n t e f f e c t o f g a m m a - t h u j a p l i c i n o n t h e h i s t a m i n e c o n t r a c t i o n 6 8 1 5 . T h e r e l a x a t i o n p r o d u c e d b y g a m m a - t h u j a p l i c i n i n r e s e r p i n i z e d a o r t i c s t r i p s . . . 7 2 1 6 . T h e r e l a x a t i o n o f t h e t y r a m i n e c o n t r a c t i o n b y g a m m a - t h u j a p l i c i n i n r e s e r p i n i z e d a o r t i c s t r i p s 7 2 X APPENDIX LIST OF TABLES TABLE PAGE A. Per cent increase i n the duration - 5 0 of the response to norepinephrine 0 . 8 x lO-Tjyiin the presence of a potentiator. 9 1 B. Per cent increase i n the height of contraction of the norepinephrine 0 . 8 x 1 0 ~ 7 M response i n the presence of a potentiator 9 2 C. Actual height of contraction produced by the combination of norepinephrine plus potentiator . . 9 3 D. Beta-hydroxy t h u j a p l i c i n as an agonist 9 4 E. Beta-hydroxy t h u j a p l i c i n as an aagonist- i n the presence of EDTA 9 5 F. The e f f e c t of beta-hydroxy t h u j a p l i c i n on the tyramine response aft e r pretreatment with i p r o n i a z i d 9 6 G. The e f f e c t of i p r o n i a z i d on the beta-hydroxy t h u j a p l i c i n response 9 7 H. The e f f e c t of cocaine on the beta-hydroxy t h u j a p l i c i n response 9 8 I. Gamma-thujaplicin as an agonist 9 9 J . The e f f e c t of cocaine on the gamma-thujaplicin response 1 0 0 K. The relaxant effects of GT on the histamine contraction 1 0 1 L. The e f f e c t of propranolol on the gamma-thujaplicin-induced relaxation of the histamine contraction 1 0 2 ACKNOWLEDGEMENTS The a u t h o r i s i n d e b t e d t o Dr. J.E. H a l l i d a y f o r h i s gu i d a n c e and encouragement t h r o u g h o u t t h e co u r s e o f t h i s work. INTRODUCTION The Thujaplicins The t h u j a p l i c i n s are isopropyl derivatives of tropolone which occur i n the heartwood of western red cedar (Thuja p l i c a t a D. Don). They include the three isomers alpha-, beta- and gamma-thujaplicin and beta-hydroxy t h u j a p l i c i n . The term tropolone i s used to designate the seven-membered carbon ring compound, 2-hydroxy-2,4,6-cycloheptatrien-l-one. Derivatives of tropolone are r e l a t i v e l y rare i n nature and the t h u j a p l i c i n s are the simplest of these. The chemical and physical properties of the tropolones have been well investigated (1) but information on t h e i r bio-l o g i c a l a c t i v i t y i s ..limited. Antifungal experiments have shown the t h u j a p l i c i n s to be f u n g i c i d a l to rot producing fungi i n wood. In vivo work on b a c t e r i a l infections was not as success-f u l since the t h u j a p l i c i n s are i n e f f e c t i v e i n the presence of blood. The pharmacology of beta-thujaplicin has been studied largely by Japanese workers (2). This isomer i s i s o l a t e d from the tree Formosan hinoki and i s c a l l e d h i n o k i t i o l by the Japanese. Lee (2) found the actions of beta-thujaplicin to be largely depressant i n nature i n both i n vivo and in" v i t r o studies. However, i n small doses on mice u t e r i , i t was a stimulant (3) though i n larger doses the depressant effects were dominant. Beta-hydroxy t h u j a p l i c i n Alpha-thujaplicin 3 The pharmacology o f t h e sodium s a l t o f g a m m a - t h u j a p l i c i n was s t u d i e d by H a l l i d a y ( 4 ) . I n mice the i n t r a p e r i t o n e a l LD 50 was found t o be 162±3.2 mg/Kg and t h e CD 50 ( c o n v u l s i v e dose) was 73.5±1.8 mg/Kg. Both s t i m u l a n t and d e p r e s s a n t e f f e c t s were seen i n mammals. I n t e s t s on d e c e r e b r a t e c a t s and r a t s t h e s i t e o f the c o n v u l s i v e a c t i o n was shown t o be c e r e b r a l . The a c t i o n o f g a m m a - t h u j a p l i c i n (GT) i s not a n a l e p t i c s i n c e i t does no t s h o r t e n t h e s l e e p i n g time f o r v a r i o u s h y p n o t i c s . : I n some c a s e s , the s l e e p i n g time was i n c r e a s e d . I n the a n e s t h e t i z e d c a t GT i n a dose from 25 t o 50 mg/Kg produced a temporary f a l l i n b l o o d p r e s s u r e accompanied by a d e c r e a s e d h e a r t r a t e . T h i s h y p o t e n s i v e e f f e c t was not b l o c k e d by a t r o p i n e o r b i l a t e r a l s e c t i o n o f the vagus nor was t h e response t o c a r o t i d o c c l u s i o n o r e p i n e p h r i n e a f f e c t e d . T h i s f a l l i n b l o o d p r e s s u r e was a t t r i b u t e d t o a p o s s i b l e d i r e c t m y o c a r d i a l d e p r e s s i o n produced by i t s exposure t o a sudden h i g h c o n c e n t r a t i o n o f GT. On g u i n e a p i g i l e u m and r a b b i t i n t e s t i n e , t h e e f f e c t s o f GT a r e a l s o i n h i b i t o r y . Spontaneous c o n t r a c t i o n s a re i n -h i b i t e d and the e f f e c t s o f many s t i m u l a n t s i n c l u d i n g a c e t y l -c h o l i n e , h i s t a m i n e , n i c o t i n e , b a r i u m c h l o r i d e and 5-hydroxy-t r y p t a m i n e a r e i n h i b i t e d t o v a r y i n g d e g r e e s . The s t i m u l a n t e f f e c t o f n i c o t i n e was i n h i b i t e d t o the g r e a t e s t degree which might i n d i c a t e a d e p r e s s a n t e f f e c t on m y e n t e r i c g a n g l i a as w e l l as on the i n t e s t i n a l muscle i t s e l f . O nly d e p r e s s a n t e f f e c t s were demonstrated i n f r o g s o v e r th e e n t i r e dose range. K a t s u r a (5) suggested t h a t b e t a -4 t h u j a p l i c i n was a n e r v e - m u s c l e p o i s o n r e l a t e d i n i t s a c t i o n t o camphor, t h u j o n e and o t h e r t e r p e n e compounds. GT seems t o f o l l o w t h e same p a t t e r n i n i t s m i x t u r e o f s t i m u l a n t and d e p r e s s a n t e f f e c t s . I n 1965, t h e e f f e c t s o f GT on t h e i s o l a t e d g u i n e a p i g a t r i a were i n v e s t i g a t e d by Ko ( 6 ) . GT was f o u n d t o have a s l o w b u t p r o l o n g e d s t i m u l a t o r y e f f e c t on b o t h t h e i n o t r o p i c and c h r o n o t r o p i c a c t i o n s o f t h e a t r i a . GT a l s o p o t e n t i a t e d t h e e f f e c t s o f a d r e n e r g i c amines on t h e a t r i a i n a d o s e - r e s p o n s e p a t t e r n s i m i l a r t o t h o s e f o r c o c a i n e and EDTA ( e t h y l e n e -d i a m i n e t e t r a a c e t i c a c i d ) . T h i s p o t e n t i a t i o n o f c a t e c h o l a m i n e s was n o t a f f e c t e d by r e s e r p i n i z a t i o n . GT a l s o p o t e n t i a t e d t h e r e s p o n s e o f t h e a t r i a t o h i s t a m i n e a l t h o u g h t h i s p o t e n t i a t i o n was g r e a t l y r e d u c e d o r a b o l i s h e d i n r e s e r p i n i z e d a t r i a . I t was c o n c l u d e d t h a t t h e s e r e s u l t s d i d n o t s u p p o r t t h e a s s u m p t i o n t h a t GT p o t e n t i a t i o n o f a d r e n e r g i c r e s p o n s e s i s due t o i n h i b i -t i o n o f t h e enzyme c a t e c h o l - O - m e t h y l t r a n s f e r a s e . I t a p p e a r s t h a t GT has a n o n - s p e c i f i c s e n s i t i z i n g e f f e c t on t h e a t r i a l m u s c l e . Some p h a r m a c o l o g i c a l and m i c r o b i o l o g i c a l e f f e c t s o f b e t a - h y d r o x y - t h u j a p l i c i n (BHT) were s t u d i e d by S a n d e r s and H a l l i d a y i n 1961 ( 7 ) ( 8 ) . When a d m i n i s t e r e d t o m i c e , BHT ex-h i b i t e d b o t h s t i m u l a t o r y and d e p r e s s a n t e f f e c t s , d e p e n d i n g on t h e d o s e . A t a d o s e o f 100 mg/Kg, d e p r e s s a n t e f f e c t s o n l y were e v i d e n t . I n t h e d o s e r a n g e o f 150 t o 200 mg/Kg, a com-b i n a t i o n o f s t i m u l a n t and d e p r e s s a n t e f f e c t s were s e e n . The 5 i n t r a p e r i t o n e a l LD 50 i n m i c e was 155+4.5 mg/Kg and t h e CD 50 ( c o n v u l s i v e dose) was 163+6.2 mg/Kg. The s t i m u l a n t e f f e c t s i n m i c e were m a n i f e s t e d by h y p e r e x c i t a b i l i t y and i n l a r g e r , d o s e s by c o n v u l s i o n s . The d e p r e s s a n t e f f e c t s o f s m a l l d o s e s p r o d u c e d h y p o t h e r m i a and h y p o k i n e s i a . When BHT was i n j e c t e d i n t r a v e n o u s l y o r i n t r a - a r t e r i a l l y i n r a b b i t s a s h a r p r i s e i n b l o o d p r e s s u r e was p r o d u c e d . T h i s r e s p o n s e was u n r e l a t e d t o t h e EEG p a t t e r n and may be e i t h e r c e n t r a l o r p e r i p h e r a l i n n a t u r e . B a r b i t u r a t e s l e e p i n g t i m e was n o t d e c r e a s e d by BHT b u t r a t h e r was i n c r e a s e d as was a l s o shown f o r GT p r e v i o u s l y . BHT showed b a c t e r i o s t a t i c and f u n g i s t a t i c e f f e c t s f o r many o r g a n -isms, s t u d i e d by S a n d e r s i n ' V i t r o b u t was n o t f o u n d a c t i v e i n i n v i v o t e s t s . A d r e n e r g i c N e u r o n s C u r r e n t knowledge c o n c e r n i n g t h e c e l l u l a r s i t e s and mechanisms o f s y n t h e s i s , s t o r a g e and r e l e a s e o f c a t e c h o l a m i n e s has b e e n d e r i v e d f r o m s t u d i e s o f b o t h a d r e n a l m e d u l l a r y t i s s u e and a d r e n e r g i c a l l y i n n e r v a t e d o r g a n s . The s t r u c t u r e o f a d r e n -e r g i c s y n a p s e s has been s t u d i e d by t h e c o n v e n t i o n a l l i g h t and e l e c t r o n m i c r o s c o p i c a l t e c h n i q u e s and t h e d i s t r i b u t i o n o f c a t e c h o l a m i n e s has been i n v e s t i g a t e d by f l u o r e s c e n c e t e c h n i q u e s . N o r e p i n e p h r i n e , t h e a d r e n e r g i c t r a n s m i t t e r o c c u r s i n n e a r l y a l l p a r t s o f t h e a d r e n e r g i c n e u r o n , a l t h o u g h t h e c o n c e n t r a t i o n v a r i e s c o n s i d e r a b l y . The h i g h e s t c o n c e n t r a t i o n o c c u r s i n 6 c h a r a c t e r i s t i c enlargements, so-called v a r i c o s i t i e s , within the terminal f i b r e network. In other parts of the neuron the amine l e v e l i s considerably lower. The uneven amine d i s t r i -bution seems to be due to the accumulation of amine granules, the intraneuronal amine-storing organelles within the v a r i -c o s i t i e s . I t i s estimated that there are about 1000 amine granules within a v a r i c o s i t y and about 15,000 norepinephrine molecules per granule (9). Upon stimulation of the adrenergic neuron, norepinephrine i s released quantally. The amine granules appear to be synthesized i n the c e l l bodies and trans-ported down the axon to the v a r i c o s i t i e s of the nerve terminals. The free cytoplasmic f r a c t i o n of norepinephrine i s very small due to the presence of the enzyme monoamine oxidase (MAO) i n the cytoplasm. In the granules, the norepinephrine (NE) appears to be bound with adenosine triphosphate (ATP). This cate-cholamine-nucleotide s a l t probably represents the reserve or stable pool of catecholamine storage that has been postulated to e x i s t i n active equilibrium with one or more mobile pools. The exact mechanism whereby NE i s released from the storage granules upon nerve stimulation i s not known. The nerve impulse may d i r e c t l y or i n d i r e c t l y influence the granule to give o f f part of i t s content into the synaptic c l e f t . In th i s case, the granule may be assumed to be i n very close contact or even fused together with the c e l l membrane. A l t e r -natively, the nerve impulse may cause release of a hypothetical transmitter pool attached to s p e c i f i c s i t e s of the c e l l mem-7 brane. Reloading of these s i t e s could then be achieved speci-f i c a l l y by the amine granules. Reuptake of the released NE transmitter by the adrener-gic nerves appears to be an important mechanism i n the termin-ation of a c t i v i t y as well as i n replenishing of the NE stores. I t has been shown that about 60% of infused NE i n physiological quantities i s taken up by the adrenergic nerves. I t has been postulated that the uptake mechanism for accumulating amines within these nerves consists of two major components: active transport through the nerve c e l l membrane and incorporation into the storage granule complex. Both of these mechanisms can be s e l e c t i v e l y blocked by certa i n drugs. P r o t r i p t y l i n e and desipramine are potent i n h i b i t o r s of the "membrane pump" while reserpine i s a potent blocker of the uptake of NE into the granules (10) . Reserpine and Catecholamine Storage Reserpine i s capable of blocking adrenergic neurotrans-mission by i n h i b i t i n g amine uptake by the storage granules which leads to depletion of the store. The NE released from the stores undergoes oxidative deamination by MAO before i t leaves the c e l l . This reserpine-sensitive uptake mechanism i s ATP and Mg + + dependent. During recovery from a single large dose or repeated small doses of reserpine, however, normal function reappears long before the tissue levels of 8 monoamines have increased markedly. A major f r a c t i o n of NE appears to be present i n reserve pools without immediate functional importance. Functional changes appear to be better correlated with changes of a small NE f r a c t i o n (functional pool) which recovers from the effects of reserpine much e a r l i e r than the major f r a c t i o n . The time required for t o t a l recovery of the NE levels i n d i f f e r e n t tissues after a large dose of reserpine i s about the same as the calculated l i f e - s p a n of amine storage granules for most species. This suggests that the action of reserpine on the storage mechanisms of the granules i s largely i r r e v e r -s i b l e and that the recovery^is mainly due to a steady down-transport o f newly formed amine granules. These newly formed granules may be of. importance also for the early functional recovery af t e r reserpine but at present i t i s not possible to exclude a reversible e f f e c t on a small functional pool of the granules. A reserpine-resistant uptake mechanism by amine gran-ules has also been demonstrated (11). Metaraminol seems to use this reserpine-resistant mechanism to a greater extent than does NE, while dopamine seems to use both mechanisms. i A functional response to sympathetic stimulation can be ob-tained a f t e r reserpine when a large dose of dopamine i s given af t e r MAO i n h i b i t i o n . Under these conditions dopamine appears to u t i l i z e a reserpine-resistant mechanism, and to be trans-9 formed to NE by beta-hydroxylation i n the granules (see Figure 2). This NE can then be. released on nerve stimulation, pre-sumably from a small functional pool. Inactivation of Transmitter The released transmitter may be inactivated by various mechanisms. , (a) Membrane pump: Recapture of the NE by the membrane pump appears to be an important mechanism under normal circum-stances . When the membrane pump i s blocked, the normally swift relaxation of the effector c e l l s a f t e r cessation of a stimulation period f a i l s ; at higher impulse frequencies the nerve f i b r e s are f a i r l y rapidly exhausted, and furthermore, the released amount of NE per impulse i s decreased. Under normal conditions the active NE reuptake, combined with NE synthesis, generally appears to keep the readily available transmitter pool constant and s u f f i c i e n t . (b) Diffu s i o n to the blood: The released transmitter may di f f u s e from the synaptic gap into the blood stream. This may occur when the blood flow i s high as i n muscular exercise or when in a c t i v a t i n g mechanisms are blocked. C i r c u l a t i n g NE seems to be metabolized mainly by l i v e r catechol-O-methyl-transferase (COMT). (c) Local enzymatic destruction: This i n a c t i v a t i o n mechanism presumably plays only a minor quantitative r o l e . According to G i l l e s p i e and Kirpekar (12), MAO and COMT together T y r o s i n e CH-CH-N^ COOH T y r o s i n e H y d r o x y l a s e Dopa C H - C H - N H , COOH Dopamxne Dopa ^ D e c a r b o x y l a s e CH-CH-NH % . a. v N o r e p i n e p h r i n e Dopamine 6 - H y d r o x y l a s e CH-CH-NH OH F i g u r e 2. B i o s y n t h e s i s o f N o r e p i n e p h r i n e . 