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The effects of certain drugs on the uptake and release of ³H-noradrenaline in rat whole brain homogenates Pylatuk, Karen Lee 1974

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T H E E F F E C T S O F C E R T A I N D R U G S O N T H E U P T A K E A N D R E L E A S E O F 3 H - N O R A D R E N A L I N E I N R A T W H O L E B R A I N H O M O G E N A T E S b y K A R E N L E E P Y L A T U K B . S c . ( P h a r m . ) , 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 7 1 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 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 a n d T o x i 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 4 [ 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 r e q u i r e m e n t s f o r 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 f o r r e f e r e n c e and s tudy . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g 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 g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d that c o p y i n g 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 g a i n s h a l l not be a l l o w e d w i thou t my w r i t t e n p e r m i s s i o n . an advanced degree at the U n i v e r s i t y o f B r i t i s h Co lumb ia , I a g ree that Department The U n i v e r s i t y o f B r i t i s h Co lumbia Vancouver 8, Canada i i ABSTRACT E m p l o y i n g an in. v i t r o method adap t e d from Snyder and C o y l e (1969) and u s i n g r a t whole b r a i n homogenate, t w e l v e d r u g s ( c o -c a i n e , t y r a m i n e , f o u r t r i c y c l i c a n t i d e p r e s s a n t s , and s i x a n t i -h i s t a m i n e s ) were s t u d i e d w i t h r e s p e c t t o t h e i r e f f e c t s on i n -h i b i t i o n o f n e u r o n a l uptake o f H - n o r a d r e n a l i n e (NA) and on r e -l e a s e o f the amine from p r e s y n a p t i c n e r v e t e r m i n a l s . To d i s t i n g u i s h between t h e s e p a r a t e a c t i o n s on c a t e c h o l -amine r e l e a s e and i n h i b i t i o n o f t h e u p t a k e p r o c e s s , two b a s i c p r o c e d u r e s were used. I n t h e f i r s t , homogenate was p r e i n c u -3 b a t e d w i t h H-NA p r i o r t o a d d i t i o n o f t h e d r u g i n o r d e r t o l o a d t h e n e r v e e n d i n g s w i t h NA so t h a t t h e e f f e c t s o f d r u g s on r e -l e a s e c o u l d be measured. The second p r o c e d u r e i n v o l v e d p r e -i n c u b a t i n g homogenate w i t h t h e v a r i o u s d r u g s f o l l o w e d by a d d i -3 t i o n o f t h e H-NA and f u r t h e r i n c u b a t i o n i n o r d e r t o a s s e s s t h e i n h i b i t o r y e f f e c t s o f t h e drugs on NA u p t a k e . From t h e f o r m e r e x p e r i m e n t s , a l l d r u g s t e s t e d were found t o produce some r e l e a s e o f NA a l t h o u g h t y r a m i n e was by f a r t h e most p o t e n t d r u g i n t h i s r e s p e c t . T r i p e l e n n a m i n e and c o c a i n e were o b s e r v e d t o produce t h e l e a s t r e l e a s e o f t h e t w e l v e d r u g s . Of t h e r e m a i n i n g compounds, w h i c h were s i g n i f i c a n t l y l e s s po-t e n t t h a n t y r a m i n e , t h e t r i c y c l i c a n t i d e p r e s s a n t s were g e n e r -a l l y more e f f e c t i v e i n p r o d u c i n g r e l e a s e t h a n t h e a n t i h i s t a m i n -i c s . When t h e p o t e n c i e s o f t h e s e compounds were c o r r e l a t e d w i t h t h e i r r e s p e c t i v e l i p i d s o l u b i l i t i e s , o n l y t y r a m i n e dev-i a t e d g r e a t l y from t h e e s t a b l i s h e d l i n e a r r e l a t i o n s h i p . T h i s indicated that, unlike the other drugs which appeared to be causing NA release through a nonspecific mechanism r e l a t e d to l i p o p h i l i c i t y , tyramine i s acting by a s p e c i f i c mechanism, probably i n v o l v i n g accumulation of t h i s amine by the NA uptake mechanism followed by displacement and subsequent release of bound i n t r a c e l l u l a r NA. The studies of i n h i b i t i o n of NA uptake again demonstrated tyramine to be the most potent of the twelve drugs although i n t h i s case i t d i d not d i f f e r s i g n i f i c a n t l y from cocaine and tripelennamine. The remaining compounds also showed a de-3 creased accumulation of H-NA but were less potent than t y r -amine (although a l l drugs produced i n h i b i t i o n of uptake of NA' at a lower dose than that required for release of the amine). Tyramine again deviated from the l i n e a r r e l a t i o n s h i p between i n h i b i t o r y potency and p a r t i t i o n c o e f f i c i e n t , but so d i d co-caine and tripelennamine. Chlorpheniramine and diphenhydra-mine also d i d not seem to f i t the c o r r e l a t i o n although the d i s -crepancy was less pronounced than for the other three compounds. I t thus appears that drugs such as tyramine, cocaine and t r i -pelennamine are i n h i b i t i n g accumulation of NA by a s p e c i f i c i n t e r a c t i o n with the neuronal uptake process, whereas the other compounds studied may be acting i n a noncompetitive, nonspec-i f i c manner or with mixed e f f e c t s . Only tyramine, besides blocking the uptake mechanism competitively, also appears to act as a substrate for the transport system and therefore can enter the nerve terminal to bring about d i r e c t release. Signatures of Examiners V TABLE OF CONTENTS PAGE ABSTRACT i i LIST OF TABLES v i i LIST OF FIGURES , i x LIST OF ABBREVIATIONS x i i INTRODUCTION 1 The Transport and Storage of Noradrenaline . . 1 Neuronal Uptake of Noradrenaline 3 Other Types of Uptake Processes 5 Properties and C h a r a c t e r i s t i c s of Uptake-^ . . . 6 I n h i b i t i o n of Uptake^ 12 The Release of Noradrenaline from Nerve End- 1 ings 18 Drug E f f e c t s on Noradrenaline Release 22 Nonspecific Membrane E f f e c t s : L i p i d S o l u b i l i t y and i t s Relationship to Potency 28 Background and Objectives of the Present Study. 29 MATERIALS AND METHODS 3 2 Anima4si>?al.s. 3 2 Chemicals and Drugs 32 Tissue Preparation 33 Incubation Procedure 34 Determination of R a d i o a c t i v i t y 37 Calculations 39 v i PAGE RESULTS . 41 Uptake i n the Absence of Test Drugs 41 E f f e c t s of Drugs on the E f f l u x of Noradrenaline: Time-Effect Studies . 46 Ef f e c t s of Drugs on the E f f l u x of Noradrenaline: Concentration-Effect Studies 52 E f f e c t s of Drugs on the I n h i b i t i o n of Norad-renaline Uptake: Time-Effect Studies . . 62 E f f e c t s of Drugs on the I n h i b i t i o n of Norad-renaline Uptake: Concentration-Effect Studies 71 A Co r r e l a t i o n of Drug E f f e c t s with L i p i d S o l u b i l -i t i e s of the Compounds 81 DISCUSSION 87 SUMMARY AND CONCLUSIONS 109 BIBLIOGRAPHY 113 v i i LIST OF TABLES TABLE PAGE I. K i n e t i c Constants for Noradrenaline Uptake i n Rat Heart 7 I I . I n h i b i t i o n of Noradrenaline Uptake (Uptake^) by Sympathomimetic Amines i n the Rat I s o l -ated Heart 13 I I I . Modified Krebs-Henseleit Buffer 36 IV. Bray's S c i n t i l l a t i o n Solvent 38 V. Volumes of NA Solutions Added to the Incu-bation Mixture 41 VI. Accumulation of Noradrenaline i n the Absence of Drug Treatment 42 VII. The % Release of Noradrenaline by 10~ 4 M Drugs at Various Incubation Times 47 VIII. The % Release of Noradrenaline a f t e r Twenty Minutes Incubation with Varying Concentra-tions of the Test Drugs 54 IX. The Relative Potencies, i n Decreasing Order, for Drugs Producing E f f l u x of T r i t i a t e d Noradrenaline From Rat Brain Homogenate^ *v— A f t e r Incubation for Twenty Minutes ... ~.. ... ,.. 61 X. The % I n h i b i t i o n of T r i t i a t e d Noradrenaline Uptake by 10 M Drugs at Various Incuba-t i o n Times 64 XI. The % I n h i b i t i o n of T r i t i a t e d Noradrenaline Accumulation aft e r Forty Minutes Incubation with Varying Doses of the Test Drugs . . . . 72 XII. Relative Potencies, i n Decreasing Order, for Drugs Producing I n h i b i t i o n of Uptake of T r i t -i a ted Noradrenaline aft e r Forty Minutes In-cubation 79 XIII. A Comparison of Drug Potencies i n Decreasing Order for E f f e c t s on Both E f f l u x of Norad-renaline and I n h i b i t i o n of Uptake of the Catecholamine 80 v i i i TABLE XIV. XV. XVI. X V I I . X V I I I . PAGE L o g a r i t h m s o f t h e O c t a n o l / W a t e r P a r t i t i o n C o e f f i c i e n t s f o r t h e Twelve Drugs . . . . . . 82 I n h i b i t i o n o f C a t e c h o l a m i n e Uptake by t h e T e s t Drugs i n V a r i o u s T i s s u e s and S p e c i e s . 99 I n h i b i t i o n o f N o r a d r e n a l i n e Uptake i n t o Synaptosomes P r e p a r e d from S e v e r a l B r a i n R e g i o n s 104 S p e c i e s D i f f e r e n c e s i n C a t e c h o l a m i n e Up-t a k e i n the P e r f u s e d H e a r t s o f V a r i o u s V e r -t e b r a t e s 105 A f f i n i t y C o n s t a n t s f o r (+)-NA Uptake by C e r -e b r a l C o r t e x o f V a r i o u s S p e c i e s 106 i x L IST OF FIGURES FIGURE PAGE 1. The b a s i c e v e n t s o c c u r r i n g i n s y n a p t i c t r a n s m i s s i o n 2. A w o r k i n g h y p o t h e s i s f o r t h e e f f e c t o f i n -o r g a n i c i o n s on u p t a k e and s t o r a g e o f NA by p e r i p h e r a l a d r e n e r g i c n e r v e e n d i n g s . . . . 9 3 3. A c c u m u l a t i o n o f H - n o r a d r e n a l i n e i n the ab-sence o f d r u g t r e a t m e n t , e m p l o y i n g f o u r c o n -c e n t r a t i o n s o f c a t e c h o l a m i n e : 0.05 p.M, 0.27 pM, 0.70 uM, and 2.0 pM 43 4. Time f o r peak a c c u m u l a t i o n o f 0.05 jaM n o r a d -r e n a l i n e by r a t b r a i n homogenate 44 5a. The ti m e c o u r s e o f e f f l u x o f n o r a d r e n a l i n e from r a t b r a i n homogenate f o l l o w i n g i n c u b a -t i o n w i t h 10 M a m i t r i p t y l i n e , i m i p r a m i n e , and t r i p e l e n n a m i n e 48 5b. The t i m e c o u r s e o f e f f l u x o f n o r a d r e n a l i n e from r a t b r a i n homogenate f o l l o w i n g i n c u b a -t i o n w i t h 10~ M p r o m e t h a z i n e , c h l o r p h e n i r -amine, and c o c a i n e 49 5c. The ti m e c o u r s e o f e f f l u x o f n o r a d r e n a l i n e from r a t b r a i n homogenate f o l l o w i n g i n c u b a -t i o n w i t h 10 M t y r a m i n e , phenindamine, and t r i p r o l i d i n e 50 5d. The ti m e c o u r s e o f e f f l u x o f n o r a d r e n a l i n e from r a t b r a i i j homogenate f o l l o w i n g i n c u b a -t i o n w i t h 10" M n o r t r i p t y l i n e , d e s i p r a m i n e , and d i p h e n h y d r a m i n e 51] 6. R e l a t i v e e f f i c a c y , i n d e c r e a s i n g o r d e r , f o r r e l e a s e o f n o r a d r e n a l i n e from r a t b r a i n hom-ogenate a f t e r i n c u b a t i o n f o r 20 m i n u t e s w i t h e q u i m o l a r c o n c e n t r a t i o n s o f t h e t w e l v e d r u g s 53 7a. The e f f e c t o f v a r y i n g c o n c e n t r a t i o n s o f ami-t r i p t y l i n e , i m i p r a m i n e , and t r i p e l e n n a m i n e on e f f l u x o f H - n o r a d r e n a l i n e from r a t b r a i n homogenate a f t e r i n c u b a t i o n f o r 20 mi n u t e s . 57 X FIGURE PAGE 7b. The e f f e c t of v a r y i n g c o n c e n t r a t i o n s of p r o - 58 methaz i n e , ^ c h l o r p h e n i r a m i n e , and c o c a i n e on e f f l u x of H - n o r a d r e n a l i n e from r a t b r a i n homogenate a f t e r i n c u b a t i o n f o r 20 minutes . 