11 account for about 15 per cent of the i n a c t i v a t i o n of infused NE i n physiological doses. I t i s generally agreed that MAO exerts i t s function mainly intraneuronally, whereas COMT i s important for i n a c t i v a t i o n of e x t r a c e l l u l a r NE. Tyramine Tyramine i s the prototype of i n d i r e c t l y acting sympa-thomimetic amines. I t i s generally believed that the sympathomimetic effects of tyramine are caused by a release of norepinephrine from the adrenergic nerve endings. This hypo-thesis may explain why the response::: to i n d i r e c t l y acting amines i s l o s t a f t e r NE depletion by denervation and by reser-pine. Furthermore, there i s chemical evidence that tolerable doses of i n d i r e c t l y acting amines released NE into the perfusate of the i s o l a t e d heart, into the venous ef f l u e n t of the heart i n vivo or the perfused kidney i n • s i t u (13). I n d i r e c t l y acting amines also release NE into the general c i r c u l a t i o n after administration of adrenergic blocking drugs ( 1 3 ) . Though i t i s obvious that tyramine releases NE into the c i r c u l a t i o n i t i s questionable whether the quantities of NE released can f u l l y account for the powerful sympathomimetic effects observed. It has been demonstrated that the action of the NE released i s enhanced by the tyramine present. Thus r e l a t i v e l y small amounts of norepinephrine may become e f f e c t i v e ( 1 4 ) ( 1 5 ) . The mechanism by which tyramine potentiates NE may be s i m i l a r to that of cocaine, that i s , i n h i b i t i o n of NE removal bv uptake i : ••'.t< 12 s i t e s . Although storage of NE administered to the heart v i a the c i r c u l a t i o n i s blocked by pretreatment of the animal with reserpine, the removal mechanism which operates by uptake of NE into the c e l l i s not affected by reserpine. Under these conditions drugs i n h i b i t i n g NE removal from the e x t r a c e l l u l a r space are s t i l l e f f e c t i v e . Tyramine i n h i b i t s both the increase 3 i n H -NE concentration of various organs after i n j e c t i o n of 3 H -NE and the reuptake of NE released by nerve stimulation (16). I t i s therefore l i k e l y that tyramine i n h i b i t s reuptake of NE released by tyramine's own action as well. Thus, sympathomim-e t i c e f f e c ts of i n d i r e c t l y acting amines such as tyramine may be explained by a combination of t h e i r a b i l i t i e s to release NE and to i n h i b i t i t s removal from the synaptic c l e f t . Evidence for a d i r e c t vasoconstrictor action of tyramine was obtained i n noninnervated vascular smooth muscle, the human umbilical vein which contains no demonstrable catechol-amines (17) and also i n the perfused ear of rabbits prettreated with reserpine (18). These results do not necessarily contra-d i c t the hypothesis that tyramine acts by NE release on other organ systems since there are alternative explanations for a d i r e c t e f f e c t of tyramine on vascular tissue. In these studies, the p o s s i b i l i t y has not been ruled out that tyramine acted on tryptamine.receptors rather than on adrenergic receptors. Tryptamine receptors are stimulated by i n d i r e c t l y acting sympathomimetic amines (19). There i s also the pos-s i b i l i t y that the responses of vascular smooth muscle may have 13 been e l i c i t e d by a metabolic product of tyramine for example dopamine or norsynephrine which have d i r e c t action. These products have been produced by metabolism i n vascular smooth muscle (20)(21) but i t i s not l i k e l y they would be produced rapidly enough or i n large enough quantity to account for the stimulation observed. Cocaine Cocaine i n h i b i t s uptake by tissues of sympathomimetic amines such as norepinephrine, epinephrine, nordefrin and metaraminol. By blocking the uptake of these drugs by adrenergic nerve endings higher concentrations of these catecholamines are permitted to accumulate i n the region of the receptor and thus a potentiation of t h e i r e f f e c t s i s seen. Cocaine also i n h i b i t s NE release by tyramine (16). This i n h i b i t i o n does not seem to be related to the membrane-s t a b i l i z i n g e f f e c t of the l o c a l anesthetics since pseudo-cocaine and tetracaine do not a f f e c t NE release caused by tyramine. Cocaine may i n h i b i t the penetration of tyramine into the c e l l or a l t e r n a t i v e l y i t may i n h i b i t NE release from nerve endings by an action on the c e l l membrane. The f i r s t suggestion i s the most l i k e l y since doses of cocaine that i n h i b i t pharmacologic responses and NE release by tyramine do not i n t e r f e r e with the NE release produced by stimulation of the sympathetic nerves. The i n h i b i t i o n of NE uptake by 14 cocaine i s competitive (22) and.a competitive antagonism between cocaine and tyramine was also demonstrated. I t would seem that prevention of norepinephrine and tyramine entry into the nerve terminals by cocaine may have a similar mech-anism of action. Beta-adrenergic Blocking Agents Before 1957 there were no known s p e c i f i c beta-adrenergic blocking agents (23) . L i t t l e recognition had been given to the c l a s s i f i c a t i o n of adrenergic receptors which Ahlquist had proposed i n 1948. In 1957 however, the discovery of the unique adrenergic blocking c h a r a c t e r i s t i c s of dichloroisoproterenol (DCI) by Slater and Powell of the L i l l y Research Laboratories greatly increased the knowledge and understanding of adrenergic blockade and prompted acceptance of Ahlquist's c l a s s i f i c a t i o n . A search for s i m i l a r compounds was immediately stimulated. Powell and Slater described DCI as having agonistic as well as antagonistic actions on several systems. Of p a r t i -cular importance was the demonstration that DCI antagonized the i n h i b i t o r y effects of sympathomimetic amines on the tracheobronchial system, on blood vessels, on the uterus and on the i n t e s t i n e , but blocked neither the vasopressor e f f e c t of sympathomimetic amines, nor the adrenergically induced contraction of the n i c t i t a t i n g membrane. DCI depressed the frog heart and decreased the p o s i t i v e inotropic and chrono-trop i c e ffects of epinephrine. Moran and Perkins (1958) CI OH C H C H - N H X C H , - C H CH CI D i c h l o r o i s o p r o t e r e h o l OH I C H CH. C H - N H 2 - CH P r o n e t h a l o l CH - CH 7. CH - NH 1 P r o p r a n o l o l g u r e 3 . B e t a - a d r e n e r g i c b l o c k i n g a g e n t s 16 confirmed the blockade by DCI of adrenergically induced vaso-d i l a t a t i o n , and i n addition, showed that DCI produced a highly s p e c i f i c cardiac adrenergic blockade i n the dog heart and i n the i s o l a t e d perfused heart of the rabbit. A disad-vantage of DCI, however, was that i t also had sympathomimetic eff e c t s of i t s own. For example, i n the dog heart DCI i n i -t i a l l y produced increases i n heart rate, force of contraction and vasodilatation before the depression of the heart occured. In 1962, Black and associates described the adrenergic blocking properties of pronethalol (24) (nethalide, A l d e r l i n ) , a drug with actions s i m i l a r to those of DCI except for a lesser degree of d i r e c t sympathomimetic a c t i v i t y . At the same time, Dornhorst and colleagues (23) described preliminary results with pronethalol i n patients. Pronethalol was found to antagonize cardiac stimulation,'adrenergically induced vasodilatation, bronchodilatation and i n t e s t i n a l relaxation. I t was useful i n reducing exercise tachycardia and antagonized arrhythmias induced by cardiac glycosides. Pronethalol could likewise prevent arrhythmias induced by adrenaline during hydrocarbon anesthesia and p o t e n t i a l therapeutic uses might have been i n management of angina, hypertrophic obstructive cardiomyopathy as well as i n phaeochromocytoma. The anti-arrhythmic e f f e c t of pronethalol did not seem to be due t o t a l l y to beta-adrenergic blockade. For example, d i g i t a l i s arrhythmias were equally prevented by 17 d e x t r o - r o t a t o r y and r a c e m i c p r o n e t h a l o l w h i l e d e x t r o - r o t a t o r y p r o n e t h a l o l had v e r y l i t t l e b e t a - a d r e n e r g i c b l o c k i n g a c t i o n . I t was suggested t h a t t h e r e a re s e v e r a l a n t i - a r r h y t h m i c mech-anisms i n v o l v e d such as b e t a - r e c e p t o r b l o c k a d e , l o c a l anes-t h e s i a and a q u i n i d i n e - l i k e a c t i o n o r d i r e c t m y o c o r d i a l d e p r e s -s i o n . S t a n d a e r t (25) su g g e s t s t h a t some o f t h e a n t i - a r r h y t h m i c p r o p e r t i e s o f p r o n e t h a l o l might be based on an a c t i o n o f t h e drug on t h e t e r m i n a l f i l a m e n t s o f the a d r e n e r g i c n e r v e s , a d r u g - i n d u c e d d e p r e s s i o n o f a d r e n e r g i c nerve t e r m i n a l s and a consequent r e d u c t i o n i n a d r e n e r g i c t r a n s m i s s i o n . Such an e f f e c t i s a p o t e n t i a l h a z a r d i n t h e use of p r o n e t h a l o l f o r the a n a l y s e s o f drug r e a c t i o n s . S t u d i e s on t h e t o x i c i t y o f p r o n e t h a l o l i n a n i m a l s (23) showed t h a t a c u t e t o x i c i t y produced tremors and c o n v u l s i o n s . T h i s was c o r r e l a t e d w i t h the e v i d e n c e t h a t the b r a i n concen-t r a t i o n s o f p r o n e t h a l o l were o f t h e o r d e r o f one hundred times the b l o o d c o n c e n t r a t i o n . These a c u t e t o x i c . s i g n s appeared a t doses seven t o t e n ti m e s g r e a t e r than t h o s e needed f o r e f f e c -t i v e b e t a - a d r e n e r g i c r e c e p t o r b l o c k a d e . W h i l e tremors and c o n v u l s i o n s have n o t been d e s c r i b e d i n man, e f f e c t i v e doses a r e o f t e n a s s o c i a t e d w i t h u n p l e a s a n t s u b j e c t i v e s i d e - e f f e c t s . M i l d d i s o r i e n t a t i o n , s l i g h t i n c o o r d i n a t i o n , nausea and vom-i t i n g have been d e s c r i b e d ( 2 6 ) . S i n c e t h e t o x i c s i g n s i n a n i m a l s a r e seen w i t h t h e i n a c t i v e d e x t r o - r o t a t o r y isomer as w e l l as w i t h the " a c t i v e 18 l e v o - r o t a t o r y i s o m e r , i t has been assumed t h a t t h e e f f e c t s o f p r o n e t h a l o l on the c e n t r a l nervous system a r e p r o b a b l y not due t o b e t a - a d r e n e r g i c r e c e p t o r b l o c k a d e . A more s e r i o u s t o x i c m a n i f e s t a t i o n was found i n m ice. S t u d i e s showed t h a t p r o n e t h a l o l was a m o d e r a t e l y p o t e n t c a r c i n o g e n . i n t h i s s p e c i e s . M a l i g n a n t tumours, m a i n l y thymic lymphosarcomata, began t o appear a f t e r 10 weeks c o n t i n u o u s d o s i n g ( 2 7 ) . P r o n e t h a l o l was t h e n withdrawn from f u r t h e r c l i n i c a l i n v e s t i g a t i o n . I n i t s p l a c e , B l a c k and a s s o c i a t e s (28) i n t r o d u c e d -a more p o t e n t and more s p e c i f i c compound p r o p r a n o l o l . P r o p r a n o l o l i s o v e r t e n t i m e s as a c t i v e as p r o n e t h a l o l as a b e t a - a d r e n e r g i c b l o c k i n g agent (2 9 ) . However, t h i s g r e a t e r a c t i v i t y i s not r e f l e c t e d i n enhanced s i d e a c t i o n s f o r the sympathomimetic e f f e c t s o f p r o p r a n o l o l , a r e s a i d t o be m i n i m a l . I t i s t h e most p r o m i s i n g o f t h e c a r d i a c a d r e n e r g i c b l o c k i n g agents t e s t e d . The c o n t r a c t i l i t y o f the myocardium i s r e g u -l a t e d t o an i m p o r t a n t e x t e n t by the i m p u l s e t r a f f i c i n the c a r d i a c s y m p a t h e t i c n e r v e s , which d e t e r m i n e s th e r a t e a t which the n e u r o t r a n s m i t t e r i s r e l e a s e d from the nerve t e r m i n a l s and, t h e r e f o r e , the q u a n t i t y o f n o r e p i n e p h r i n e a c t i n g upon the myocardium. D u r i n g m u s c u l a r e x e r c i s e o r o t h e r c o n d i t i o n s i n w h i c h th e r e q u i r e m e n t s f o r c a r d i a c performance are augmented, t h e r e i s an i n c r e a s e i n the a c t i v i t y o f the c a r d i a c s y m p a t h e t i c n e r v e s . A c c o r d i n g l y , i t might be a n t i c i p a t e d t h a t b e t a -1 9 adrenergic blockade would l i m i t the cardiac output during muscular exercise and thereby place a l i m i t on exercise per-formance (30)• S i g n i f i c a n t symptomatic improvement has been reported i n angina pectoris using o r a l propranolol. Propranolol has been found useful i n antagonizing arrhythmias induced by catecholamines and d i g i t a l i s . A t r i a l f i b r i l l a t i o n and a t r i a l f l u t t e r are likewise antagonized but treatment of ve n t r i c u l a r arrhythmias has not been so successful. Propranolol has decreased the mortality rate of acute myocardial i n f a r c t i o n somewhat and i t also brings about a reduction i n frequency of anginal attacks i n patients with idiopathic hypertrophic sub-ao r t i c stenosis. Deleterious effects related to the pharmacologic action of beta blockade are not uncommon. Severe hypotension or the sudden appearance of increasing cardiac f a i l u r e have been re-ported, usually i n patients with moderately advanced cardiac disease and whose hearts were presumably c r i t i c a l l y dependent upon sympathetic support to maintain compensation. The acute toxic effects and the LD 50 of propranolol are si m i l a r to those of pronethalol but propranolol has not been found to be carcinogenic. Daily treatment of mice with propranolol over a period of eighteen months did not produce thymic tumours (28). 20 In c l i n i c a l practice two per cent of the patients ex-perienced minor side effects which usually did not require discontinuation of the drug. Two cases of nonthrombocytopenic purpura which cleared on cessation of the drug were reported. Marked bronchoconstriction may occur i n asthmatic patients. Beta-adrenergic blocking drugs may be of considerable therapeutic effectiveness i n certain circumstances but they are potent agents and must be used with considerable care. They have provided pharmacologists with a research tool which has further f a c i l i t a t e d . i n v e s t i g a t i o n s of adrenergic mechanisms. Alpha-adrenergic Blocking. Agent's - The 2-Halogehoethylamines The adrenergic blocking actions of the 2-halogenoethyl-amines were f i r s t noted by Nickerson and Goodman i n 1945 when they showed that N,N-dibenzyl-2-chloroethylamine (Dibenamine) antagonized the pressor actions of injected adrenaline i n the cat. Following t h i s discovery a considerable amount of work was carried out on related 2-halogenoethylamines and many were found to possess si m i l a r adrenergic blocking properties (31). These compounds are of substantial value as pharmacological tools i n the understanding of drug-receptor interactions. Phenoxybenzamine has been most extensively investigated i n man and i s the only member of the series available for c l i n i c a l use. 21 Phenoxybenzamine F i g u r e 4. A l p h a - a d r e n e r g i c b l o c k i n g agents A p r o l o n g e d d u r a t i o n o f a c t i o n and the completeness w i t h which the s e l e c t e d r e s p o n s e s are a n t a g o n i z e d a r e c h a r -a c t e r i s t i c o f t h e s e b l o c k i n g a g e n t s . The a d r e n e r g i c b l o c k , once e s t a b l i s h e d , cannot be overcome by h i g h c o n c e n t r a t i o n s o f n o r e p i n e p h r i n e . However, d u r i n g the e a r l y s t a g e s o f the b l o c k a d e , n o r e p i n e p h r i n e can p r e v e n t , more o r l e s s c o m p l e t e l y , 22 the production of the i r r e v e r s i b l e actions of the 2-halogeno-ethylamines. This suggests that there i s i n i t i a l l y compe-t i t i o n between the blocker and the norepinephrine for attach-ment to the receptor s i t e . The law of mass action applies to th i s r e l a t i o n s h i p . In due course this relationship i s l o s t and complete block ensues. I t i s apparent that the 2-halogen-oethylamines are acting at the same receptor s i t e as norepine-phrine and may be described as competitive i r r e v e r s i b l e antagonists of norepinephrine (32) . This term i s meant to imply that the blocker acts at the.same s i t e as norepinephrine but i t i s i r r e v e r s i b l e or non-equilibrium i n it s ' action. This block once established cannot be removed from an iso l a t e d tissue by washing except over a period of several days. The persistence and completeness of the blockade produced appear to be dependent upon a r e l a t i v e l y stable bonding to tissue constituents. An attempt has been made to relate the p e r s i s -tence of action of these compounds to t h e i r presence i n the body by administering to mice phenoxybenzamine la b e l l e d at 14 the methylene of the benzyl group with a C atom and following the d i s t r i b u t i o n of radioactive emission (32). Accumulation of large- amounts, however, was not demonstrable i n f a t depots, r a d i o a c t i v i t y being found i n the soft tissues, p a r t i c u l a r l y the l i v e r and kidneys, even afte r twenty-four hours. About half of the t o t a l a c t i v i t y was excreted i n the urine. 23 The 2-halogenoethylamines are unique i n t h a t b i o l o g i c a l a c t i v i t y i s dependent on c h e m i c a l r e a c t i v i t y , t h e p r o d u c t o f c h e m i c a l and p h y s i c a l p r o p e r t i e s . The b l o c k i n g a c t i o n on the a l p h a r e c e p t o r f o r c a t e c h o l a m i n e s i s not e x e r t e d by t h e p a r e n t compound but by the ethyleneimonium i o n which i s formed .from i t s r e a c t i o n i n aqueous s o l u t i o n . The r e l a t i v e l y slow o n s e t o f a c t i o n o f 2-halogenoethylamines even a f t e r i n t r a v e n o u s a d m i n i s t r a t i o n i s p r o b a b l y due t o t h e time r e q u i r e d f o r t h e f o r m a t i o n o f t h e r e a c t i v e i n t e r m e d i a t e which t h e n a c t s as an a l k y l a t i n g agent on a r e c e p t o r c o n s t i t u e n t . The e x a c t n a t u r e o f t h e g r o u p i n g on o r n e a r ' t h e a l p h a - a d r e n e r g i c r e c e p t o r w i t h which t h e s e compounds r e a c t i s not known. An analogy has been drawn between th e mode o f a c t i o n o f 2-halogenoethylamines and r e l a t e d n i t r o g e n mustard compounds wh i c h a c t by a l k y l a t i o n ..also. The s p e c i f i c i t y o f t h e 2-halogenoethylamines f o r t h e a l p h a - a d r e n e r g i c r e c e p t o r i s h i g h , b u t n o t a b s o l u t e , t h e d i s -s o c i a t i o n c o n s t a n t f o r t h i s r e c e p t o r b e i n g s i m p l y lower than t h a t o f o t h e r r e c e p t o r s . F o r example, Boyd (34) found phen-oxybenzamine t o be e x t r e m e l y p o t e n t a g a i n s t t h e e x c i t a t o r y a c t i o n s o f added a c e t y l c h o l i n e and o f c h o l i n e r g i c nerve s t i m -u l a t i o n . He s u g g e s t s t h a t the use o f autonomic drugs as a n a l y t i c a l t o o l s i s n o t r e l i a b l e u n l e s s combined w i t h b o t h h i s t o c h e m i c a l e x a m i n a t i o n o f the p r e p a r a t i o n and assay o f t h e s u b s t a n c e s r e l e a s e d upon s t i m u l a t i o n o f the n e r v e s . 24 The s t r u c t u r a l c o n f i g u r a t i o n s which favour a n t i a d r e n -e r g i c a c t i o n a l s o favour a n t i h i s t a m i n i c a c t i o n i n the 2-halogenoethylamines ( 3 5 ) . The attachment of the compounds to the histamine r e c e p t o r i s not so s t r o n g as t h a t to the al p h a - a d r e n e r g i c r e c e p t o r and i s p a r t i a l l y r e v e r s i b l e by washing i n an i s o l a t e d p r e p a r a t i o n . Antagonism to 5-hydroxy-tryptamine i s a l s o seen ( 3 6 ) . Although blockade of responses to histamine and 5-hydroxytryptamine r e q u i r e the same b a s i c chemical c o n f i g u r a t i o n as does a l p h a - a d r e n e r g i c b l o c k i n g a c t i v i t y , potency w i t h r e s p e c t to these p r o p e r t i e s v a r i e s w i d e l y . The 2-halogenoethylamines do not a l t e r f u n c t i o n of ad r e n e r g i c nerves, t r a n s m i s s i o n through autonomic g a n g l i a nor the b a s i c response mechanisms of e f f e c t o r c e l l s . They have no e f f e c t on the enzyme ca t e c h o l — O - m e t h y l t r a n s f e r a s e ( 3 7 ) . Many of these compounds i n h i b i t monoamine oxidase t o v a r y i n g degrees but t h i s e f f e c t does not p a r a l l e l the a l p h a - a d r e n e r g i c b l o c k i n g e f f e c t s ( 3 5 ) . The p r e s s o r response t o a d r e n a l i n e i n the i n i t i a l stages of treatment of an animal with 2-halogenoethylamines may be enhanced r a t h e r than antagonized. T h i s e f f e c t i s t a c h y p h y l a c t i c i n untreated.animals but absent i n animals p r e t r e a t e d with r e s e r p i n e . I t i s r e s t o r e d when such animals are i n f u s e d with n o r e p i n e p h r i n e . The plasma l e v e l of nore-p i n e p h r i n e has been shown to r i s e a f t e r a d m i n i s t r a t i o n of 25 phenoxybenzamine even a f t e r adrenalectomy. The mechanism of t h i s e f f e c t i s not c l e a r . The r i s e i n catecholamines i s d i f -f i c u l t t o i n t e r p r e t because of the marked i n c r e a s e i n sympa-t h e t i c nerve a c t i v i t y induced r e f l e x l y by the p e r i p h e r a l blockade ( 3 8 ) . I t i s l i k e l y t h a t the i n i t i a l a c t i o n of 2-halogenoethylamines i s to d i s p l a c e the catecholamines from jtheir storage s i t e s and to loosen t h e i r attachment to the c e l l r e c e p t o r s . Phenoxybenzamine has a l s o been used to block up-take of n o r e p i n e p h r i n e by t i s s u e storage mechanisms ( 3 9 ) . In g e n e r a l , 2-halogenoethylamines are p o o r l y s o l u b l e i n water. When g i v e n o r a l l y , a b s o r p t i o n i s poor and i r r e g u l a r , only twenty to t h i r t y per cent of phenoxybenzamine being absorbed i n the o r a l form." I f g i v e n p a r e n t e r a l l y , the intravenous route should be used as the products of decompo-s i t i o n are a c i d and very i r r i t a t i n g by other p a r e n t e r a l r o u t e s . They must be i n j e c t e d s l o w l y and w e l l d i l u t e d to a v o i d d i s -turbance of the c e n t r a l nervous system. Phenoxybenzamine g i v e n o r a l l y i s s a i d to produce a prolonged i n c r e a s e i n blood flow i n p a t i e n t s s u f f e r i n g from p e r i p h e r a l v a s c u l a r d i s o r d e r s . I t can be used i n the d i a g n o s i s and p r e o p e r a t i v e management of pheochromocytoma. Good e f f e c t s have been shown i n management of h y p e r t e n s i v e d i s e a s e but i r r e g u l a r i t y of a b s o r p t i o n by mouth makes i t d i f f i c u l t t o c o n t r o l the dose-response r e l a t i o n s h i p . In treatment of shock, intravenous phenoxybenzamine can prevent the i r r e v e r -s i b l e phase by b l o c k i n g p e r i p h e r a l v a s o c o n s t r i c t i o n . 26 The c l i n i c a l a p p l i c a t i o n o f t h e s e p o t e n t drugs has been d e l a y e d and h i n d e r e d because o f the u n p l e a s a n t s i d e e f f e c t s t hey produce. R e l a x a t i o n o f p e r i p h e r a l a r t e r i o l e s causes o r t h o s t a t i c h y p o t e n s i o n and r e f l e x t a c h y c a r d i a . There i s c o n g e s t i o n o f t h e n a s a l mucosa and m i o s i s . The a d r e n e r g i c component of s w e a t i n g may c e a s e , w h i l e t h e l o s s o f h e a t on exposure t o c o l d may i n c r e a s e g r e a t l y due t o f a i l u r e o f t h e s k i n v e s s e l s t o c o n s t r i c t . I n j e c t i o n i n t o a h y povolemic p a t i e n t may cause a sharp f a l l i n b l o o d p r e s s u r e . Large doses produce s t i m u l a t i o n o f the c e n t r a l nervous system r e s u l t i n g i n nausea, v o m i t i n g , h y p e r v e n t i l a t i o n , motor e x c i t a b i l i t y and c o n v u l s i o n s . Phenoxybenzamine g i v e n i n s m a l l e r doses can produce a f e e l i n g o f s l e e p i n e s s and weakness. Due t o t h e i r r i t a n c y o f t h e s e compounds, o r a l use may be accompanied by v o m i t i n g , c o l i c o r d i a r r h e a . C a t e c h o l - O - M e t h y l t r a n s f e r a s e A l t h o u g h the m e t a b o l i s m o f c a t e c h o l a m i n e s such as e p i n e p h r i n e , n o r e p i n e p h r i n e and dopamine was thought a t one time t o proceed m a i n l y by d e a m i n a t i o n , e v i d e n c e o f a n o t h e r pathway, O - m e t h y l a t i o n , has been found ( 4 0 ) . I n 1957, A x e l r o d demonstrated i n v i t r o t h a t e p i n e p h r i n e was O-methylated t o metanephrine i n an e x t r a c t o f r a t l i v e r i n c u b a t e d w i t h S - a d e n o s y l m e t h i o n i n e . I n v i v o O - m e t h y l a t i o n o f n o r e p i n e p h r i n e and e p i n e p h r i n e was demonstrated by A x e l r o d ' e t a l i n .1958 (41) . The enzyme c a t e c h o l - O - m e t h y l t r a n s f e r a s e (COMT) r e q u i r e s t h e c o - f a c t o r S - a d e n o s y l m e t h i o n i n e f o r a c t i v i t y as w e l l as •u , » + + » , + + ^ ++' „ ++ „ + + r , + + • a d i v a l e n t c a t i o n such as Mg , Mn , Co , Ca , Zn , Fe o r N i + + J A l l c a t e c h o l s a r e m e t h y l a t e d r e g a r d l e s s o f t h e s i d e c h a i n on t h e a r o m a t i c n u c l e u s i n c l u d i n g n o r e p i n e p h r i n e , dopamine, dopa, .3,4-dihydroxymandelic a c i d and 3 , 4 - d i h y d r o x y -p h e n y l a c e t i c a c i d . The enzyme does n o t show s p e c i f i c i t y toward d e x t r o o r l e v o i s o m e r s . Monophenols are not m e t h y l a t e d ( 4 2 ) . O - m e t h y l a t i o n o c c u r s m a i n l y on t h e meta p o s i t i o n b u t a l s o on t h e p h e n o l i c group p a r a t o e l e c t r o n - d e f i c i e n t as w e l l as n o n d e f i c i e n t s i d e c h a i n s (43) . P h e n o l i c s u b s t r a t e s h a v i n g t h r e e a d j a c e n t hydroxy groups such as g a l l i c a c i d and p y r o g a l l o l a r e m e t h y l a t e d on t h e m i d d l e hydroxy group r e g a r d l e s s of whether th e hydroxy group o c c u p i e s a p a r a o r a meta p o s i t i o n (44). The s p e c i f i c i t y f o r c a t e c h o l s and the r e q u i r e m e n t f o r d i v a l e n t c a t i o n s s u g g e s t s t h a t the c a t i o n may s e r v e as a c h e l a t i n g agent t o b r i n g t h e enzyme, S - a d e n o s y l m e t h i o n i n e , and s u b s t r a t e t o g e t h e r i n a b r i d g e complex. The m e t h y l group o f S - a d e n o s y l -m e t h i o n i n e , w h i c h i s e l e c t r d p h i l i c would t h e n be t r a n s f e r r e d t o t h e hydroxy by a n u c l e o p h i l i c d i s p l a c e m e n t r e a c t i o n . S i n c e i n t h e case o f most c a t e c h o l s t h e meta hydroxy group i s s t r o n g l y n u c l e o p h i l i c , t h e t r a n s f e r t a k e s p l a c e on t h e 3 p o s i t i o n . The O - m e t h y l a t i o n o f c a t e c h o l a m i n e s i s an i n a c t i -v a t i o n p r o c e s s s i n c e the p h y s i o l o g i c a l a c t i v i t y o f t h e 28 O-methylated•products i s much less than that of the parent compound. The r e l a t i v e importance of in a c t i v a t i o n by COMT was found to depend on whether the catecholamines are c i r c u l a t i n g or bound and on the tissue and species involved. In experi-ments on recovery of infused norepinephrine into cat spleen G i l l e s p i e and Kirpekar (11) showed that 80 per cent of NE was bound to nerve endings thus l i m i t i n g binding to a l l other s i t e s to 20 per cent. At most, l o c a l COMT may inactivate about f i f t e e n per cent of infused NE. As mentioned previously reuptake by the sympathetic neurons i s probably the most im-portant mechanism for i n a c t i v a t i o n of both c i r c u l a t i n g and l o c a l l y released NE. In man, a l l of the endogenous NE and epinephrine (E) not taken up by tissue binding i s metabolized and excreted as O-methylated-deaminated products. The d a i l y excretion of endogenous O-methylated metabolites ranges from 2-4 mg for 3-methoxy-4 hydroxymandelic acid (VMA), 100-300 micrograms for normetanephrine, and 100 to 200 micrograms for metane-phrine. Most of the VMA presumably arises from the deamination of NE within the sympathetic nerves followed by O-methylation, probably outside the nerves. The VMA i n the urine probably represents the amount of NE produced and metabolized before i t had a chance to produce a physiological e f f e c t . The nor-metanephrine largely represents the amount of ph y s i o l o g i c a l l y active NE that was discharged from the sympathetic nerves. on H O N o r e p i n e p h r i n e M o n o a m i n e o x i d a s e C a t e c h o l - O - m e t h y l -t r a n s f e r a s e CHjO \ \ CH CHXNHV M o n o a m i n e o x i d a s e N o r m e t a n e p h r i n e OH A l d e h y d e d e h y d r o g e n a s e 3 , 4 - d i h y d r o x y p h e n y l g l y c o l a l d e h y d e 3 , 4 , - d i h y d r o x y m a n d e l i c a c i d A l d e h y d e r e d u c t a s e CM CII^ OM HO 3 , 4 - d i h y d r o x y p h e n y l g l y c o l ^ OH 3 - m e t h o x y - 4 - h y d r o x y p h e n y l g l y c o l A l d e h y d e d e h y d r o g e n a s e 3 - m e t h o x y - 4 - h y d r o x y p h e n y l -g l y c o l a l d e h y d e C a t e c h o l - O - m e t h y l -t r a n s f e r a s e OH CMcOotL HO 3 - m e t h o x y - 4 - h y d r o x y -m a n d e l i c a c i d ( V M A ) F i g u r e 5 . P a t h w a y s o f m e t a b o l i s m o f n o r e p i n e p h r i n e . 