7c. The e f f e c t of v a r y i n g c o n c e n t r a t i o n s of tyramine, phenindamine, and t r i p r o l i d i n e on e f f l u x of H - n b r a d r e n a l i n e from r a t b r a i n homogenate a f t e r i n c u b a t i o n f o r 20 minutes 59 7d. The e f f e c t of v a r y i n g c o n c e n t r a t i o n s of n o r t r i p t y l i n e , d esipramine, and diphenhy-dramine on e f f l u x o f H - n o r a d r e n a l i n e from r a t b r a i n homogenate a f t e r i n c u b a t i o n f o r 20 minutes 60 3 8a. The time course of i n h i b i t i o n of H-norad-r e n a l i g e uptake i n t o r a t b r a i n homogenate by 10" M t r i p e l e n n a m i n e , a m i t r i p t y l i n e , and imipramine 66 3 8b. The time course of i n h i b i t i o n of H-norad-r e n a l i g e uptake i n t o r a t b r a i n homogenate by 10" M c o c a i n e , c h l o r p h e n i r a m i n e , and promethazine 67 3 8c. The time course of i n h i b i t i o n o f H-norad-r e n a l i g e uptake i n t o r a t b r a i n homogenate by 10" M tyramine, phenindamine, and t r i -p r o l i d i n e 68 3 8d. The time course of i n h i b i t i o n of H-norad-r e n a l i g e uptake i n t o r a t b r a i n homogenate by 10" M n o r t r i p t y l i n e , desipramine, and diphenhydramine 69 9. R e l a t i v e e f f e c t i v e n e s s , i n d e c r e a s i n g o r d e r , of equimolar c o n c e n t r a t i o n s of the t e s t com-pounds f o r i n h i b i t i o n of uptake of t r i t i a -ted n o r a d r e n a l i n e a f t e r f o r t y minutes i n c u -b a t i o n 70 10a. The e f f e c t of v a r y i n g c o n c e n t r a t i o n s of t r i -pelennamine, a m i t r i p t y l i n e , _ a n d imipramine on i n h i b i t i o n of uptake of H- n o r a d r e n a l i n e i n t o r a t b r a i n homogenate a f t e r 40 minutes i n c u b a t i o n 74 x i FIGURE 10b. 10c. lOd. 11. PAGE The e f f e c t of varying concentrations of co-caine, chlorpheniramine, and promethazine on i n h i b i t i o n of uptake of H-noradrenaline into r a t brain homogenate afte r 40 minutes incubation 75 The e f f e c t of varying concentrations of tyramine, phenindamine, a n d ^ t r i p r o l i d i n e on i n h i b i t i o n of uptake of H-noradrenaline into r a t brain homogenate afte r 40 minutes incubation 76 The e f f e c t of varying concentrations of d i -phenhydramine, n o r t r i p t y l i n e , and desipra-mine on i n h i b i t i o n of uptake of H-norad-renaline into r a t brain homogenate af t e r 40 minutes incubation 77 The r e l a t i o n s h i p between drug potency with respect to e f f e c t s on e f f l u x of H-norad-renaline (EC50) and the l i p i d s o l u b i l i t y of the compound (calculated as the logarithm of the octanol/water p a r t i t i o n c o e f f i c i e n t . . 84 The r e l a t i o n s h i p between drug potency with respect to i n h i b i t o r y e f f e c t s on H-norad-renaline uptake (IC50) and the l i p i d solu-b i l i t y of the compound (calculated as the logarithm of the octanol/water p a r t i t i o n co-e f f i c i e n t 85 x i i L IST OF ABBREVIATIONS ATPase — a d e n o s i n e t r i p h o s p h a t a s e cAMP — c y c l i c a d e n o s i n e 3 1,5 1-monophosphate COMT — 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 CPM — c o u n t s p e r minute DA -- dopamine ( 3 , 4 - d i h y d r o x y p h e n y l e t h y l a m i n e ) DBH — d o p a m i n e - ^ - h y d r o x y l a s e DPM — d i s i n t e g r a t i o n s p e r minute EC50 — c o n c e n t r a t i o n r e q u i r e d f o r h a l f - m a x i m a l (50%) e f -f e c t on r e l e a s e 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 a t e g — average g r a v i t y ID50 or IC50 — dose (or c o n c e n t r a t i o n ) r e q u i r e d f o r h a l f -maximal (50%) i n h i b i t i o n o f u p t a k e Km — M i c h a e l i s c o n s t a n t Log P — l o g a r i t h m o f t h e o c t a n o l / w a t e r p a r t i t i o n c o e f f i c i e n t Tf — l o g a r i t h m o f t h e p a r t i t i o n c o e f f i c i e n t o f a s u b s t i t u e n t group MAO -- monoamine o x i d a s e NA — n o r a d r e n a l i n e P — p e l l e t P/M r a t i o — p a r t i c l e ( o r p a r t i c u l a t e ) / m e d i u m r a t i o POBZ — phenoxybenzamine POPOP — p - b i s - ^ 2 - ( 5 - p h e n y l o x a z o l y l ) ] -benzene PPO — 2 , 5 - d i p h e n y l o x a z o l e r — c o r r e l a t i o n c o e f f i c i e n t ( c o n t i n u e d ) x i i i rpm — revolutions per minute S — supernatant SAR — s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p S.E.M. — standard error of the mean Vmax — maximal rate of an enzyme reaction A C K N O W L E D G M E N T S T h e a u t h o r g r a t e f u l l y a c k n o w l e d g e s t h e a s s i s t a n c e o f D r . J . H . M c N e i l l w h o s e e n c o u r a g e m e n t , a d v i c e , a n d h e l p f u l c r i t i c i s m h a v e p r o v e d i n v a l u a b l e t h r o u g h o u t t h e c o m p l e t i o n o f t h e e x p e r i m e n t a l s t u d i e s a n d p r e p a r a t i o n o f t h i s m a n u s c r i p t . 1 INTRODUCTION The T r a n s p o r t and S t o r a g e o f N o r a d r e n a l i n e I t has become e v i d e n t i n r e c e n t y e a r s t h a t some form o f m e d i a t e d t r a n s p o r t system p r o b a b l y e x i s t s f o r e v e r y p r o p o s e d n e u r o t r a n s m i t t e r s u b s t a n c e and t h a t f o r each neuron t y p e , t h e system appears t o be a s s o c i a t e d w i t h t h e t r a n s m i t t e r w h i c h i s m a n u f a c t u r e d and r e l e a s e d t h e r e . I n t h i s r e v i e w , however, em-p h a s i s w i l l be p l a c e d on t h e t r a n s p o r t o f o n l y n o r a d r e n a l i n e ('NA). I t i s t h i s p r o c e s s w h i c h has r e c e i v e d t h e most a t t e n t i o n and which has been t h e most t h o r o u g h l y c h a r a c t e r i z e d because o f t h e e v i d e n c e t h a t u p t a k e and b i n d i n g o f t h i s c a t e c h o l a m i n e w i t h o u t any e n z y m a t i c d e g r a d a t i o n r e p r e s e n t t h e p r i m a r y mech-anism f o r t e r m i n a t i o n o f t h e a c t i o n o f NA a f t e r i t s r e l e a s e from t h e p r e s y n a p t i c n e r v e t e r m i n a l s . I n a d d i t i o n , much o f t h e i n -f o r m a t i o n about t h e t r a n s p o r t systems i s a r e s u l t o f p h a r m a c o l -o g i c a l r e s e a r c h on d r u g s w h i c h were found t o i n t e r a c t w i t h t h e p r o c e s s e s . F i g u r e 1 d e p i c t s s c h e m a t i c a l l y t h e b a s i c e v e n t s w h i c h a r e pr o p o s e d t o o c c u r i n n o r a d r e n e r g i c s y n a p t i c t r a n s m i s s i o n . The t r a n s m i t t e r s u b s t a n c e i s b e l i e v e d t o be s t o r e d w i t h i n s y n a p t i c v e s i c l e s o r g r a n u l e s (a) i n t h e n e r v e t e r m i n a l . These a r e h i g h -l y s p e c i a l i z e d s u b c e l l u l a r p a r t i c l e s i n which t h e NA appears t o be c o n c e n t r a t e d i n a 4:1 