30 The d i s t r i b u t i o n o f COMT i s w i d e s p r e a d . I t i s found i n g l a n d s , b l o o d v e s s e l s , s y m p a t h e t i c and p a r a s y m p a t h e t i c n e r v e s and g a n g l i a and a l l ar e a s o f the b r a i n . I n homogenates COMT i s c o n f i n e d m a i n l y t o t h e s o l u b l e s u p e r n a t a n t f r a c t i o n . A s m a l l amount o f t h i s enzyme i s a l s o p r e s e n t i n the m i c r o s o m a l f r a c t i o n o f the l i v e r . C a t e c h o l - O - M e t h y l t r a n s f e r a s e I n h i b i t i o n by T r o p o l o n e s W i t h t h e d i s c o v e r y o f t h e r o l e o f COMT i n m e t a b o l i s m o f the c a t e c h o l a m i n e s , i n h i b i t o r s o f t h i s enzyme were sought. On the b a s i s o f s i m p l e s t r u c t u r a l a n a l o g i e s ; i t c o u l d be seen t h a t a v a r i e t y o f p o l y p h e n o l s s h o u l d compete w i t h c a t e c h o l s f o r COMT. I t was e s t a b l i s h e d t h a t p y r o g a l l o l c o u l d compete w i t h c a t e c h o l a m i n e s i n the m e t h y l a t i o n r e a c t i o n s . However, p y r o g a l l o l a c t s c o m p e t i t i v e l y , i s a good s u b s t r a t e f o r t h e enzyme and i s b i o c h e m i c a l l y l a b i l e . I t was t h e r e f o r e n o t a s a t i s f a c t o r y i n h i b i t o r f o r q u a n t i t a t i v e s t u d i e s i n v i v o . I n a p r e l i m i n a r y paper i n 1961, B e l l e a u and Burba r e p o r t e d on a p r o t o t y p e o f t h e t r o p o l o n e s , 4 - m e t h y l t r o p o l o n e . A t a concen--4 t r a t i o n o f 3 x 10 . M, 4 - m e t h y l t r o p o l o n e produced 90 p er c e n t i n h i b i t i o n o f m e t h y l a t i o n o f c a t e c h o l a m i n e s i n a r a t l i v e r enzyme p r e p a r a t i o n . They r e p o r t e d t h e i n h i b i t i o n o f COMT t o be n o n - c o m p e t i t i v e b u t l a t e r work showed i t t o be o f a com-p e t i t i v e n a t u r e . 31 Musacchio and Goldstein i n 1962 (45) studied the effects of beta-thujaplicin (4-isopropyltropolone) on the metabolism of catecholamines i n vivo i n rats . They found a decrease i n O-methylated products i n the urine and an i n -crease i n NE content i n the spleen and heart. Mavrides et a l i n 1963 (46) v e r i f i e d the competitive nature of COMT i n h i b i t i o n by 4-methyltropolone on rat l i v e r preparations and i n i n vivo work i n the rat showed a decreased excretion of la b e l l e d metanephrine i n the urine a f t e r 4-methyltropolone. Further work was done by Belleau and Burba i n 1963 (47), on a long series of substituted tropolones which produced detailed data on i n h i b i t i o n constants toward the enzyme COMT, Beta-thujaplicin, gamma-thujaplicin, 4-methyltropolone and 7-hydroxy-4-isopropyltropolone (beta-hydroxy t h u j a p l i c i n , BHT) a l l showed i n h i b i t o r y a c t i v i t y . Their r e s u l t s indicated that the presence of ringsubstituents or t h e i r nature had only a marginal e f f e c t on the a f f i n i t y of the tropolone ring for the enzyme. Belleau has theorized that since the tropolone ring i s v i r t u a l l y i s o s t e r i c with the catechol r i n g , these i n h i b i -tors might displace catechols from the enzyme surface. The chelating properties of the tropolones would also f i t the complex formation envisioned between the catechol r i n g , a divalent ion, S-adenosylmethionine and COMT (see Figure 6). 3 2 F i g u r e 6. A p o s t u l a t e d complex formed between the t r o p o l o n e r i n g , a d i v a l e n t c a t i o n , S - a d e n o s y l m e t h i o n i n e and c a t e c h o l - O - m e t h y 1 t r a n s f e r a s e . I n h i s s t u d i e s on i n h i b i t i o n c o n s t a n t s , B e l l e a u ob-s e r v e d t h a t i n b o t h the i n h i b i t o r and s u b s t r a t e s e r i e s , t h e p r e s e n c e o f r i n g s u b s t i t u e n t s produced p a r a l l e l m a r g i n a l v a r i a t i o n s on t h e a f f i n i t y f o r COMT. T h i s i s e v i d e n c e t h a t 33 the active s i t e s responsible for the binding of catechols may be the same as those involved i n the binding of tropolones. If the tropolones were to act simply as metal ion scavengers as i s the case with ethylenediamine te t r a a c e t i c acid (EDTA) t h e i r s p e c i f i c i t y would be limited. Belleau tested the p o s s i b i l i t y that tropolones could act by p a r t i a l l y depriving COMT of i t s magnesium ions and found that a f i v e - f o l d r e l a t i v e ++ increase i n [Mg ] had no e f f e c t on the in h i b i t e d rate i n the presence of 4-methyltropolone.- This would indicate that the tropolones form a complex with the enzyme active s i t e s nor-mally occupied by the substrate and some degree of s p e c i f i c i t y would be expected. Another possible chelation mechanism whereby the tro -polones would potentiate catecholamines i s by chelation of copper ions involved i n oxidation reactions. In iso l a t e d preparations of rabbit a o r t i c s t r i p s , when EDTA i s added to the bath i t potentiates the epinephrine contraction. When 4-methyltropolone i s added i n addition, further potentiation occurs. I t would appear therefore that the potentiating e f f e c t of the tropolones cannot be explained by the removal of copper ions by chelation. Murnaghan and Mazurkiewicz (1963) (48) investigated the influence of 4-methyltropolone on the t o x i c i t y of cate-cholamines i n mice. They found that 4-methyltropolone i n small doses (10 mg/Kg) increased the percentage mortality of 34 mice i n j e c t e d w i t h e p i n e p h r i n e b u t i n l a r g e r doses (100 mg/Kg) 4- m e t h y l t r o p o l o n e e x e r t e d a p r o t e c t i v e a c t i o n . The i n c r e a s e i n m o r t a l i t y was a t t r i b u t e d t o i n h i b i t i o n o f c a t e c h o l - O -m e t h y l t r a n s f e r a s e and the p r o t e c t i v e a c t i o n was thought t o be due t o a t r a n s i t o r y b l o c k a d e o f th e a l p h a r e c e p t o r s . As an a l p h a b l o c k e r 4 - m e t h y l t r o p o l o n e i s one hundred t i m e s l e s s e f f e c t i v e than, p h e n t o l a m i n e and i s t r a n s i e n t and n o n - s p e c i f i c . 4 - m e t h y l t r o p o l o n e d i d n ot p o t e n t i a t e n o r e p i n e p h r i n e and t h i s was a t t r i b u t e d t o r a p i d b i n d i n g w i t h t i s s u e components. S i m i l a r l y , the COMT i n h i b i t o r p y r o g a l l o l p o t e n t i a t e s e p i n e -p h r i n e and n o t n o r e p i n e p h r i n e . The t o x i c i t y o f i s o p r o t e r e n o l was p o t e n t i a t e d by low and h i g h doses o f 4 - m e t h y l t r o p o l o n e . T h i s e f f e c t was a t t r i b u t e d t o COMT i n h i b i t i o n . E x p e r i m e n t s on the c a t ' s b l o o d p r e s s u r e i n d i c a t e d t h a t the 4 - m e t h y l t r o p o l o n e had some b e t a - r e c e p t o r b l o c k i n g a c t i o n as i t reduced the v a s o d e p r e s s o r e f f e c t s o f i s o p r o t e r e n o l . Murnaghan p u b l i s h e d more complete r e s u l t s o f i n v i v o and i n v i t r o e f f e c t s o f some t r o p o l o n e s i n 1964 (49). B e t a -t h u j a p l i c i n and t r o p o l o n e p a r a l l e l l e d t he e f f e c t s o f 4-m e t h y l t r o p o l o n e on e p i n e p h r i n e m o r t a l i t y i n mice. On the r a b b i t ' s a o r t i c h e l i c a l s t r i p , t r o p o l o n e , 4 - m e t h y l t r o p o l o n e , b e t a - t h u j a p l i c i n and p a p a v e r i n e i n low c o n c e n t r a t i o n p o t e n -t i a t e d the e p i n e p h r i n e response b u t a n t a g o n i z e d i t i n h i g h e r c o n c e n t r a t i o n s . Murnaghan i n v e s t i g a t e d the p o s s i b i l i t y t h a t the t r o p o l o n e s c o u l d s e n s i t i z e smooth muscle t o a v a r i e t y o f s t i m u l a n t s . He found t h a t the t r o p o l o n e s i n low c o n c e n t r a t i o n s 35 c o u l d p o t e n t i a t e the s t i m u l a n t e f f e c t s o f p o t a s s i u m and barium i o n s on a o r t i c s t r i p s and a n t a g o n i z e them i n h i g h c o n c e n t r a t i o n s . L i k e w i s e , t h e t r o p o l o n e s c o u l d p o t e n t i a t e c a r b a c h o l , h i s t a m i n e and barium i o n s on the g u i n e a - p i g i l e u m . C o n s e q u e n t l y , Murnaghan reasoned t h a t t h e p o t e n t i a t i o n o f e p i n e p h r i n e on i s o l a t e d t i s s u e s appeared t o be due t o a non-s p e c i f i c s e n s i t i z a t i o n produced by a v a r i e t y o f compounds. However, i t i s p o s s i b l e t h a t i n h i b i t i o n o f COMT may have p l a y e d a r o l e i n t h e e p i n e p h r i n e i n d u c e d m o r t a l i t y e x p e r i -ments on mice. 36 METHODS AND MATERIALS Isolated Rabbit A o r t i c Strips The studies were carried out i n v i t r o using as a vas-cular smooth muscle preparation the s p i r a l l y - c u t s t r i p of rabbit thoracic aorta. This preparation i s highly sensitive to many drugs. The thinness of the s t r i p , about 0.4 mm, permits r e l a t i v e l y rapid d i f f u s i o n of drugs from the surrounding medium into deeper layers when the surrounding medium i s well oxygenated. The s t r i p s usually have n e g l i g i b l e inherent tone and never show rhythmic contractions. H i s t o l o g i c a l examination of the rabbit thoracic artery shows an e s s e n t i a l l y c i r c u l a r orientation of the muscle i n this vessel. There i s p r a c t i c a l l y no l o n g i t u d i n a l l y oriented smooth muscle. The c i r c u l a r l y oriented muscle constitutes almost half of the contents of the vessel wall, a high f r a c -t i o n for an aorta (50) . Male, albino rabbits of from three to f i v e pounds weight were used. The rabbit was k i l l e d by a blow on the base of the s k u l l and the descending thoracic aorta was re-moved and placed i n Krebs-bicarbonate solution. Fat and connective tissue were removed and the whole length of the excised vessel was cut along a close s p i r a l . The r e s u l t i n g s t r i p was 2.0 to 2.5 millimeters wide and i t s smooth muscle fi b r e s (the c i r c u l a r l y arranged fibres of the i n t a c t aorta), were oriented at about f i f t e e n degrees r e l a t i v e to the long 37 axis of the s t r i p . Shorter s t r i p s of two centimeters long were cut from the long s t r i p and mounted i n the muscle baths. The muscle baths, of a capacity of ten m i l l i l i t e r s , were maintained at a temperature of 37.5°C and were aerated by a flow of gas of 95 per cent oxygen and 5 per cent carbon dioxide. The Krebs-bicarbonate solution used i n the muscle baths contained 0.0,1 M glucose. The solution was freshly made before each experiment by mixing i n the following order: 100 ml of a 10-fold concentrated s a l t solution (82.60 gm Na.Cl, .4.22 gm KC1, 3.36 gm CaCl 2 , 1.94 gm-KH2P04, and 3.50 gm MgS04-7H20 per l i t e r ) , 900 ml d i s t i l l e d water, 192 ml of 1.3% NaHGO^ and 2.15 gm glucose. Adequate washout of drugs was accomplished by passing about 60 ml of bathing f l u i d from the reservoir flask through the muscle baths by an overflow method. The ao r t i c s t r i p s were connected by surgical clamps and thread to recording levers under a tension of four grams. Heart/smooth muscle modules Model #271 manufactured by the Harvard Apparatus Co. were used and the maximum magnification of six times the muscle response was recorded throughout on a Harvard Chart Mover using a chart speed of 3 millimeters per minute. A s p i r a l l y cut rabbit a o r t i c s t r i p attached to a lever under four grams tension immediately stretches about forty per cent followed by a further gradual increase i n length which may vary from.two to ten per cent. This gradual c 38 i n c r e a s e approximates an e x p o n e n t i a l c u r v e w i t h a h a l f time o f t e n t o t w e n t y - f i v e minutes so t h a t e l o n g a t i o n i s e s s e n -t i a l l y complete i n one and a q u a r t e r t o t h r e e h o u r s . T h i s g r a d u a l e l o n g a t i o n does n ot r e s u l t from a g r a d u a l l o s s o f i n h e r e n t tone b u t r a t h e r from a p h y s i c a l p r o c e s s i n which c e r t a i n u n d e f i n e d s t r u c t u r a l elements o f the a o r t i c s t r i p s l o w l y l e n g t h e n under t e n s i o n . A g r a d u a l i n c r e a s e i n s e n s i -t i v i t y t o s t i m u l a t i n g drugs o c c u r s w i t h t h e s t r e t c h i n g b u t p r o b a b l y i s not dependent on i t (51) . I n t h e s e e x p e r i m e n t s a s t r e t c h i n g t ime o f two hours was a l l o w e d b e f o r e any drugs were added. When a s t i m u l a t i n g drug was added t h e time r e q u i r e d f o r a c o n t r a c t i o n t o r e a c h i t s f u l l h e i g h t v a r i e d from 5 t o 15 m i n u t e s , depending on the drug used, i t s c o n c e n t r a t i o n , and the i n d i v i d u a l a o r t i c s t r i p . Once t h e f u l l c o n t r a c t i o n has been a c h i e v e d the tone i s h e l d c o n s t a n t f o r s e v e r a l min-u t e s e x c e p t when the drug i s i n a c t i v a t e d i n s o l u t i o n as i s n o r e p i n e p h r i n e , o r when t a c h y p h y l a x i s o c c u r s as w i t h h i g h c o n c e n t r a t i o n s o f t y r a m i n e . A f t e r washout o f a s t i m u l a t i n g d r u g , the time r e q u i r e d f o r r e l a x a t i o n o f a s t r i p t o t h e b a s e l i n e may v a r y from about 10 minutes a f t e r a low c o n t r a c -t i o n t o a l m o s t one hour a f t e r a maximal c o n t r a c t i o n o b t a i n e d w i t h h i g h c o n c e n t r a t i o n s o f n o r e p i n e p h r i n e o r h i s t a m i n e . I n some c a s e s , e s p e c i a l l y a f t e r t h e t h u j a p l i c i n s , b a s a l tone was n o t reached two hours a f t e r a c o n t r a c t i o n , even a f t e r 39 repeated washings. This r e l a t i v e l y long relaxation time of a o r t i c s t r i p s following wash-out of stimulating drugs i s one of t h e i r disadvantages, since i t l i m i t s the number of tests that can be carried out on any one s t r i p within a given time. The s e n s i t i v i t y of the s t r i p s to d i f f e r e n t stimulating drugs and the r e l a t i v e heights of the maximal contractions obtainable with them varied considerably. In the case of tyramine, GT, and BHT the s e n s i t i v i t y of s t r i p s from d i f f e r e n t aortas and even from d i f f e r e n t sections of the same aorta may vary widely. For example, i t was found that the part of the aorta proximal to the heart was much more sensitive to drugs than the remaining d i s t a l portion. When thi s was discovered, only that portion of the aorta proximal to the heart was used i n further work. This aided i n reducing v a r i a b i l i t y of res-ponses. I t was also found that p r i o r use of one of the t h u j a p l i c i n s on a s t r i p greatly reduced the response of any agonist subsequently used on that s t r i p . The second response of the t h u j a p l i c i n i t s e l f was also decreased or more usually e n t i r e l y blocked. Consequently, only one test per s t r i p could be done using BHT or GT. Drugs which cause relaxation l i k e isoproterenol can-not be tested on untreated a o r t i c s t r i p s since these s t r i p s possess l i t t l e or no tone and are therefore already i n a relaxed state. For quantitative studies on the relaxing e f f e c t s of such drugs, the s t r i p s must f i r s t be brought into a steady state of tone by adding a drug causing contraction. 