s t o i c h i o m e t r i c r a t i o w i t h a d e n o s i n e t r i p h o s p h a t e and may be bound t h e r e i n t o a s o l u b l e p r o t e i n 2 F i g u r e 1. The b a s i c events o c c u r r i n g i n s y n a p t i c t r a n s m i s s i o n . The v a r i o u s p r o c e s s e s which may be i n v o l v e d are d e s i g n a t e d as f o l l o w s : (a) s t o r a g e w i t h i n s y n a p t i c v e s i c l e s , (b) r e -l e a s e i n t o the s y n a p t i c c l e f t , (c) c o m b i n a t i o n w i t h a r e -c e p t o r , (d) d i f f u s i o n i n t o the b l o o d stream, (e) metabol-ism e x t r a c e l l u l a r l y by COMT, ( f ) p o s t s y n a p t i c or e x t r a -neuronal uptake f o l l o w e d by i n t r a c e l l u l a r metabolism, and (g) uptake i n t o the p r e s y n a p t i c nerve t e r m i n a l , (adapted from I v e r s e n , 1967) (Shore, 1972). Depolarization i n the nerve ending brings about release of these v e s i c u l a r contents into the synaptic c l e f t (b). Proposed mechanisms for t h i s release w i l l be discussed i n a l a t e r section. In the c l e f t , the NA can combine with a receptor (c) to exert i t s p h y s i o l o g i c a l or pharmacological ac-t i o n . Once a transmitter i s released from the presynaptic t e r -minals, an e f f i c i e n t process for i t s removal from the region of the receptor must be a v a i l a b l e . Various mechanisms may be i n -volved i n terminating the transmitter's pharmacological action and any interference with the processes would be expected to prolong and potentiate the e f f e c t s of nerve stimulation. A small amount may d i f f u s e into the blood stream (d); another f r a c t i o n may be metabolized by catechol O-methyltransferase (COMT) (e), but the greatest f r a c t i o n appears to be removed from the synaptic c l e f t by means of a membrane transport system. This occurs most commonly into the presynaptic nerve terminal (g) although there i s evidence that uptake into postsynaptic or other c e l l s occurs, followed by i n t r a c e l l u l a r metabolism (f) (Iversen, 1965 c,). Neuronal Uptake of Noradrenaline Before proceeding, i t should be noted that the term "up-take" i s commonly used i n several d i f f e r e n t contexts but s t r i c t -l y speaking, only r e f e r s to the mechanism whereby e x t r a c e l l u l a r amine i s transferred into the i n t r a c e l l u l a r space of a t i s s u e . In contrast, "storage" or "accumulation" may also involve trans-port into and retention i n the storage granules as well as be-ing affected by metabolism or release from the nerve terminal. However, since catecholamine uptake i s commonly studied by meas-uring the accumulation of exogenous amine, the term "uptake" may also be used to r e f e r to the more complex phenomenon, the meaning depending on the context i n which the term i s used. The f i r s t suggestion that catecholamines might be taken up and stored i n tissue binding s i t e s was made by Burn i n 1932. Since then, a great deal of research has been done i n the area to elucidate the properties as well as the p h y s i o l o g i c a l and pharmacological s i g n i f i c a n c e of uptake processes. Although several groups of investigators had shown that exogenous catecholamines, i n large doses, could be accumulated i n various peripheral t i s s u e s , i t was not u n t i l l a b e l l e d amines of high s p e c i f i c a c t i v i t y became available that doses comparable to normal p h y s i o l o g i c a l concentrations could be used. Employing 3 3 r e l a t i v e l y small doses of H-adrenaline and H-noradrenalme, Axelrod e_t _al. (1959) and Whitby and coworkers (1961) were the f i r s t to demonstrate that the uptake and binding of these sub-stances p e r i p h e r a l l y represented an important mechanism for th e i r i n a c t i v a t i o n . This uptake was shown to occur to the greatest extent i n tissues with a r i c h sympathetic innervation. S i m i l a r l y , many other researchers have provided a d d i t i o n a l e v i -dence that uptake and retention of NA occurs p r i m a r i l y i n ad-renergic neurons of both the peripheral and c e n t r a l nervous sys-tems by using histochemical and autoradiographic methods. A 5 more d e t a i l e d account of the evidence for neuronal uptake of NA may be found i n several review papers by Iversen (1965a; 1967; 1971b). Although early studies of uptake i n brain presented prob-lems because of the presence of a "blood-brain b a r r i e r " to cat-echolamines, more recently the preparation and i s o l a t i o n of synaptosomes have provided researchers with a new to o l for studying the transport processes which occur across a neuronal membrane (De Belleroche and Bradford, 1973). Both Gray and Whittaker (1962) and DeRobertis and coworkers (1962), separate-l y i s o l a t e d brain nerve endings from the mitochondrial f r a c t i o n of r a t brain homogenates employing a c e l l f r a c t i o n a t i o n tech-nique. These endings or "synaptosomes" may be described as the pinched-off nerve terminals from brain. During tissu e rupture, the presynaptic nerve terminals are torn from the post-synaptic membrane together with the postsynaptic thickening and at the same time e i t h e r bud o f f from the axon or break away from i t . In the l a t t e r case, the damaged membrane r a p i d l y reseals to form a continuous surface. Synaptosomes contain synaptic v e s i c l e s , one or more mitochondria, and usually carry a post-synaptic thickening. ' Other Types of Uptake Processes I t has become evident that other uptake processes e x i s t i n addition to neuronal uptake of NA (which i s commonly denoted as "Uptake^ "),. More recently, i t has been demonstrated that an up-6 take process e x i s t s which transports catecholamines across mem-branes of non^neuronal tissues and t h i s has become known as "Uptake 2" (Iversen, 1965c). G i l l e s p i e (1973) has presented a thorough review on t h i s t o p i c . Another mechanism has been des-cribed which brings about r e d i s t r i b u t i o n of catecholamine from the axoplasm into the i n t r a c e l l u l a r storage v e s i c l e