40 S i n c e i s o p r o t e r e n o l causes the r e l a x a t i o n o f tone produced by the p r i o r a d d i t i o n o f n o r e p i n e p h r i n e and s i n c e the s t r i p s have n e g l i g i b l e i n h e r e n t t o n e , t h e i s o p r o t e r e n o l must r e l a x t h e v e r y same muscle c e l l s w hich the n o r e p i n e p h r i n e c o n t r a c t e d . These muscle c e l l s t h e r e f o r e seem t o have b o t h a l p h a - and b e t a - a d r e n e r g i c r e c e p t o r s ( 5 2 ) . I t must be r e c o g n i z e d t h a t r esponses o f t h e r a b b i t t h o r a c i c a o r t a t o c e r t a i n drugs may d i f f e r q u a n t i t a t i v e l y and q u a l i t a t i v e l y from t h e responses o f the smooth muscle o f p e r i p h e r a l v a s c u l a r beds, w h i c h a r e o f much g r e a t e r i m p o r t a n ce i n c o n t r o l l i n g b l o o d p r e s s u r e i n t h e i n t a c t a n i m a l . F o r example, a o r t i c s t r i p s a r e c o n t r a c t e d by h i s t a m i n e and a c e t y l -c h o l i n e , whereas t h e s e drugs d i l a t e most p e r i p h e r a l v a s c u l a r beds i n most s p e c i e s . The response o f t h e smooth muscle o f the r a b b i t a o r t i c s t r i p s t o drugs i n v i t r o i s not c o m p l i c a t e d by many o f t h e v a r i a b l e s w h i c h m o d i f y t h e response o f v a s c u l a r smooth muscle i n the i n t a c t a n i m a l o r i n p e r f u s e d t i s s u e s . Because o f i t s s i m p l i c i t y , t h i s p r e p a r a t i o n i s t h e r e f o r e most u s e f u l f o r i n v e s t i g a t i n g t h e modes and mechanisms o f t h e l o c a l a c t i o n o f drugs on v a s c u l a r smooth muscle. 41 RESULTS The e f f e c t of beta-hydroxy t h u j a p l i c i n , gamma-thujaplicin, ethylenediamine te t r a - a c e t i c acid and pyr o g a l l o l on the response of the is o l a t e d aorta to norepinephrine Various tropolone derivatives are capable of i n h i b i t i n g the enzyme catechol-O-methyltransferase (COMT). Some of the i n i t i a l experiments with the th u j a p l i c i n s on iso l a t e d aorta were concerned with the effects of these compounds on the response to norepinephrine (NE). An attempt was made to see i f there was any potentiation of. the NE response which might be attributable to i n h i b i t i o n of COMT. In these experiments the t h u j a p l i c i n s were compared with another COMT i n h i b i t o r , pyrogallol,~and another chelating agent, ethylenediamine-te t r a - a c e t i c acid (EDTA). _7 A control response to NE 0.8 x 10 M was f i r s t re-corded and the s t r i p was allowed to relax to i t s normal length. The appropriate concentration of the drug to be -7 tested was placed m the bath, then NE 0.8 x 10 M was added and the response was recorded. The results were tabu-lated as the actual height of the response as well as the per cent increase i n the height over the control NE response. The per cent increase i n the Duration - 50 (the time required for the s t r i p to relax to half of the maximum height attained) 42 was also tabulated (see Tables I and I I ) . I t was found that the presence of any of these four substances i n the bath s i g n i f i c a n t l y enhance the height and duration of contraction produced by NE (see Figures 7 and 8). Beta-hydroxy t h u j a p l i c i n (BHT) and EDTA showed similar maximum effe c t s on prolongation of the NE response, however the pro-longation by EDTA increased almost l i n e a r l y with concentration whereas prolongation by BHT reached a maximum and began to decline with higher concentrations of BHT. When graphed as per cent increase i n height against concentration, EDTA po-te n t i a t i o n r i s e s s t e a d i l y whereas BHT potentiation f i r s t decreases then levels o f f . Gamma-thujaplicin (GT) and pyr o g a l l o l are less e f f e c t i v e potentiators of NE as far as prolongation of response i s concerned, than are BHT and EDTA. When expressed as per cent increase i n height over the norepinephrine control, p y r o g a l l o l and GT show very similar responses, p y r o g a l l o l being s l i g h t l y more e f f e c t i v e than GT. The subsequent finding that the th u j a p l i c i n s had an action of t h e i r own on the iso l a t e d aorta, together with the fact that chelating agents, whether COMT i n h i b i t o r s or not, can potentiate the action of NE on iso l a t e d preparations, made i t evident that i t was impossible to relate the poten-t i a t i o n to COMT i n h i b i t i o n . The results show, however, that while potentiation and prolongation of NE effects i s produced by a l l four compounds, there are apparently differences i n the ways i n which they inte r a c t with NE. 43 Table I: Per cent increase i n the duration - 50 of the res-_7 ponse to norepinephrine 0.8 x 10 M i n the pres-ence of a potentiator (mean + standard error)(%) Concentration of potentiator x 10 M Potentiator 0.8 1.6 3.2 4.8 n % increase n % increase n % increase n % increase GT 3 87H5 3 471±116 3 680±139 3 888151 EDTA 4 27614 3 878±196 3 2577±486 3 39001352 BHT 1 2426 2 4428+115 6 40691254 4 3272+412 Pyrogallol 3 166±24 4 10531358 - \ 6 12611188 See Figure 7 T a b l e I I : P e r c e n t i n c r e a s e i n the h e i g h t o f c o n t r a c t i o n o f -7 the n o r e p i n e p h r i n e 0.8 x 10 M response i n t h e pre s e n c e o f a p o t e n t i a t o r (mean ± s t a n d a r d e r r o r ) C o n c e n t r a t i o n o f p o t e n t i a t o r x 10 M P o t e n t i a t o r 0.8 n % i n c r e a s e n % 1.6 i n c r e a s e 3. n % 2 i n c r e a s e 4 n % .8 i n c r e a s e GT 3 97±48 3 112±23* 3 237±77* 3 221±93* EDTA 4 307±86* 3 317±96* 2 470±312 3 710179* BHT . 1 390 4 250129* 6 110+33* 5 137140* P y r o g a l l o l 2 95±23 5 262±13* 7 240169* See F i g u r e *p < 0.01 -8 St u d e n t ' s t -t e s t , p a i r e d d a t a , o n e - t a i l e d , compared t o n o r e p i n e p h r i n e c o n t r o l . 45 5000 -i 0\° O m i C O •rH -P (0 u C -H <D Ui m Q) U O C •H -P C tu u 4000 4 3000 2000 H CM 1000 0.8 1.6 3.2 4.8 Concentration of potentiating, agent (x 10~ 5 M) Figure 7. The per cent increase i n duration-50 of a norepine-phrine 0.8 x 10~7M response i n the presence of a potentiator. 46 0 0.8 : 1.6 3.2 _ 5 4.8 Concentration of potentiating agent (x 10 M) • Figure 8. Potentiation of the height of the norepinephrine response expressed as the per cent increase over the norepinephrine control response. Concentration of norepinephrine 0.8 x 10-7 Molar. 47 Beta-Hydroxy Thujaplicin Beta-hydroxy t h u j a p l i c i n as an agonist In preliminary experiments i t was found that beta-hydroxy t h u j a p l i c i n (BHT) i t s e l f could produce a contraction of the i s o l a t e d rabbit a o r t i c s t r i p . I t was decided to i n -vestigate t h i s c.agonistic e f f e c t . The r e l a t i v e s e n s i t i v i t y of each a o r t i c s t r i p was f i r s t determined by i t s response to norepinephrine (NE) 5 x _7 10 M. After the maximum contraction was recorded, the NE was washed from the bath and the s t r i p was allowed to relax to i t s normal state of tone.. The BHT was then added to the muscle bath and the response was recorded. A new s t r i p had to be used for each BHT response since a second BHT response could not be produced i n any of the s t r i p s tested. The res-ponse to BHT i s a slow gradual contraction (see Figure 9). NE BHT 10 min I 1 5 x 10 7 M 5 x 10" 5 M • •>  Figure 9. Beta-hydroxy t h u j a p l i c i n contraction. 48 _5 The concentration of BHT 1 x 10 M produced no contraction i n the four s t r i p s tested. At the concentra--5 ti o n of BHT 5 x 10 M, six out of eight s t r i p s tested res-ponded with a contraction of an average of 6.1 mm (43.4% of the height of the average NE control contraction)(see Table -4 I I I ) . At the concentration of BHT of 1 x 10 M, the average contraction height was 10.2 mm (70.1% of the NE control res-_3 ponse). Two s t r i p s tested at the concentration, of 1 x 10 M BHT showed an average height of 5.3 mm which i s low i n ab-solute terms but because the NE controls were r e l a t i v e l y low (mean 3.8 mm), showed a high per cent increase of 112.3%. The wide v a r i a b i l i t y i n s e n s i t i v i t y of the s t r i p s plus the lack, of response of some of the s t r i p s to BHT renders these results s t a t i s t i c a l l y i n s i g n i f i c a n t . E f f e c t of EDTA on the beta-hydroxy t h u j a p l i c i n response Further study of the'agonistic e f f e c t s of BHT was undertaken with EDTA 2.7 xllO M present i n the bathing f l u i d . EDTA i s an e f f i c i e n t and p r a c t i c a l drug for prevention of the oxidation of NE. At thi s concentration EDTA prevents any detectable oxidation of NE over a period of 30 minutes and removes only i n s i g n i f i c a n t amounts of calcium and magnesium ions from solution. Also i t does not of i t s e l f d i r e c t l y a l t e r the response of a r t e r i a l s t r i p s to drugs. If the agonistic e f f e c t of BHT i s due to a release of NE from tissue stores, then perhaps EDTA w i l l potentiate the eff e c t s of BHT by allowing the NE to remain i n contact for a longer time 49 Table I I I : Beta-hydroxy t h u j a p l i c i n as an agonist (mean i standard error) n BHT BHT NE control BHT% concentration contraction 5 x 10 M (Molar) (mm± s.e.) 8 5 x l O " 5 6.1 + 2.3 15.8 + 1.7 43.4 + 13.9 4 1 X i o - 4 10.2 + 3.4 16.0 + 1.5 70.1 + 5.3 2 1 X l O " 3 5.3 + 4.3 3.8 + 2.3 112.3 + 45.7 with the tissue receptors. A l t e r n a t i v e l y , i f BHT potentiates endogenous NE by chelating with ions involved with NE o x i -dation a difference i n response should be seen with EDTA pres-ent. With EDTA i n the bath the average height of the BHT -5 5 x 10 M response i s 7.2 mm or an average 50.8% of the NE -5 controls, Without EDTA the BHT response to 5 x 10 M aver-ages 6.1 mm or 43.4% of the NE controls (see Table IV). This small difference i n the height of the BHT contractions i n the presence of EDTA i s not s t a t i s t i c a l l y s i g n i f i c a n t . I t would seem that nonspecific chelation i s not a great source of loss for BHT. 50 Table IV: Beta-hydroxy t h u j a p l i c i n as an agonist i n the _5 presence of EDTA 2.7 x 10 M (mean ± standard error) n BHT BHT NE control BHT% -7 concentration contraction 5 x 10 M (molar) •' (mm±s.e.) 13.. 5 x 10 5 M 7.2 ± 1 . 2 17.2 ± 2.2 50.8 ± 13.6* * -5 Student's t - t e s t compared to BHT 5 x 10 M without EDTA 0.2 > p < 0.1; not s i g n i f i c a n t . The e f f e c t of phenoxybenzamine on the beta-hydroxy t h u j a p l i c i n response It had been found i n previous experiments that phenoxy-benzamine would relax a contraction induced by BHT when the blocker was administered at the height of the contraction. The effects of th i s alpha-adrenergic blocker were then further investigated. A control response to NE was f i r s t recorded. The s t r i p s — 6 were then incubated with phenoxybenzamine 10 gm/ml (2.9 x — 6 10 M) for 30 minutes. Alpha-blockade was then confirmed by -7 the lack of response to another addition of NE 5 x 10 M. BHT was then added to the bath (see Table V). 51 Table V: The BHT response aft e r alpha-adrenergic blockade Control Phenoxy- Test NE BHT 10 M response benzamine after response aft e r S t r i p to NE — fi alpha- alpha-blockade 5 x 10 M . 2.9 x 10 °M blockade 1 6 mm x 30 min 0 mm 0 mm 2 5.5 mm x 30 min 0 mm 0 mm 3 13.5 mm x 30 min 0 mm 0 mm 4 15.5 mm no treatment 14.5 mm Phenoxybenzamine blocked the c o n t r a c t i l e response to BHT i n the s t r i p s tested with the blocker. A normal BHT contraction was produced i n the absence of alpha-blockade i n the control s t r i p . These results suggest that the agonistic e f f e c t of BHT may be mediated by the alpha adrenergic recep-tors however phenoxybenzamine has been known to block other types of receptors as well (34,35) and i s not absolutely s p e c i f i c i n i t s a f f i n i t y for alpha receptors. The e f f e c t of pronethalol on the beta-hydroxy t h u j a p l i c i n  response The beta-adrenergic blocking agent pronethalol was tested for any effects on the response of the i s o l a t e d rabbit a o r t i c s t r i p to BHT. A control response to NE was f i r s t 52 recorded and the s t r i p s were washed and incubated with pro-— 6 — 6 nethalol 10 gm/ml (3.8 x 10 M) for t h i r t y minutes, This concentration of pronethalol has been shown to block the relaxant effects of the beta-adrenergic amine isoproterenol - 4 on the a o r t i c s t r i p . The response to BHT 10 M was then tested (see Table VI). Table VI: The e f f e c t of pronethalol on the beta-hydroxy t h u j a p l i c i n response . Control response Pronethalol Response to %BHT P t o NE 5 x 10-7M • 3.8 x 10"6M BHT 10~ 4 M NE 16 mm x 30 min 8.0 mm 50.0 13.5 mm x 30 min 6.0 mm 44.5 15.5 mm x 30 min 7.0 mm 45.2 12.0 mm no treatment .11.0 mm 91.6 mean: . 15.0 mm mean with mean with s.e.: 0.75 mm beta block: 7.0 mm beta block: 46.7% s.e. 0.6 mm s-.e.: 1.8% The average response to BHT 10 M i s 70.1% of the NE control or 10.2 mm (Table I I I ) . In the presence of the pro-nethalol blockade i n this experiment the s t r i p s contracted to a l e v e l of only 46.7% of the NE control response or an average 5 3 h e i g h t o f 7 . 0 m m . P e r h a p s t h e p r o n e t h a l o l i s s h o w i n g s o m e o f i t s o w n r e l a x a n t p r o p e r t i e s w h i c h w o u l d t e n d t o a n t a g o n -i z e t h e v a s o c o n s t r i c t o r a c t i o n o f B H T . P r o n e t h a l o l h a s b e e n s a i d t o h a v e a d e p r e s s a n t e f f e c t o n ^ a d r e n e r g i c n e r v e t e r m i n -a l s ( 2 5 ) . T h e . e f f e c t o f b e t a - h y d r o x y t h u j a p l i c i n o n t h e t y r a m i n e  r e s p o n s e I f B H T i s p o t e n t i a t i n g t h e e f f e c t s o f n o r e p i n e p h r i n e e i t h e r b y i n h i b i t i n g a n e n z y m e s u c h a s c a t e c h o l - O - m e t h y l -t r a n s f e r a s e , o r , l e s s s p e c i f i c a l l y , b y p r e v e n t i n g t h e o x i d a -t i o n o f n o r e p i n e p h r i n e b y c h e l a t i o n w i t h m e t a l i o n s w h i c h c a t a l y z e t h i s o x i d a t i o n , B H T w o u l d b e e x p e c t e d t o a u g m e n t o r p r o l o n g t h e r e s p o n s e o f t h e a o r t i c s t r i p t o t y r a m i n e . I n t h i s f i r s t s e t o f e x p e r i m e n t s w i t h B H T a n d t y r a -m i n e , a N E c o n t r o l w a s f i r s t r e c o r d e d , t h e n t h e r e s p o n s e t o - 4 t y r a m i n e 5 x 1 0 M a l o n e w a s r e c o r d e d . T h e r e s p o n s e t o t h e - 4 - 5 c o m b i n a t i o n o f t y r a m i n e 5 x 1 0 M p l u s B H T 3 . 2 x 1 0 M t o g e t h e r w a s t h e n r e c o r d e d , a s s h o w n i n F i g u r e 1 0 . N E T y r a m i n e B H T p l u s T y r a m i n e 5 x 1 0 M 5 x 1 0 M 3 . 2 x 1 0 M 5 x 1 0 M F i g u r e 1 0 . B e t a - h y d r o x y t h u j a p l i c i n p l u s t y r a m i n e ; 54 Using t h i s method the response to the tyramine plus BHT combination was almost i d e n t i c a l to the f i r s t control tyramine response. Because tyramine i s said to act i n d i r -e c t l y by releasing norepinephrine from tissue stores, i t s response can become tachyphylactic, each response becoming smaller than the l a s t . This i s a factor which may have hidden or cancelled out any potentiating e f f e c t of BHT on the tyra-mine response. The experiment was adjusted to avoid such an e f f e c t by using paired s t r i p s of aorta from the same animal. -7 In the control s t r i p , the response to NE 5 x 10 M was re--4 corded, then the response to tyramine 5 x 10 M was recorded. -7 In the test s t r i p , the response to NE 5 x 10 M was recorded, -4 -5 and then the response to tyramine 5 x 10 M plus BHT 5 x 10 M combined was recorded (see Tables VII, VIII, and IX). When the responses to tyramine and to tyramine plus BHT are expressed r e l a t i v e to the norepinephrine control ( i . e . norepinephrine control equals unity), the average res-ponse to tyramine alone i s 0.50 and the average response to tyramine plus BHT i s 0.62--a difference of a f r a c t i o n of 0.12 or 12%. These results are not s t a t i s t i c a l l y meaningful. When considering these results we must also take into account that the effects may be additive rather than poten-t i a t i v e . Also, when the results are viewed simply as height of contraction and•ignoring the differences i n responsiveness to the tes t dose of NE, the results are reversed: the 55 tyramine average response i s 8.1 mm while the tyramine plus BHT response averages 7.0 mm. The e f f e c t of BHT on the duration of action of tyramine i s not appreciable. The duration"..