s (Carlsson, H i l l a r p and Waldeck, 1963; von Euler and Lishajko, 1963). I t i s now cl e a r that t h i s v e s i c u l a r uptake system i s very much d i f -ferent from other characterized uptake processes. F i n a l l y , i t i s now known that s p e c i a l i z e d uptake processes also e x i s t for every other proposed mammalian neurotransmitter substance. These processes andotheirtpfoperties have been reviewed by Iversen (1971a; 1971b). Properties and C h a r a c t e r i s t i c s of Uptake^ The neuronal uptake of NA has been studied extensively and consequently, a good deal of information i s now a v a i l a b l e on t h i s system. From the many studies i t has become apparent that Uptake^ displays almost i d e n t i c a l properties i n the NA-contain-ing neurons of both the c e n t r a l and peripheral nervous systems (Iversen, 1973). Early experiments which showed that NA i n peripheral t i s -sues was accumulated against a concentration gradient were per-formed by Axelrod e_t a l . .(,1959) and Whitby and coworkers (1961). Similar findings using brain ti s s u e were presented by Dengler and coworkers (1961b) who suggested that a saturable membrane 7 t r a n s p o r t p r o c e s s was i n v o l v e d . A k i n e t i c s t u d y o f t h e u p t a k e system i n i s o l a t e d , p e r f u s e d r a t h e a r t ( I v e r s e n , 1963) c o n f i r m e d t h e s u g g e s t i o n o f D e n g l e r e_t a l . (1961b) t h a t t h e p r o c e s s obeyed M i c h a e l i s - M e n t o n k i n e t i c s by m e a s u r i n g i n i t i a l r a t e s o f u p t a k e a t v a r i o u s amine c o n c e n t r a t i o n s . As w e l l , he showed t h a t the u p t a k e p r o c e s s was s t e r e o c h e m i c a l l y s e l e c t i v e f o r t h e n a t u r a l l y -o c c u r r i n g (-)-isomer o f NA and t h a t c o c a i n e a c t e d as a p o t e n t c o m p e t i t i v e i n h i b i t o r o f NA u p t a k e . The k i n e t i c c o n s t a n t s d e r -i v e d from t h i s s t u d y a r e g i v e n i n T a b l e I . T a b l e I K i n e t i c C o n s t a n t s f o r N o r a d r e n a l i n e Uptake i n Rat H e a r t (from I v e r s e n , 1963) M i c h a e l i s C o n s t a n t 7 ( K m ) Vmax - S.E. (M x 10" ) (ng/min/g o f h e a r t ) ( - ) - N o r a d r e n a l i n e 6.64 ( - ) - N o r a d r e n a l i n e 2.66 ( + ) - N o r a d r e n a l i n e 13.90 234 - 5.1 196 - 20.2 295 - 8.4 C o y l e and Snyder (1969b) examined the s t e r e o s p e c i f i c i t y i n d i f -f e r e n t a r e a s o f r a t b r a i n and a l s o f o u n d a marked p r e f e r e n c e o f t h e u p t a k e system f o r (-)-NA i n a l l r e g i o n s e x c e p t t h e c o r -pus s t r i a t u m where dopamine i s the major c a t e c h o l a m i n e . 3 The temperature-dependence o f t h e u p t a k e o f H-NA was shown by Z i a n c e and c o w o r k e r s (1972a) and C o l b u r n e_t a l . (1968). I n t h e p e r f u s e d h e a r t , any s i n g l e m e t a b o l i c i n h i b i t o r seems t o 8 have l i t t l e i n f l u e n c e on NA u p t a k e . I n t h e absence o f b o t h g l u c o s e and oxygen, however, u p t a k e o f NA i s i n h i b i t e d b u t can be r e s t o r e d by p r o v i d i n g e i t h e r agent (Wakade and F u r c h g o t t , 1968). I n c o m p a r i s o n , i n b r a i n t i s s u e , u p t a k e i s i n h i b i t e d by m e t a b o l i c i n h i b i t o r s (White and Keen, 1970) and s t i m u l a t e d by g l u c o s e and oxygen ( D e n g l e r e t a l . , 1962). I n b o t h p e r i p h e r a l and c e n t r a l n e u r o n s , t h e r e i s a marked dependence of t h e u p t a k e system f o r sodium i o n s i n t h e e x t e r n a l medium, w i t h a l m o s t c o m p l e t e i n h i b i t i o n o f t h e p r o c e s s i n t h e absence o f sodium ( I v e r s e n and K r a v i t z , 1966; B o g d a n s k i e t a l . . 1968; C o l b u r n e t a l . , 1968; B o g d a n s k i and B r o d i e , 1969; Keen and B o g d a n s k i , 1970; B o g d a n s k i e t al_. , 1970). Optimum u p t a k e was shown t o o c c u r a t e s s e n t i a l l y p h y s i o l o g i c a l l e v e l s o f b o t h sod-ium ( N a + ) and p o t a s s i u m ( K + ) i o n s ; c o m p l e t e absence o f K + mark-e d l y d i m i n i s h e d u p t a k e w h i l e r a i s i n g t h e e x t e r n a l K + concen-t r a t i o n a l s o d e c r e a s e d t h e r a t e o f NA u p t a k e t h r o u g h c o m p e t i t i o n w i t h N a + ( I v e r s e n and K r a v i t z , 1966; B o g d a n s k i e t a l . , 1968; C o l b u r n et _ a l . , 1968; B o g d a n s k i and B r o d i e , 1969; White and Keen, 1970; B o g d a n s k i e t a l . , 1970). I v e r s e n and K r a v i t z (1966) f o u n d t h a t N& u p t a k e i n r a t h e a r t was n o t a f f e c t e d by t h e absence o f c a l c i u m i o n s ( C a + + ) and t h i s was s u p p o r t e d by B o g d a n s k i and B r o -d i e (1969) and Keen and B o g d a n s k i (1970). _ 3 Ouabain, 10 M,^was a l s o shown t o i n h i b i t t h e u p t a k e o f 3 H^NA ( B o g d a n s k i , T i s s a r i and B r o d i e , 1968; C o l b u r n e t a l . , 1968; B o g d a n s k i and B r o d i e , 1969; White and Keen, 1971). Because o f t h i s o u a b a i n s e n s i t i v i t y and N a + - and K +-dependence of NA u p t a k e , i t has been s u g g e s t e d t h a t t h e p r o c e s s i s c a r r i e r - m e d i a t e d w i t h OUTSIDE INSIDE High[Na+] Low[Na+] F i g . 