-50 for tyramine ( i . e . the time required for the s t r i p to relax to half of the maximum height attained) was 28 minutes, while the duration-50 for tyramine plus BHT was 31.3 minutes. Table VII: The e f f e c t of BHT on the tyramine response -4 . NE control Tyramine control Tyramine 5 x 10 M paired 5 x K T V . 5 x 10~ 4 M _ BHT 5 x 10" 5 M A. 9.0 mm 5.0 mm B. 9.0 mm 6.0 mm A. 8.0 mm 3.0 mm B. 4.5 mm 3.0 mm A. 24.0 mm 19.0 mm B. 15.5 mm 11.0 mm A. B. 16.0 mm 18.5 mm 5.5 mm 8.0 mm mean: 13.1 mm s.e.: 2.3 mm mean: 8.1 mm s.e.: 4.3 mm mean: 7.0 mm s .e.: 1.7 mm Table VIII: The e f f e c t of BHT on the tyramine response (data from table VII expressed r e l a t i v e to NE control: NE control equals unity). 56 NE control 5 x 10~7 M Tyramine, control 5 x 10~4 M Tyramine 5 x 10 M plus BHT 5 x 10-5 M 1 1 1 1 mean: S • 6 • i 0.55 0.38 0.74 0.34 0.50 0.10 mean: 0.67 0.67 0.71 0.43 0.62 0.06 Table IX: Comparative Durati6n-r.50s from paired s t r i p s Tyramine 5 x 10 -4 M Tyramine 5 x 10 -4 M p l U S BHT 5 X 10 M mean: 32 min 26 min 26 min 28 min 2 min mean: 25 min 35 min 34 min 31.3 min 3.2 min 57 The e f f e c t of beta-hydroxy t h u j a p l i c i n oh the tyramine  response afterpretreatment with i p r o n i a z i d With the intention of producing more meaningful results with the tests of the action of BHT on the tyramine response, i p r o n i a z i d was used to i n h i b i t monoamine oxidase. Z e l l e r -5 M and Barsky (53) have shown that pretreatment with 10 ipr o n i a z i d for 30 minutes, completely and i r r e v e r s i b l y i n -h i b i t e d monoamine oxidase. Furchgott (54) found that such pretreatment with i p r o n i a z i d markedly potentiated the response to tyramine, frequently increasing s e n s i t i v i t y to this drug over ten-fold. This indicates that the oxidation of tyramine by monoamine oxidase i s o r d i n a r i l y the main process by which tyramine i s inactivated i n the ao r t i c smooth muscle c e l l s . By p a r a l l e l reasoning, because ip r o n i a z i d pretreatment does not potentiate epinephrine or norepinephrine, t h e i r i n a c t i -vation must be p r i n c i p a l l y due to processes other than o x i -dation by monoamine oxidase. Ao r t i c s t r i p s were incubated with i p r o n i a z i d 3.6 x -4 -4 10 M (10 gm/ml) for 30 minutes and then washed. EDTA -4 -5 3.6 x 10 M (10 gm/ml) was present i n the bathing f l u i d . A response to tyramine alone was recorded, the s t r i p was washed and allowed to relax, then the response to tyramine _5 plus BHT 5 x 10 M together was recorded (see Table X and Figure 11). 5 8 T a b l e X : T h e e f f e c t o f b e t a - h y d r o x y t h u j a p l i c i n o n t h e t y r a m i n e r e s p o n s e a f t e r p r e t r e a t m e n t w i t h i p r o n i a z i d ( m e a n ± s t a n d a r d e r r o r ) T y r a m i n e c o n t r o l T y r a m i n e p l u s P e r c e n t i n c r e a s e C o n c e n t r a t i o n C o n t r a c t i o n B H T 5 x 1 0 5 M o v e r c o n t r o 1 o f t y r a m i n e n (mm ± s . e . ) (mm ± s . e . ) (% ± s . e . ) 1 0 ~ 5 M 3 8 . 3 ± 1 . 2 1 1 . 7 ± 0 . 9 * 4 3 . 3 ± 1 2 . 3 * 5 x 1 0 5 M - 4 1 0 M 1 5 . 8 ± 3 . 4 1 3 . 6 ± 5 . 7 5 x 1 0 ~ 4 M 4 1 9 . 9 1 3 . 1 1 8 . 0 ± 4 . 2 * 1 5 . 9 ± 5 . 8 * 1 8 . 5 ± 3 . 3 1 6 . 5 1 1 2 . 1 * 1 4 . 5 1 5 . 8 * - 4 ( S t r i p s w e r e p r e t r e a t e d w i t h i p r o n i a z i d 3 . 6 x - 1 0 M a n d E D T A - 5 2 . 7 x - 1 0 M w a s p r e s e n t i n t h e b a t h i n g f l u i d ) t - t e s t , p a i r e d , o n e - t a i l e d * p < 0 . 0 1 , s i g n i f i c a n t c o m p a r e d t o t y r a m i n e c o n t r o l . n e q u a l s n u m b e r o f a n i m a l s . O n c o m p a r i s o n w i t h t h e t y r a m i n e r e s p o n s e i n t h e p r e -v i o u s w o r k , t h e t y r a m i n e r e s p o n s e h e r e i s l a r g e r a f t e r t h e m o n o a m i n e o x i d a s e i n h i b i t o r a n d a d e q u a t e r e s p o n s e s c o u l d b e p r o d u c e d b y s m a l l e r d o s e s o f t y r a m i n e . T h e u s e o f i p r o n i a z i d a i L s o p e r m i t t e d t h e u s e o f t y r a m i n e t w i c e i n t h e s a m e s t r i p w i t h o u t a r e d u c t i o n i n t h e s e c o n d r e s p o n s e s o t h a t p a i r i n g o f t h e s t r i p s w a s n o t n e c e s s a r y a n d e a c h s t r i p c o u l d s e r v e a s 59 BHT Tyramine Figure 11. The e f f e c t of beta-hydroxy t h u j a p l i c i n on the tyramine response a f t e r pretreatment with i p r o n i a z i d . as i t s own control. This reduced the v a r i a b i l i t y of the results somewhat. The results are expressed as the per cent increase of the tyramine plus BHT response, over that of the control tyramine response.. At the lowest concentration of _5 tyramine tested, 10 M, the per cent increase over the control was 43.3%. As the concentration of tyramine was increased, the per cent increase over the control was gradu--4 a l l y decreased to zero at tyramine 5 x 10 M. Perhaps the contractions here were near maximal, or that portion of the contraction o r i g i n a l l y potentiated by, or caused by the BHT had been surpassed by the tyramine alone. The responses to the combination of tyramine plus BHT for the three lowest -5 -5 -4 concentrations of tyramine, 10 , 5 x 10 , and 10 M were a l l s t a t i s t i c a l l y shown to be s i g n i f i c a n t l y larger than the control tyramine responses at these concentrations. From these results i t can be stated that BHT can augment the tyra-60 mine response of the rabbit a o r t i c muscle up to a maximum concentration of tyramine whereupon BHT can no longer increase the tyramine response. This type of pattern does not follow that expected by a simple potentiation due to metal chelation or enzyme i n h i b i t i o n unless at the l e v e l l i n g off point the muscle i s contracting to i t s maximum c a p a b i l i t y . Other evidence against the augmentation of the tyramine response being due to i n h i b i t i o n of the oxidation of nore-pinephrine can be found i n the work of Furchgott (50) . EDTA 2.7 x 10 ^ M was present i n the bathing f l u i d and at the -5 concentration of 10 M has been shown to prevent any detec-table oxidation of norepinephrine over a period of 30 minutes. A method for determining whether an observed potentiation of the norepinephrine response by a drug i s due to some mode of action other than i n h i b i t i o n of oxidation i n solution i s to use s u f f i c i e n t EDTA i n the solution to i n h i b i t the oxidation e s s e n t i a l l y completely and then test the e f f e c t of the drug on the norepinephrine response. Therefore, from the results obtained i n the presence of EDTA i t can be stated that poten-t i a t i o n of tyramine by BHT must be due to a mechanism other than protection of norepinephrine from oxidation by metal chelation--for instance, by catechol-O-methyltransferase i n -h i b i t i o n or simply the addition of the two agonistic e f f e c t s . 61 The e f f e c t . o f i p r o n i a z i d on the beta-hydroxy t h u j a p l i c i n  response. Tests were carried out which p a r a l l e l e d the f i r s t studies of the BHT response with the v a r i a t i o n that these a o r t i c s t r i p s were f i r s t incubated with ip r o n i a z i d 10 4 gm/ml -4 (3.6 x 10 M) for 30 minutes. A problem arose here i n some s t r i p s when the muscle tone gradually increased during incu-bation with the i p r o n i a z i d and could not be reversed with repeated washings. The tone was presumably due to a b u i l d up of endogenous NE r e s u l t i n g i n leakage of NE from the nerve endings a f t e r i n h i b i t i o n of monoamine oxidase. The presence of greater than normal tone at the s t a r t of an experiment makes further contractions appear smaller i n height compared to contractions i n s t r i p s i n which low i n i t i a l tone exists and can be a source of v a r i a t i o n i n results obtained. After -7 the i p r o n i a z i d incubation, a control response to NE 5 x 10 M was recorded. The response to the concentration of BHT to be tested was then recorded (see Table XI). When compared to the r e sults for the BHT responses without i p r o n i a z i d , no s i g -n i f i c a n t difference i s found. The e f f e c t of cocaine.on the beta-hydroxy t h u j a p l i c i n response The agonistic effects of BHT on i s o l a t e d rabbit a o r t i c s t r i p may be a d i r e c t action or an i n d i r e c t action. Tyramine i s believed to act i n d i r e c t l y by release of NE from nerve ending storage s i t e s . Cocaine has been shown to relax a 62 Table XI: The e f f e c t of ipr o n i a z i d on the beta-hydroxy t h u j a p l i c i n response BHT BHT NE control % BHT -7 n concentration contraction 5 x 10 M (mm .+. s.e.) .-. (mm ± • s. e.) . NE 4 5 x 10~ 5 M 4.6+1.1 13.8 ± 1 . 5 34.2 ± 4.3 8 1 x 10" 4 M 8.4 ± 1.7 11.9 ± 1.1 71.9 ± 15.7 -4 Strips were incubated with i p r o n i a z i d 3.6 x 10 M for 30 minutes p r i o r to te s t s . tyramine-induced contraction i n rabbit a o r t i c s t r i p s (55). The e f f e c t of cocaine was tested on BHT-induced contractions i n an attempt to investigate the mode of action of BHT. -7 A control response to norepinephrine 5 x 10 M was recorded and the s t r i p was washed. When the s t r i p had relaxed to normal tone, the test concentration of BHT was added to the bath. When the response to the "BHT was maximal, cocaine 1.75 x 10 ^ M (5 x 10 ^ gm/ml) was added. In four s t r i p s tested with BHT, three s t r i p s showed a further contraction of 0.5 mm upon addition of cocaine (see Figure 12 and Table XII). In no instance was the r e s u l t that of rapid relaxation as seen when cocaine i s added during a tyramine-induced contrac-63 Figure 12. The e f f e c t s of cocaine on the responses to tyramine and beta-hydroxy t h u j a p l i c i n t i o n . The presence of the s l i g h t l y increased contraction pro-duced by cocaine suggests the involvement of norepinephrine release by BHT since cocaine alone has no stimulant e f f e c t on the a o r t i c s t r i p but i t can potentiate the effects of any norepinephrine present. The mechanism of action of BHT must d i f f e r from that of tyramine but the mechanism of tyramine has not been c l e a r l y described. I t i s s t i l l uncertain whether the postulated release of norepinephrine by tyramine depends primarily on some action associated with the axon membrane, which might become more permeable to the trans-mitter, or some other process such as i n h i b i t i o n of nore-pinephrine uptake (56). 64 Table XII: The e f f e c t of cocaine on the beta-hydroxy t h u j a p l i c i n response (mean ± standard e r r o r ) . NE control 5 x 10~ 7 M response ; (mm+s.e.) BHT response -4 10 M (mm+s.e.) Cocaine 1. plus BHT 10 _ 4M 75 x 10 _ 6M (mm±s.e.) 11.4 ± 1 .0 8.8 ± 3.0 9.1 ± 2 .9 n = 4 Gamma-Thuj a p l i c i n Gamma-thujaplicin as an agonist Gamma-thujaplicin was tested for i t s agonistic effects on the is o l a t e d thoracic aorta of the rabbit using the s p i r -a l l y cut s t r i p s previously described. The response of each s t r i p to a test dose of norepine-phrine was f i r s t recorded. . The NE was then washed from the bath and the s t r i p was permitted to relax to i t s o r i g i n a l length. Gamma-thujaplicin (GT) was then added and the res-ponse was recorded. The results are shown here both as the mean height of contraction and also as a mean per cent of the control NE response (see Table XIII and Figure 13). 65 T a b l e X I I I : G a m m a - t h u j a p l i c i n as an a g o n i s t (mean I s t a n d a r d e r r o r ) . . GT GT NE c o n t r o l c o n c e n t r a t i o n c o n t r a c t i o n 5 x 10 M (mm± s-.e.) . . . . (mm±s.e.) NE-4 5 X l O " 5 M 1.8. ± 0.3 6.8 ± 1.4 28 .2 ± 5. 8 6 5 X l O " 4 M 3.4 ± 0.9 17,3 ± 1.2 19 .4 ± 4. 5 3 1 X l O " 3 M 2.8 ± 1.0 20.2 ± 7.9 16 .7 ± 7. 7 5 X l O " 3 M p r e c i p i t a t i o n from s o l u t i o n When 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 c o n t r o l NE r e s -ponse the l a r g e s t response i s seen a t the l o w e s t e f f e c t i v e -5 c o n c e n t r a t i o n o f GT 5 x 1.0 M. Wi t h h i g h e r c o n c e n t r a t i o n s of GT the a g o n i s t i c e f f e c t s d e c r e a s e . When e x p r e s s e d s i m p l y as t h e h e i g h t o f c o n t r a c t i o n , -4 the most e f f e c t i v e c o n c e n t r a t i o n o f GT i s 5 x 10 M. However, t h i s d a t a does n ot acco u n t f o r the d i f f e r e n c e i n s e n s i t i v i t y o f t h e i n d i v i d u a l s t r i p s . _3 A t t h e c o n c e n t r a t i o n 5 x 10 M, t h e GT p r e c i p i t a t e d from s o l u t i o n as a y e l l o w s o l i d . The c o n t r a c t i o n r e c o r d e d a t t h i s c o n c e n t r a t i o n o f GT was much more r a p i d than t h o s e a t t h e o t h e r c o n c e n t r a t i o n s and i t does n ot f o l l o w the same time c o u r s e so i s p r o b a b l y due t o some o t h e r f a c t o r s such as h y p e r t o n i c i t y o r pH. 66 NE Control GT Figure 13. Gamma-thujaplicin as an agonist. The e f f e c t of cocaine on the ;gamma-thu japTicin response Experiments on the e f f e c t of cocaine on the gamma-thujaplicin-induced contractions of rabbit a o r t i c s t r i p s p a r a l l e l to those previously described with BHT and cocaine were performed. In eight s t r i p s tested with GT, two s t r i p s showed a further contraction of 0.5 mm,upon addition of cocaine. There was no relaxation of the s t r i p by cocaine as seen with a tyramine-induced contraction (see Table XIV). The beta-adrenergic effects of gamma-thujapTicin Other studies i n thi s laboratory have shown that GT has a stimulant action on is o l a t e d a t r i a which i s at least p a r t i a l l y due to beta-adrenergic receptor stimulation. I t was therefore of in t e r e s t to tes t GT for beta-adrenergic effects i n the rabbit a o r t i c s t r i p preparation. 67 Table XIV: The e f f e c t of cocaine on the gamma-thujaplicin response (mean + standard e r r o r ) . n NE control response GT response Cocaine _fi _ 7 -4 1 - 7 5 x 1 0 M 5 x 10 - M (mm+s.e.) 1 x 10 M. (mm±s.e.) plus GT 1 x 10"4M (mm±s.e.) 16.2 ± 2.2 5.6 ± 1 . 4 5.8 .± 1.4 Beta-adrenergic stimulation produces relaxation i n aor t i c s t r i p s and so i n order to test for this a c t i v i t y the s t r i p must f i r s t be brought into a state of tone. Histamine — 6 — 6 in a concentration of 5.5 x 10 M(10 gm/ml) was found to give a reproducible tone which was maintained for about an hour. In each test a histamine response was f i r s t produced, then the s t r i p was washed and was permitted to relax to i t s normal length. This step acted as a control for the h i s t a -mine response. A second histamine contraction was then pro-duced and when the maximum height of contraction was reached, -5 GT.5 x 10 M was added. The degree of relaxation produced i n 25 minutes by GT was tabulated and expressed as a per cent of the o r i g i n a l contraction. In preparations from 10 animals tested, GT produced relaxation i n 8 and no change i n 2. There was always a s l i g h t i n i t i a l contraction before the relaxation i n the 8 animals where relaxation occurred (see Figure 14 and Table XV). 