2. A working hypothesis f o r the e f f e c t of inorganic ions on uptake and storage of NA by p e r i p h e r a l adrenergic nerve endings. "C" denotes the membrane c a r r i e r . Arrows rep-resented by "X" i n d i c a t e i n h i b i t o r y f a c t o r s . Brackets i n -dicate coupled reactions. (Bogdanski and Brodie, 1969) 10 (Na + K )-dependent ATPase playing a c e n t r a l r o l e to maintain the i o n i c gradient (Colburn e_t a_l. , 1968; Bogdanski e_t _al. , 1968; Bogdanski and Brodie, 1969; Bogdanski et a l . , 1970). Ac-cording to t h i s theory, NA i s transported across the c e l l mem-brane by a c a r r i e r with the energy for transport being provided by the inward movement of Na + down a concentration gradient which i s maintained by ATPase (Fig. 2). However, t h i s mechanism has been challenged. White and Keen (1971) found that low con-centrations of ouabain i n h i b i t e d ATPase a c t i v i t y to the same ex-tent as 1 mM ouabain, yet d i d not depress NA uptake as the higher concentrations did. They also presented evidence that ion grad-ients do not provide energy for the uptake process,by a l t e r i n g the i n t e r n a l and external ion concentrations of synaptosomes (White and Keen, 1970) and a k i n e t i c analysis by White and Paton (1972) suggested that Na + d i d not increase the a f f i n i t y of a car-r i e r for NA. A d d i t i o n a l evidence for a membrane c a r r i e r transport system for NA comes from the demonstrated s t r u c t u r a l s p e c i f i c i t y of the process. Many s t r u c t u r a l analogs can act as substrates for the NA uptake system and from experiments on these compounds the s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s (SAR) for optimum binding to the uptake s i t e s can be obtained. S i m i l a r l y , various i n h i b i t o r s of the uptake process have been studied and the SAR for i n h i b i t i o n has also been determined. I t i s important to d i f f e r e n t i a t e be-tween substrates and competitive i n h i b i t o r s of NA uptake since the l a t t e r compounds may be able to bind with the c a r r i e r s i t e s yet may lack the s t r u c t u r a l features necessary for transport into 11 the c e l l . To show that a substance i s transported into the neuron would require d i r e c t measurement of the accumulation of t h i s substance i n the t i s s u e . I t should also be remembered that some drugs which i n h i b i t NA uptake do not f i t the SAR for i n -h i b i t i o n and may act i n a manner which i s not competitive. Com-pounds which are taken up as substrates have the p o t e n t i a l to a f f e c t intraneuronal binding or storage of the transmitter. In some of the e a r l i e s t experiments which demonstrated the importance of tissu e uptake of catecholamines, i t was observed that the uptake of NA was more important as a mechanism for the i n a c t i v a t i o n of c i r c u l a t i n g catecholamines than that of adren-a l i n e (Axelrod e_t jal. , 1959; Whitby e_t a l . , 1961). Iversen (1965c; 1965d) showed that adrenaline was accumulated by the same mechanism as was NA and also determined k i n e t i c constants for adrenaline uptake. From observations such as these which i n d i -cated that s t r u c t u r a l analogs of NA may also be a c t i v e l y accum-ulated by the neuron, a seri e s of experiments res u l t e d i n the SAR for substances to serve as substrates for the NA uptake pro-cess. This has been outlined by Iversen (1971b). B r i e f l y , the basic s t r u c t u r a l requirements for phenethylamine d e r i v a t i v e s to serve as a substrate are (1) absence of bulky N-substituents, (2) presence of phenolic hydroxyl groups ( e s p e c i a l l y i n the meta-position), and (3) absence of bulky methoxyl groups on the phenyl r i n g . 12 I n h i b i t i o n o f U p t a k e ^ I n h i b i t o r s o f NA u p t a k e f a l l i n t o two g e n e r a l c a t e g o r i e s , t h e f i r s t c o m p r i s i n g s y n p a t h o m i m e t i c amines s t r u c t u r a l l y r e - 1 l a t e d t o NA (some o f which may a l s o a c t as s u b s t r a t e s ) and t h e second c o n s i s t i n g o f d r u g s n o t r e l a t e d i n s t r u c t u r e t o NA and h a v i n g ' J d i v e r s e p h a r m a c o l o g i c a l a c t i o n s . The f o r m e r group was s t u d i e d e x t e n s i v e l y by b o t h Horn (1973) i n r a t b r a i n homogenates and Burgen and I v e r s e n (1965) i n r a t i s o l a t e d h e a r t . The s t r u c t u r a l r u l e s o b t a i n e d f o r i n h i b -i t i o n o f NA u p t a k e by t h e p h e n e t h y l a m i n e s d e r i v a t i v e s appeared to h o l d t r u e f o r b o t h c e n t r a l and p e r i p h e r a l n e u r o n s . These a u t h o r s f o u n d t h a t p h e n o l i c h y d r o x y l groups and «*-methylation o f th e s i d e c h a i n l e d t o an i n c r e a s e i n a f f i n i t y f o r t h e u p t a k e s i t e s , whereas m e t h y l a t i o n o f t h e p h e n o l i c h y d r o x y l g r o u p s , h y d r o x y l a t i o n o f t h e s i d e c h a i n , o r N - m e t h y l a t i o n l e d t o de-c r e a s e d a f f i n i t y . A c o m p a r i s o n o f t h e a f f i n i t i e s of t h e s e ana-l o g s a r e shown i n T a b l e I I (Burgen and I v e r s e n , 1965). Amphetamine i s one o f t h e s t a n u c t u r a l a n a l o g s w h i c h appears t o be a p o t e n t i n h i b i t o r o f NA u p t a k e b u t n o t a s u b s t r a t e (Thoenen e t a l . , 1968; I v e r s e n , 1971a, 1971b, 1973) a l t h o u g h Thoenen's paper s u g g e s t s t h a t t h e u p t a k e o f amphetamine may oc-c u r y e t be masked by a r a p i d o utward d i f f u s i o n , t h u s p r e v e n t i n g a c c u m u l a t i o n . A z z a r o e t a l . , (1974) s u g g e s t e d t h a t amphetamine does e n t e r t h e neuron t h r o u g h t h e amine t r a n s p o r t system s i n c e c o c a i n e and d e s i p r a m i n e b o t h r e d u c e d i t s a c c u m u l a t i o n i n synap-tosomes o f r a t b r a i n t i s s u e . Much of t h e work of t h i s r e s e a r c h 13 Table II I n h i b i t i o n of Noradrenaline Uptake (Uptake ) by Sympathomimetic Amines i n the Rat Isolated Heart ( A f f i n i t i e s were determined by d i r e c t analysis of uptake k i n e t -i c s . The ID50 i s the drug concentration producing 50% in h i b -i t i o n of noradrenaline uptake; a f f i n i t i e s are r e l a t i v e to phen-ethylamine=100) (Burgen and Iversen, 1965) Drug ID50 Relative (M) A f f i n i t y (-)-Metaraminol 7. 6 X 10"? 1,440 Dopamine 1. 7 X io-l 650 (+_) -ot-Methyldopamine 1.8 X 10~-7 610 (+•) -Amphetamine 1.8 X 1 0 1 610 (+)-Hydroxyamphetamine 1.8 X io-l 610 (-)-Nordefrin 2.0 X io-l 550 (-)-Noradrenaline 2. 7 X 1 0 7 407 (+)-Nordefrin 4. 2 X 1 0 7 260 Tyramine 4. 5 X 1 0 7 245 (±) -Amphetamine 4. 6 X io-l 240 Metatyramine 5.1 X 10~1 215 (+)-Methylamphetamine 6. 7 X io-l 165 (+)-Noradrenaline 6. 7 X io-l 165 (+)-Prenylamine 7.4 X 1 0 7 149 N-Methyldopamine 7. 6 X 1 0 7 145 (+)-Buphenine 8. 5 X io-l 130 (+_)-Propylhexedrine 8. 5 X 1 0 6 130 (-)-Adrenaline 1.0 X 1 0 6 110 Mephentermine 1.0 X i o i 110 PHENETHYLAMINE 1.1 X 1 0 6 100 (+)-Octopamine 1.3 X 1 0 6 85 Tranylcypromine 1.3 X 1 0 6 85 (+)-Noradrenaline 1.4 X 10 t 78 (+)-Cyclopentamine 1.4 X 1 0 _ 6 78 (^ f) -Adrenaline 1.4 X 1 0 _ 6 78 Noradrenalone 1. 5 X i o i 73 2-(Napth-2-yl)ethylamine 1.5 X 1 0 _ 6 73 (+_) -Phenylpropanolamine 2. 0 X i(d 55 (±)-3,4-Dichloroisoprenaline 2.0 X 1 0 6 55 (-)-Ephedrine 2. 2 X 1 0 l 6 50 Hordenine 2.5 X 1 0 6 45 (+) -oc-Ethylnoradr enaline 3. 2 X io~l IO" 6 34 (-) -Amphetamine 3.7 X 30 (continued) 14 Table I I (continued) Drug ID50 R e l a t i v e (M) A f f i n i t y P h e n e l z i n e 3.8 x 1 0 _ 6 29 ( j O-Phenylethanolamine 4.8 x 1 0 ~ 6 23 (-)-Phenylephrine 5.6 x 1 0 _ 6 20 Tuaminoheptane 5.6 x 1 0 _ 6 20 ( + ) _ N - E t h y l n o r a d r e n a l i n e 9.2 x 10_ 5 12 2-(p-Methoxyphenyl)ethylamine 1.0 x 10_ 5 11 ( jO-Methoxyphenamine 1.1 x 10_ 5 10 (+)-Oxedrine 1.2 x 10_ 5 £9 5- Hydroxytryptamine 2.0 x 10_^ 5.5 ( + ) - I s o p r e n a l i n e 2.5 x 10_ 5 4.5 ( + ) - N - B u t y l n o r a d r e n a l i n e 3.5 x 10_ 5 3.2 ( + ) - N - I s o b u t y l n o r a d r e n a l i n e 4.0 x 10_ 5 2.6 (+)-Metanephrine 4.3 x 10 . 2.6 (_)_Dopa 6.0 x 1 0 " 4 1.2 (+)-Normetanephrine 2.0 x 1 0 _ 4 0.55 6- Hydroxydopamine 2.0 x 1 0 _ 4 0.55 2-(3,4HDimethoxyphenyK)ethylamine 2.0 x 10_ 3 0.55 (+)-Methoxamine 1.0 x 10_„ 0.11 M e s c a l i n e 1.5 x 10 0.007 15 group has been concerned with the complex actions of amphet-amine (Ziance and Rutledge, 1972; Wenger and Rutledge, 1974). Another analog of NA which i s known to be a potent i n h i b -i t o r of the uptake process i s tyramine. Like amphetamine, i t i s classed as an i n d i r e c t l y - a c t i n g sympathomimetic agent and has been shown to displace NA from tissue stores (Hertting, Axelrod and Patrick, 1961). I t has been postulated that tyramine uses the NA uptake system to gain entry to the adrenergic nerve t e r -minals where i t can bring about the release of endogenous NA (Furchgott e_t _al. , 1963). The accumulation of l a b e l l e d tyramine had been demonstrated by Axelrod and coworkers (1962). In sup-port of the theory that tyramine u t i l i z e s the same uptake system as does NA, a seri e s of studies has shown the e f f e c t s of various drug pretreatments on the uptake of NA and tyramine (McNeill and Brody, 1968; Commarato et a l . , 1969a; Commarato et a l . , 1969b; McNeill and Commarato, 1969), demonstrating that drugs which po-t e n t i a t e responses to NA w i l l decrease responses to tyramine, presumably by preventing the uptake of both amines. Among those compounds which are not s t r u c t u r a l l y r e l a t e d to NA, cocaine i s probably the most studied and best-known exam-ple and, as such, i s often used as a reference compound i n com-parison studies. Dengler e_t al.(l(9J53fal)) presented evidence that cocaine i n h i b i t e d NA uptake and that t h i s was r e l a t e d to the pharmacological potentiating e f f e c t of cocaine on the NA res-ponse. S i m i l a r l y , McNeill and Brody (196-;6) found a r e l a t i o n s h i p between cocaine and other i n h i b i t o r s of NA uptake and t h e i r po-t e n t i a t i o n of the NA a c t i v a t i o n of cardiac phosphorylase. Co-16 c a i n e 1 s competitive i n h i b i t i n g e f f e c t has been shown by many other researchers including Hertting, Axelrod and Whitby (1961), and 31 vers en (1963; 1965b). In addition to cocaine, other compounds of a wide v a r i e t y of structures also appear to block the uptake of NA. One such group i s the t r i c y c l i c antidepressants. Axelrod, Hertting and Potter (1962) demonstrated that imipramine had the a b i l i t y to 3 block the entry of H-NA into storage s i t e s . Similar findings for t h i s and other t r i c y c l i c antidepressants have appeared throughout the l i t e r a t u r e (Hertting, Axelrod and Whitby, 1961; Iversen, 1965b; Callingham 1966; McNeill and Brody, 1968; Horn, Coyle and Snyder, 1971; Mundo et a l . , 1974; Squires, 1974). Mundo and coworkers (1974) found that the accumulation of lab-e l l e d antidepressants by r a t i s o l a t e d a t r i a seemed to be a pas-sive and unsaturable process. Another group of drugs which have an i n h i b i t o r y e f f e c t on the uptake of NA are the antihistamines (Johnson e_t al_. , 1965; Isaac and Goth, 1965; Isaac and Goth, 1967; McNeill and Brody, 1968; Snyder, 1970; Horn, Coyle and Snyder, 1970). The adren-ergic blocking agents represent yet another group of compounds which were found to act as i n h i b i t o r s of uptake (Hertting, Ax-elrod and Whitby, 1961; Dengler et a l . , 1961a; Iversen, 1965b). In addition to the aforementioned drugs, i n h i b i t i o n of uptake of NA has been implicated as an action of the monoamine oxidase i n -h i b i t o r s (Iversen, 1965b; Hendley and Snyder, 1968), phenothia-zines such as chlorpromazine (Axelrod, Hertting and Potter, 1962; Hertting, Axelrod and Whitby, 1961; Dengler et a l . , 1961a; I v e r s e n , 1965b; Snyder, 1970; Horn e t a l . , 1 9 7 1 ), t h e a d r e n e r g i c neurone b l o c k i n g d r u g s , , g u a n e t h i d i n e and b r e t y l i u m ( H e r t t i n g , A x e l r o d and Whitby, 1961; D e n g l e r e t a l . , 1 9 6 1 a ; I v e r s e n , 1965b), amantadine ( H e r b l i n , 1972), e t h a n o l (Roach e t a l . , 1 973), v a r -i o u s a n t i p a r k i n s o n i a n a gents (Snyder, 1970; Horn e t a l M 1971), and t h e n a r c o t i c a n a l g e s i c s ( C a r m i c h a e l and I s r a e l , 1973; Mon-t e l and S t a r k e , 1973 ) a l t h o u g h a r e c e n t s t u d y by C l o u e t . and..Wil-l i a m s (1974) s u g g e s t s t h a t t h e n a r c o t i c d r u g s do n o t compete w i t h t h e c a t e c h o l a m i n e s f o r the n e u r o n a l amine t r a n s p o r t system. S i n c e t h e r e i s such a d i v e r s i t y o f s t r u c t u r e s and a c t i o n s i n t h e s e compounds, i t must be emphasized t h a t t h e d r u g s may be a c t i n