68 Histamine 5.5 x 10 10 min. I I Figure 14. Relaxant e f f e c t of gamma-thujaplicin on the histamine contraction. Table XV: The relaxant e f f e c t of gamma-thujaplicin on the histamine contraction (mean i standard e r r o r ) . Tone produced by Histamine Tone after 25 min Per cent relax-5 5 x 10 -^ M "*"n P r e s e n c e °f ^ T ation produced n (mm+s.e.) 5 x 10 _ 5M(mm±s .e.) ^ Y GTn-5*,i ^ x r 5 x 1 0 M (mm+s.e.) 12 29.5 ± 3.5 23.4 ± 3.5 27.7 ± 9.1 Gamma-thujaplicin was added at the maximum contraction height produced by histamine. The relaxation produced by the gamma-t h u j a p l i c i n i s shown. During these experiments with GT and histamine, i t was noted that GT was exhibiting a blocking action against the effects of histamine on the ao r t i c smooth muscle. Any h i s t a -mine responses recorded after using GT i n the bath for i t s relaxant e f f e c t s , were lower than the histamine controls. 69 I t was also noted that i f the GT was washed from the bath before the relaxant phase of i t s action had begun, a further histamine contraction was not blocked. Perhaps there i s a time-dependent binding mechanism involved. The blocking e f f e c t seems to be a non-specific depression of the tissue as a blocking e f f e c t had previously been observed against nore-pinephrine as well. The e f f e c t of propranolol on the gamma-thujaplicin response The beta-adrenergic blocking agent, propranolol, was used for the purpose of detecting any beta receptor stimula-t i o n by GT. In thi s set of tes t s , paired s t r i p s from the same animal were used. In the control s t r i p , histamine 5.5 — 6 x 10 M was used to produce tone, and at the maximum height of contraction GT 5 x 10 ^ M was added. In the test s t r i p , propranolol 10 6 gm/ml (3.4 x 10 6 M) was present for 30 minutes i n the bathing f l u i d before the histamine and GT were added i n the same manner as i n the control s t r i p . In prelim-— 8 inary testing, a concentration as low as 10 gm/ml (3.4 x — 8 10 M) was found to completely block the effects of the beta-adrenergic amine isoproterenol. In the 4 animals tested, propranolol did not block _5 the relaxation produced by GT 5 x 10 M. This i s evidence that the relaxation produced by GT does not involve beta receptors i n the rabbit a o r t i c s t r i p . In fa c t , rather than block the relaxation produced by GT, propranolol increased 70 this relaxation. Those s t r i p s incubated with propranolol showed an 18.4 per cent greater relaxation than those s t r i p s which did not receive the beta-adrenergic blocker (see Table XVI) . Table XVI: The e f f e c t of propranolol on the gamma-thujaplicin-induced relaxation of t h e o histamine contraction (mean ± standard error) • 1  i Histamine Tone afte r GT Tone afte r GT,% relax- % relax-5.5 x 10 - 6M 5 x 10 - 5M and propranolyby GT after GT tone for 25 min 61 for 25 min^alone and (mm+s.e,.)- (mm+s.e.) • (mm+s.e.) * propranolol 8 19.1 ± 1 . 5 15.8 ± 3.6 10.7 + 2.4 23.7 ±10.1 42.1±7.9 The e f f e c t of rese r p i n i z a t i o n on the gamma-thujapljcin response Four rabbits were reserpinized by a subcutaneous i n j e c -t i o n of reserpine 2.5 mg/Kg body weight 18 hours p r i o r to the experiments. Two other animals received two doses of the reserpine, 2.5 mg/Kg per day for 2 days, the l a s t dose given 18 hours p r i o r to the experiment. The results from these two animals receiving the two doses did not d i f f e r from those i n the animals receiving only one i n j e c t i o n of reserpine. 71 Supersensitivity to norepinephrine was seen, however the tyramine response was not abolished as had been expected by the reserpinization. However, according to Furchgott the -4 concentration of tyramine used, 5 x 10 M, i s capable of producing a d i r e c t stimulant action and i s not dependent on endogenous norepinephrine (50) . -4 The response to GT 10 M on i s o l a t e d a o r t i c s t r i p s from 5 reserpinized animals was a relaxation below normal basal tone i n 4 animals and no e f f e c t i n 1 animal (see Table XVII and Figure 15). This relaxation i s an opposite e f f e c t from the contraction produced by GT i n the non-reserpinized a o r t i c s t r i p s . Table XVII: Relaxation produced by gamma-thujaplicin i n reserpinized rabbit a o r t i c s t r i p s Preparation Relaxation below basal tone number produced by GT 10""4 M 1. 5.0 mm 2 2.0 mm 3 5.0 mm 4 3.0 mm mean 3.8 mm o 72 normal basal tone GT -4 10 M Figure 15. Relaxation produced by gamma-thujaplicin i n reserpinized a o r t i c s t r i p s . The e f f e c t of GT on the tyramine response was tested i n a o r t i c s t r i p preparations from 3 reserpinized animals. In a l l 3 preparations GT produced a relaxation of the tyra-mine contraction (see Figure 16). In non-reserpinized animals GT augments the tyramine contraction s l i g h t l y . Figure 16. Relaxation by GT of tyramine response i n reserpinized s t r i p s . 73 Another property shown was a complete block of the tyramine response by GT i f GT i s used p r i o r to tyramine i n the reserpinized s t r i p s . I t appears from these results that reserpinization prevents the c o n t r a c t i l e effects of GT on rabbit a o r t i c s t r i p s but does not a f f e c t the relaxant effects of GT. This would suggest that endogenous norepinephrine i s involved i n the contraction seen with GT i n non-reserpinized s t r i p s . . 74 DISCUSSION Many tropolone derivatives have been shown to be capable of i n h i b i t i n g the enzyme COMT. The effects of gamma-thujaplicin and beta-hydroxy t h u j a p l i c i n on the response of the is o l a t e d aorta to norepinephrine were studied i n an attempt to show whether or not COMT i n h i b i t i o n was produced i n t h i s preparation. P a r a l l e l tests were carried out using a chelating agent, EDTA, and a COMT i n h i b i t o r , p y r o g a l l o l . A l l of these agents potentiated the NE response by increasing the height of contraction and by prolonging the duration of action. The potentiation of the NE response by EDTA showed an almost l i n e a r r elationship with increasing concentration for both per cent increase i n height and i n per cent increase i n duration. This potentiation i s due sol e l y to protection of NE from metal ion oxidation reactions by the chelating properties of EDTA. EDTA has no d i r e c t stimulatory e f f e c t on ao r t i c t issue. Pyrogallol and GT had similar effects on both the per cent increase i n duration and per cent increase i n height of contraction over NE controls although pyrogallol i s s l i g h t l y more e f f e c t i v e than GT.• These e f f e c t s may be complex i n v o l -ving additive effects due to chelation of metal ions and COMT i n h i b i t i o n . P yrogallol has no d i r e c t stimulatory 75 e f f e c t s on a o r t i c tissue but higher concentrations of GT were found to contract a o r t i c s t r i p s . BHT was found to be a strong potentiator of NE but with higher concentrations of BHT the potentiation began to decline. A possible explanation for this e f f e c t could be a non-specific relaxant e f f e c t by BHT which became evident i n la t e r experiments. A l t e r n a t i v e l y the concept of cross-receptors could also be used i n an attempt to explain this e f f e c t . Norepinephrine receptors may become occupied by BHT to a greater extent as the BHT concentration increases, so that what i s seen i s less of the e f f e c t of NE and more the ef f e c t of BHT alone. Such a p o s s i b i l i t y i s supported by the fact that the alpha-adrenergic blocker, phenoxybenzamine blocks the BHT-induced contraction of ao r t i c s t r i p s . The po-ten t i a t i o n of NE by BHT may be complex, involving protection for NE from metal ion oxidation reactions, COMT i n h i b i t i o n and additive e f f e c t s . Since both BHT and GT were found to have action of th e i r own on the aorta, combined with the fact that chelating agents whether COMT i n h i b i t o r s or not have a potentiating e f f e c t on the NE response of a o r t i c s t r i p s i t i s obvious that these results cannot be used as conclusive evidence for COMT i n h i b i t i o n by BHT and GT. The nature of the stimulatory action of BHT and GT on the i s o l a t e d aorta was investigated using several agents as pharmacological to o l s . This stimulatory e f f e c t on the 76 aorta could be a d i r e c t e f f e c t produced by receptor e x c i t a -t i o n of a s p e c i f i c nature or a non-specific smooth muscle stimulation. There i s also the p o s s i b i l i t y that BHT and GT act i n d i r e c t l y by releasing endogenous NE from adrenergic nerve endings. The BHT-induced contraction was found to be blocked by the alpha-adrenergic blocking drug phenoxybenzamine. This would suggest that BHT acts on the alpha-adrenergic receptors i n a d i r e c t manner but i t must be remembered that phenoxy-benzamine i s not absolutely s p e c i f i c as an alpha-adrenergic blocker and may also block at histamine, acetylcholine and 5-hydroxy-tryptamine receptor s i t e s . Likewise, phenoxybenza-mine could also block the effects of any endogenous NE i f any were released by BHT. Cocaine does not relax the BHT contraction as i s seen with cocaine and the tyramine contraction. This, however, i s not conclusive evidence for a d i r e c t action by BHT since BHT could release NE from a d i f f e r e n t pool or by a d i f f e r e n t mechanism than does tyramine. The s i t e s of action of BHT and tyramine may also be d i f f e r e n t . The fact that there i s a very s l i g h t potentiation of the BHT contraction by cocaine could suggest that endogenous NE has been released by BHT and i t s effects potentiated by cocaine. Cocaine alone has no stimulatory effects on the i s o l a t e d aorta. BHT and tyramine together produce a potentiative or an additive e f f e c t on the a o r t i c s t r i p . This e f f e c t has 77 also been seen on the guinea-pig a t r i a l preparation. Poten-t i a t i o n might r e s u l t from COMT i n h i b i t i o n by BHT allowing more NE to accumulate at receptor s i t e s . A l t e r n a t i v e l y BHT may release NE from d i f f e r e n t stores or i t s effects may just be additive due to a possible d i r e c t stimulatory e f f e c t . EDTA i s present i n these experiments so that a simple non-specific chelating e f f e c t by BHT cannot be suggested. This fact fur-ther indicates d i f f e r e n t modes of action for BHT and EDTA. The effects of GT on the a o r t i c s t r i p p a r a l l e l those of BHT. GT produced a contraction which was blocked by phenoxybenzamine. Cocaine does not relax the GT contraction as i s seen with cocaine on the tyramine response. There i s a very s l i g h t potentiation of the GT response by cocaine which may indicate endogenous NE release by GT. Cocaine has no stimulatory e f f e c t on ao r t i c tissue by i t s e l f . Since GT showed a beta-adrenergic stimulation e f f e c t on guinea-pig a t r i a , the possible beta effects on the rabbit aorta were investigated. GT was found to cause par-t i a l relaxation of the histamine contraction. However, this relaxant e f f e c t was not blocked by the beta-adrenergic block-ing agent propranolol and so i s not l i k e l y of a beta-adrenergic character. The relaxant e f f e c t i s actually i n -creased by propranolol and i s l i k e l y due to the addition of non-specific depressant effects of both these agents. Non-s p e c i f i c blocking effects to a l l stimulatory drugs used 78 throughout the experiments were seen with both BHT and GT. After r e s e r p i n i z a t i o n , GT produces a relaxation of a o r t i c smooth muscle rather than a contraction. This would seem to indicate that the presence of endogenous NE i s neces-sary for the stimulatory e f f e c t s of GT. The relaxant proper-t i e s of GT are uncovered when NE stores of the tissue have been depleted. The involvement of NE i n the GT contraction may also be indicated by the s l i g h t potentiation of thi s con-t r a c t i o n by cocaine. In the non-reserpinized a o r t i c s t r i p preparation, per-haps the COMT i n h i b i t o r y e f f e c t of GT i s allowing a slow release or leak of NE from the tissue stores permitting the NE concentration at receptor s i t e s to increase gradually. The slow time course of the BHT and GT contractions would support t h i s theory. I f BHT and GT could enter the tissue c e l l s , they might be involved i n displacing NE from storage s i t e s within the c e l l . Since the effects of BHT and GT seem to p a r a l l e l each other i n a l l tests c a r r i e d out on a o r t i c s t r i p s , i t might be assumed that t h e i r mode of action i s the same. The reversaltdf"'the'ieffect of GT by reserpinization shows that a relaxant e f f e c t i s uncovered when tissue NE i s depleted. Perhaps the nature of the e f f e c t depends on the state of the muscle when the GT or BHT i s added. For example, GT relaxes a muscle contracted by histamine but potentiates 79 a NE contraction. The r e s u l t of any test i s probably the sum of a i l i n h i b i t o r y and stimulatory factors and can vary with drugs used and with the concentrations of each. From a consideration of 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 -ships any adrenergic a c t i v i t y shown by the t h u j a p l i c i n s would be expected to involve the beta-adrenergic receptors. This would be expected due to the importance of the catechol hydroxyl groups for beta-adrenergic a c t i v i t y . For alpha-adrenergic a c t i v i t y the ca t i o n i c head of the ethylamino side chain i s important and no such group i s found i n the thuja-p l i c i n s . From these studies; the i n h i b i t o r y e f fects of GT and BHT seem to be of a non-specific depressant type rather than a beta-adrenergic e f f e c t . S p e c i f i c alpha-adrenergic a c t i v i t y i s also unli k e l y although alpha-adrenergic receptor stimu-l a t i o n could r e s u l t i n d i r e c t l y through possible release of endogenous NE by BHT and GT. Further studies should be carried out on denervated tissue to determine the r o l e , i f any, of NE i n the stimulatory effects of BHT and GT. , The main d i f f i c u l t y i n trying to understand the mechanisms of actions of drugs involving adrenergic effects i s the very incomplete knowledge concerning the function of the sympa-th e t i c transmitter apparatus. Also, great d i f f i c u l t i e s are encountered i n the interpretation of effects caused by drugs, since only a few of these appear to have a single or s p e c i f i c action. Considering the complexity of the synthesizing, 80 releasing and uptake systems i n the granules, and the tv/o-d i r e c t i o n a l transport system i n the axon membrane, i n addition to the actions on the effector c e l l s , i t i s not surprising that the opinions regarding the interpretations of drug ac-tions vary p a r t i c u l a r l y when drugs are combined. 81 SUMMARY AND CONCLUSIONS The actions of beta-hydroxy t h u j a p l i c i n (BHT) and gamma-thujaplicin (GT) on the iso l a t e d s p i r a l l y - c u t rabbit thoracic aorta have been studied. 1. The effects of a chelating agent, EDTA, a COMT i n h i b i t o r , p y r o g a l l o l , as well as BHT and GT on the response of the iso l a t e d rabbit aorta to norepinephrine were investigated. I t was found that a l l of these agents potentiated the nore-pinephrine response. I t was therefore evident that the po-ten t i a t i o n of the NE response by BHT and GT was not necessarily attributable to COMT i n h i b i t i o n . 2. Both BHT and GT were found to have an agonistic e f f e c t of the i r own on rabbit a o r t i c muscle. A slow, gradual contraction i s produced by these agents. BHT was found to be more potent an agonist than GT i n thi s preparation. The presence of EDTA i n the bath does not s i g n i f i c a n t l y a f f e c t t h i s contraction so i t i s u n l i k e l y that chelation i s an important factor. The presence of the alpha-adrenergic blocking agent phenoxybenzamine i n the bath blocks the BHT and GT contrac-tions. This alone i s not conclusive evidence that these agents act d i r e c t l y on alpha-adrenergic receptors since the alpha-adrenergic blocking agents are not absolutely s p e c i f i c for alpha receptors and also could block effects of any endogenous NE released. 82 3. Cocaine, when added to the bath at the maximum height of contraction of BHT or GT, did not cause relaxation of the contraction as i s seen with a tyramine contraction. This would indicate that the s i t e of action and perhaps also the mode of action of BHT and GT i s d i f f e r e n t from that of tyra-mine. However, since there i s a s l i g h t potentiation of the BHT and GT responses by cocaine, the involvement of endogen-ous NE i s possible. 4. BHT and tyramine together produce a potentiative or addi-t i v e e f f e c t on the a o r t i c s t r i p . Potentiation could r e s u l t from COMT i n h i b i t i o n by BHT or an increased NE release. A l t e r n a t i v e l y , the effects may be additive or a combination of these various e f f e c t s . 5. GT produces a relaxation of the histamine contraction. This relaxant e f f e c t i s not blocked by beta-adrenergic blockade and i s l i k e l y due to a non-specific depressant e f f e c t by GT. Non-specific blocking effects by both BHT and GT have been noted throughout the experiments. 6. After reserpinization of the s t r i p s GT no longer produces a contraction but relaxes them instead. I t appears that the presence of endogenous NE i s required for the c o n t r a c t i l e effects of GT. GT also produces relaxation of the tyramine contraction i n reserpinized s t r i p s . From these observations i t appears that endogenous NE plays a part i n the c o n t r a c t i l e effects of BHT and GT although the exact mechanism i s uncertain. COMT i n h i b i t i o n by BHT 83 and GT may play some role i n th i s c o n t r a c t i l e process by allowing the accumulation of NE i n the e x t r a c e l l u l a r phase re s u l t i n g or aiding i n a gradual contraction of the tis s u e . The e f f e c t of BHT and GT seems to depend on the state of contraction of the muscle and i s a sum of i n h i b i t o r y and stimulatory e f f e c t s . The depressant e f f e c t s of BHT and GT appear to be of a non-specific papaverine-like nature. BHT and GT appear to act by more than one mode of action. Coupled with the complexity of the adrenergic neurons the action of these agents i s d i f f i c u l t to inte r p r e t . B I B L I O G R A P H Y 85 1. Pawson, P.L.: Tropones and t r o p o l o n e s . Chem. Rev., 55, 9 (1955). 2. Lee, S.: P h a r m a c o l o g i c a l s t u d i e s o f h i n o k i t i o l . N i i g a t a Med. J . , 65, 566 (1951). 3. A o k i , M., Yamamoto, H., Yamaki, A., S a t o o , H.: A c t i o n o f t r o p o n o i d s on i s o l a t e d u t e r i o f some a n i m a l s . J a p . J . Pha r m a c o l . , 7, 38 (1957). 4. H a l l i d a y , J.E.: A p h a r m a c o l o g i c a l s t u d y o f gamma-t h u j a p l i c i n . J . Am. Pharm. A s s o c . , 48_, 722 (1959) . 5. K a t s u r a , S.: Ph a r m a k o l o g i s c h e untersuchungen uber h i n o k i t i o l ( m - i s o p r o p y l t r o p o l o n e ) . 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Musacchio, J.M., Goldstein, M.: Biosynthesis of nore-pinephrine and norsynephrine i n the perfused rabbit heart. Biochem. Pharmacol., 12, 1061 (1963). 22. Muscholl, E.: E f f e c t of cocaine and related drugs on the uptake of noradrenaline by heart and spleen. B r i t . J . Pharmacol., 16, 352 (1961). 23. Moran, N.C.: Development of beta adrenergic blocking drugs. Ann. N.Y. Acad. S c i . , 139, 649 (1967). 24. Black, J.W., Stephenson, J.S.: Pharmacology of a new adrenergic beta-receptor blocking compound (nethalide). Lancet, 2_, 311 (1962) . 25. Standaert, F,G., Roberts, J . : A neural action of pronethalol. Ann. N.Y. Acad. S c i . , 139, 815 (1967). 26. Alleyne, G.A.O. et a l . : E f f e c t of pronethalol i n angina p e c t o r i s . B r i t . Med. J . , 2, 1226 (1963). 87 27. Paget, G.E.: Carcinogenic action of pronethalol. B r i t . Med. J . , 2, 1266 (1963). 28. Black, J.W., Duncan, W.A.M., Shanks, R.G.: Comparison of some properties of pronethalol and propranolol. B r i t . J . Pharmacol./ 25, 577 (1965). 29. Blinks, J.R.: Evaluation of the cardiac effects of several beta-adrenergic blocking agents. Ann. N.Y. Acad. S c i . , 139., 673 (1967). 30. Epstein, S.E., Braunwald, E.: C l i n i c a l and hemodynamic appraisal of beta-adrenergic blocking drugs. Ann. N.Y.. Acad. S c i . , 139, 952 (1967). 31. T r i g g l e , D.J.: . 2-Halogenoethylamines and receptor analysis. Advances i n Drug Research, 2_, 173 (1965). 32. Graham, J.D.P.: 2-Halogenoalkylamines. Prog. Med. Chem., 2, 132 (1962) . 33. Nickerson, M.: Mechanism of the prolonged adrenergic blockade produced by haloalkylamines. Arch. Intern. Pharmacodyn., 140, 237 (1962). 34. Boyd, H. et a l . : Cholinergic-blocking action of adrenergic blocking agents i n the pharmacological analysis of autonomic innervation. B r i t . J. Pharmacol., 20^ , 418 (1963) . 35. Graham, J.D.P., Lewis, G.P.: The antihistamine and antiadrenaline properties of a series of N-Naphthyl-methyl-2-haloethylamine derivatives. B r i t . J . Pharmacol., 8_, 54 (1953). 36. Graham, J.D.P.: 5-Hydroxytryptamine, ed. by G.P. Lewis, Pergamon Press, London, 202 (1957). 37. Axelrod, J . , Tomchick, R.: Activation and i n h i b i t i o n of adrenaline metabolism. Nature (Lond.) , 184, 2027 (1959). 38. Graham, J.D.P.: The e f f e c t of reserpine on the pressor responses to i n j e c t i o n of 2-halogenoethylamines. B r i t . J . Pharmac. Chemother., 16, 77 (1961). 39. G i l l e s p i e , J.S., Kirpekar, S.M.: Removal of noradrenaline from the blood flowing through the spleen. J . Physiol. (Lond.) , 169, 100P (1963) . 88 40. Axelrod, J . : O-methylation of epinephrine and other catechols i n v i t r o and i n vivo. Science, 126, 400 (1957) . ~~ 41. Axelrod, J.,.Senoh, S., Witkop, B.: O-methylation of catecholamines i n vivo. J . B i o l . Chem., 233, 697 (1958) . 42. Axelrod, J . , Tomchick, R.: Enzymatic O-methylation of epinephrine and other catechols. J . B i o l . Chem., 233, 702 (1958). 43. Senoh, S., Daly, J . , Axelrod, J.,.Witkop, B.: Enzymatic p-0-methylation by COMT. J . Amer. Chem. S o c , 81, 6240 (1959). 44. Masri, M.S., Robbins, D.J., Emerson, O.H., De Eds, F.: Selective para or meta methylation with COMT from r a t . Nature, 202, 878 (1964). 45. Musacchio, J . , Goldstein, M.: The a l t e r a t i o n of catecholamine metabolism by tropolone i n vivo. Fed. P r o c , 21, 334 (1962). 46. Mavrides, C , Missala, K., D'lorio, A.: The e f f e c t of 4-methyltropolone on the metabolism of adrenaline. Can. J. Biochem. Physiol., 41, 1581 (1963). 47. Belleau, B., Burba, J . : Occupancy of adrenergic receptors and i n h i b i t i o n of catechol-O-methyltransferase by tropolones. J . Med. Chem., 6_, 755 (1963). 48. Murnaghan, M.F., Mazurkiewicz, I.M.: Some pharmaco-l o g i c a l properties of 4-methyltropolone. Rev. Canad. B i o l . , 22, 99 (1963) . 49. Murnaghan, M.F.: Non-specific e f f e c t s of the tropolones. Int. J . Neuropharmacol. , 3_' 8 9 (1964). 50. Furchgott, R.F.: Pharmacology of vascular smooth muscle. Pharm. Rev., 1_, 183 (1955). 51. Furchgott, R.F.: Methods i n Medical Research, ed. by H.D. Bruner, Year Book Publishers, Inc., Chicago, 8, 178 (1960). 52. Furchgott, R.F.: Dibenamine blockade i n s t r i p s of rabbit aorta and i t s use i n d i f f e r e n t i a t i n g receptors. J . Pharmacol. Exp. Therap., 111, 265 (1954). 89 Z e l l e r , E.A., B a r s k y , J . : I n v i v o i n h i b i t i o n o f l i v e r and b r a i n monoamine o x i d a s e by l - i s o n i c o t i n y l - 2 -i s o p r o p y l h y d r a z i n e . P r o c . Soc. Exper. B i o l . Med., 81, 459 (1952). F u r c h g o t t , R.F., W e i n s t e i n , P., H u e b l , H., B o z o r g m e h r i , P., Mensendiek, R.: E f f e c t o f i n h i b i t i o n o f monoamine o x i d a s e on response o f r a b b i t a o r t i c s t r i p s . Fed. P r o c . , 14, 341 (1955) . M a x w e l l , R.A., D a n i e l , A . I . , Sheppard, H., Zimmerman, J.H.: Some i n t e r a c t i o n s o f g u a n e t h i d i n e , c o c a i n e , m e t h y l p h e n i d a t e and p h e n y l a l k y l a m i n e s i n r a b b i t a o r t i c s t r i p s . J . Pharmacol. Exp. Therap., 137, No. 1, 31 (1962). von E u l e r , U.S., L i s h a j k o , F.: Mechanism o f d r u g -i n d u c e d c a t e c h o l a m i n e r e l e a s e . f r o m a d r e n e r g i c nerve g r a n u l e s . C i r c . Res., 21, S u p p l . 3, 63 (1967). A P P E N D I X 91 Table A: Perocent increase i n the duration - 50 of the _7 response to norepinephrine 0.8 x 10 M m the presence of a potentiator. -5 Concentration of potentiator x 10 M _ . ... 0.8 1.6 3.2 4.8 Potentiator . % increase % increase % increase % increase GT 67 322 675 915 78 391 924 960 118 700 441 789 EDTA 287 726 2300 3233 273 1225 3527 4433 278 683 1903 4034 268 BHT 2426 4790 3014 4300 4067 4411 2308 4664 3042 4334 3439 3623 4371 Pyroga l l o l 127 560 1033 162 850 965 209 681 1208 2122 736 1721 1903 92 Table B: Per cent increase i n the height of contraction of -7 the norepinephrine 0.8 x 10 M response i n the presence of a potentiator. Concentration of potentiator x 10 M Potentiator 0.8 1.6 3.2 4.8 % increase % increase % increase % increase GT 25 67 117 175 166 140 371 400 100 129 225 87 EDTA 200 133 160 800 400 357 780 550 500 460 780 128 BHT 390 208 110 156 225 53 250 238 56 138 330 74 350 93 154 274 Pyrogallol 71 140 154 119 110 412 173 550 110 141 105 58 95 262 93 Table C: Actual height of contraction produced by the combin-ation of norepinephrine plus potentiator. NE -_7 0.8 x 10 M (NE) - height of NE controls i n brackets mm. Concentration of potentiator ( x 10 -5 M) Potentiator 0.8 1.6 3.2 4.8 mm- .n., mm. n mm n mm n GT 5(4) 3 15(9) 3 25(11.5) 3 22(8) 3 8(3) 12(5) 17 (3.5) 20 ( 4 ) 3(1.5) 14(6) 13 ( 4 ) 22(11.5) mean 5 mm mean 13*mm mean 18*mm mean 21*mm s.e. 1. 3 mm s.e. 0.58mm s.e. 1.61mm s.e. 1.56mm EDTA 15(5) 4 21(9) 3 22(8.5) 2 20(3) 3 15(3) 16 (3.5) 22 (2.5) 21(2.5) 15(2.5) 14(2.5) 27 (3) 16(7) mean 15mm* mean 18 mm* mean 22 mm mean 22 mm* s.e. 0 . 83mm s . e . 0 . 2 9mm s.e.3.04mm s.e.2.13mm BHT 25 (5) 1 19(6) 4 24(5) 10 20(8) 13(4) 29(5) 22(6) 23 (7) 16(2.5) 30(14) 24(5.5) 22 (2) 25(10.5) 21 (10) 27(6) 29(19) 28(11) 25(13) 30 (8) 13(8) 29(17) mean 20 mm* mean 24 mm* mean 25 mm* s.e.1.82mm s.e.1.9 2mm s.e.1.21mm Pyrogal l o l 21(12) 2 36(15) 5 14(5.5) 23(10.5) 21(10) 31(6) 30(11) 13(2) 21 (10) 26(11) 21(10) 20 (13) 18(9) 38(10.5) mean 22 mm mean 23 mm* mean 25 mm* s.e.1.75mm . s...e . 2 . 2.2mm : s.e.3.12mm n = number of animals Student's t - t e s t : *p < 0.01, compared to NE control response paired data, one-tailed. 94 Table D: Beta-hydroxy t h u j a p l i c i n as an agonist Norepinephrine B H T B H T B H T B H T B H T i control response response response response ' -7 -5 -5 -4 -3 N E 5 x 10 M 10 M 5 x 10 M 10 M 10 M (mm) (mm) (mm) (mm) (mm) 8.0 4.0 8.0 15.0 0 0 0 0 0 0 0 0 15.0 4.0 26.6 19.0 0.0 0.0 14.0 0.0 0.0 20 .0 4.0 20.0 20.0 17.0 85.0 11.0 11.0 100.0 7.0 5.5 78.5 20.0 7.5 37.5 15.0 18.0 26.0 4.0 5.0 17.0 15.0 4.0 33.3 89.5 57.7 100.0 6.0 1.5 9.5 158.0 1.0 66.6 95 Table E: Beta-hydroxy t h u j a p l i c i n as an agonist i n the presence of EDTA 2.7 x 10~ 5 M (10~ 5 gm/ml) Norepinephine control 5 x 10~ 7 M 11.0 mm 18.0 mm 163.5 10.5 mm 9.5 mm 90.5 19.0 mm 9.0 mm 47.4 9.0 mm 0.0 mm 0 36.0 mm 10.0 mm 27.8 20.0 mm 3.0 mm 15.0 14.0 mm 0 mm 0 16 .0 mm 0 mm 0 20.5 mm 6.5 mm 31.7 27.5 mm 9.0 mm 32.8 9.0 mm 3.0 mm 33.3 14.0 mm 12.0 mm 85.7 16.5 mm 13.5 mm 81.7 mean : 17.2 mm mean : 7.2 mm mean: s.e. : 2.2 s.e. : 1.2 s.e.: BHT %BHT c ,«-5 NE 5 x 10 M Students t - t e s t compared to BHT 5 x 10 M without EDTA: 0.2 > p < O.i; not s i g n i f i c a n t . Table F: The e f f e c t of beta-hydroxy t h u j a p l i c i n on the tyramine response aft e r pretreatment with i p r o n i a z i d Tyramine Concentration control Contraction mm Tyramine plus BHT 5 x 1 0 - 5 M - mm Per cent increase over control % 1 0 - 5 M 9.5 12.0 26.3 6.0 10.0 66.6 9.5 13.0 36.9 5 x 1 0 - 5 M 23.0 24.0 4.4 18.0 22.0 22.2 4.5 5.5 22.2 17.5 20.5 17.3 -4 10 M 17.5 21.5 12.5 6.5 7.3 12.6 28.0 32.0 12.9 2.5 3.0 20.0 5 x 1 0 - 4 M 23.0 23.0 0 19.0 19.0 0 11.5 9.0 -26.0 23.0 — (Strips were pretreated with i p r o n i a z i d 3.6 x 10 M and EDTA _5 2.7 x 10 was present i n the bathing f l u i d . ) 97 Table G: The e f f e c t of i p r o n i a z i d on the beta-hydroxy t h u j a p l i c i n response. NE control BHT 5 x 10 M BHT 1 x 10 M . BHT % 5 x 10 M response response N E (mm) : (mm) (mm) 11.0 2.0 27.3 18.0 8.0 44.5 13.0 3.5 26.9 13.0 4.0 38.0 12.0 9.0 75.0 17.5 12.0 68.5 6.0 2.5 41.6 11.0 3.0 27.3 15.5 14.5 93.5 14.0 4.0 28.6 12.0 3.0 25.0 10.0 14.0 140.0 9.5 14.0 147.5 98 T a b l e H: The e f f e c t o f c o c a i n e on t h e b e t a - h y d r o x y t h u j a p l i c i n r e s p o n s e . — 6 NE c o n t r o l response BHT response C o c a i n e 1.75 x 10 M 5 x 1 0 - 7 M 10~ 4 M p l u s BHT 1 0 _ 4 M (mm) (mm) (mm) 14.0 12.0 10.0 9.5 4.0 3.0 14.0 14.0 4.5 3.5 14.5 14.0 9 9 T a b l e I : G a m m a - t h u j a p l i c i n a s a n a g o n i s t B H T B H T N E c o n t r o l . B H T % n c o n c e n t r a t i o n c o n t r a c t i o n 5 x 1 0 7 M N E (mm) - (mm) 4 5 x 1 0 " 5 M 2 . 0 5 . 5 3 6 . 4 2 . 0 5 . 0 4 0 . 0 1 . 0 5 . 5 1 8 . 2 2 . 0 1 1 . 0 1 8 . 2 6 5 x 1 0 ~ 4 M 5 . 0 . 1 6 . 0 3 1 . 2 5 . 0 2 1 . 0 2 3 . 8 6 . 0 1 9 . 0 3 1 . 6 2 . 0 1 3 . 0 1 5 . 4 1 . 5 1 9 . 0 7 . 9 1 . 0 1 5 . 5 6 . 5 3 1 x 1 0 ~ 3 M 1 . 0 1 0 . 5 9 . 5 4 . 5 1 4 . 0 3 2 . 2 3 . 0 3 6 . 0 8 . 3 100 Table J : The e f f e c t of cocaine on the gamma-thujaplicin response. — 6 NE control response GT response Cocaine 1.75 x 10 M 5 x 1 0 - 7 M 1 x 1 0 - 4 M plus GT 1 x 10~ 4 M (mm) (mm) (mm) 8.0 1.0 1.0 14.0 11.0 11.0 13.5 3.0 3.0 12.0 3.5 3.5 14.0 7.0 7.5 18.0 12.0 12.0 28.0 5.0 5.5 22.0 3.0 3.0 101 Table K: The relaxant effects of GT on the histamine contraction. Tone produced by Tone a f t e r 25 min Per cent relaxation histamine i n presence of GT produced by GT 5.5 x 10~6 M 5 x 10~5M 5 x 10~5 M (mm) (mm) (%.) 27.0 7.0 74.0 9.0 0 100.0 38.0 36.0 5.3 32.0 28.0 12.5 48.0 37.0 22.9 43.0 33.0 23.2 37.0 37.0 0 40.0 40.0 0 22.0 19.0 13.6 21.0 12.0 42.8 24.5 24.5 0 13.0 8.0 38.5 102 T a b l e L: The e f f e c t o f p r o p r a n o l o l on t h e g a m m a - t h u j a p l i c i n -i n d u c e d r e l a x a t i o n o f t h e h i s t a m i n e c o n t r a c t i o n P a i r e d s t r i p s : A & B H i s t a m i n e Tone a f t e r GT Tone a f t e r GT 5 . 5 x l O _ 6 M 5 x 1 0 ~ 5 M % r e l a x - % r e l a x -tone f o r 25 min (mm) (mm) , -t i a t i o n by a t i o n p l u s p r o p r a n o l o l G T * a f t e r Q T f o r 25 min (mm) a l o n e p l u s > p r o p r a n o l o l A. 22.0 B. 22.0 19.0 18.0 13.6 18.2 A. B. 21.0 20.0 12.0 10.0 42.8 50.0 A. 24.5 B. 16.0 24.5 8.0 50.0 A. 13.0 B. 14.0 8.0 7.0 38.5 50.0 

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