g a t more t h a n one s i t e i n p r o d u c i n g t h e t o t a l p h a r m a c o l -o g i c a l r e s p o n s e . As w e l l , t h e r e seems t o be no a b s o l u t e c o r r e l -a t i o n between th e p o t e n c i e s o f t h e v a r i o u s compounds as i n h i b i t -o r s o f u p t a k e and t h e i r p o t e n c i e s w i t h r e s p e c t t o o t h e r pharma-c o l o g i c a l a c t i o n s . T h e r e f o r e , a l t h o u g h the a b i l i t y t o i n h i b i t NA u p t a k e may a i d r e s e a r c h e r s i n u n d e r s t a n d i n g t h e mechanisms of a c t i o n o f d r u g s w h i c h i n t e r a c t w i t h a d r e n e r g i c mechanisms, i t must be remembered t h a t i n t e r a c t i o n s w i t h t h e u p t a k e p r o c e s s may o n l y m o d i f y t h e main p h a r m a c o l o g i c a l a c t i o n s o f t h e s e com-pounds. These o t h e r e f f e c t s may o b s c u r e t h e e x p e c t e d p o t e n -t i a t i o n o f t h e NA r e s p o n s e . F o r example, c o c a i n e and i m i p r a m i n e p o s s e s s a l o c a l a n a e s t h e t i c a c t i o n w h i c h may r e d u c e t h e e f f e c t s o f s y m p a t h e t i c s t i m u l a t i o n a t c o n c e n t r a t i o n s h i g h enough t o b l o c k u p t a k e ; d r u g s w i t h a d r e n e r g i c r e c e p t o r b l o c k i n g a c t i o n such as c h l o r p r o m a z i n e may a l s o f a i l t o p r o duce p o t e n t i a t i o n . F a i l -u r e t o p o t e n t i a t e NA may a l s o r e s u l t i f t h e i n h i b i t o r has a much 18 lower a f f i n i t y for the uptake s i t e s than NA or i f the concentra-t i o n of drug approximates the concentration needed to saturate the uptake mechanism (Iversen 1971b). These and other factors outlined by Iversen (1971b) should be considered c a r e f u l l y be-fore attempting to assess the i n h i b i t o r y capacity of various drugs by measuring the potentiation of the NA response. The Release of Noradrenaline from Nerve Endings Just as i t i s important to d i s t i n g u i s h between the uptake of NA and accumulation of t h i s catecholamine, i t i s also impor-tant to d i f f e r e n t i a t e between transmitter release and overflow. Release represents the actual output of amine produced by nerve stimulation whereas overflow, a much more complex parameter, r e f e r s to the increase i n transmitter above control l e v e l s i n the external medium of an organ or tissue preparation (Langer, 1974). This l a t t e r process may be affected by neuronal or ex-traneuronal uptake of NA, metabolism by MAO or COMT, or binding to receptors, as well as by an actual change i n transmitter re-lease. Therefore, any one or a combination of these e f f e c t s must be considered i n any study which demonstrates an increase i n transmitter overflow. In actual fact, "very l i t t l e i s known about the d e t a i l e d mechanism whereby nerve impulses and drugs bring about the re-lease of NA from nerve terminals. Although there has been i n -creased i n t e r e s t and experimentation i n t h i s area i n recent years, the number of publications i s small i n comparison to the profusion of papers published concerning the NA uptake process. The mechanism of transmitter release from sympathetic nerves appears to be s i m i l a r i n some respects to the release of acetylcholine at c h o l i n e r g i c junctions, a process which has been more extensively studied. Katz (1962) has described the release of acetylcholine as a calcium-dependent unitary event of secre-t i o n i n which multimolecular parcels of transmitter (the ves-i c l e contents) are extruded suddenly from the presynaptic mem-brane. I t has been shown that both acetylcholine and NA are stored i n granules or v e s i c l e s within the nerve endings. In keeping with t h i s , Burnstock and Holman (1966), i n experiments with guinea pig vas deferens, have implied a spontaneous release of "packets" of NA and that with nerve stimulation there i s an increase i n the number of quanta released. Much of what i s now known about NA release has resulted from experiments on "stim-ulus-secretion coupling" i n the adrenal medulla. The main e-vents occurring i n t h i s process, as outlined by Douglas (1966) appear to be f i r s t the reaction of the transmitter with the c e l l membrane r e s u l t i n g i n increased permeability of the membrane to calcium ion ( C a + + ) . This i s followed by inward movement of C a + + down i t s electrochemical gradient to the s i t e of action where i t i n i t i a t e s a process which causes release of the contents of the granules. Termination of secretion occurs upon disappear-ance of the transmitter and/or C a + + . The dependence of NA release on C a + + was demonstrated by Kirpekar and Misu (1967) i n cat spleen. They concluded that d e p o l a r i z a t i o n of post-ganglionic sympathetic f i b r e s may lead 20 to i n c r e a s e d i n f l u x of C a + + but admitted t h a t the s i t e of ac-t i o n and mechanism of the i o n i s unknown. From f i n d i n g s t h a t barium and s t r o n t i u m , but not magnesium i o n s , c o u l d r e p l a c e C a + + and t h a t sodium i o n s were not r e q u i r e d f o r r e l e a s e to oc-cur, i t was suggested t h a t the Ca was d i r e c t l y r e q u i r e d f o r the r e l e a s e mechanism and not simply f o r nerve c o n d u c t i o n . A review by Rubin (1970) supported the c r i t i c a l r o l e of C a + + i n catecholamine r e l e a s e from c e n t r a l as w e l l as from p e r i p h e r a l nerves whether r e l e a s e was due to nerve s t i m u l a t i o n , a c e t y l -c h o l i n e , or excess potassium i o n s . He noted however, t h a t tyramine-produced r e l e a s e does not seem to be C a + +-dependent. I n In attempts at d e f i n i n g more p r e c i s e l y the a c t u a l s e c r e t i o n mechanism, i t was suggested t h a t measurement of dopamine - f^-hy-d r o x y l a s e (DBH) would p r o v i d e i n f o r m a t i o n on the f a t e of s t o r -age v e s i c l e s s i n c e t h i s enzyme was known to be c o n t a i n e d w i t h i n the v e s i c l e s ( V i v e r o s e_t _al. , 1969a). I t was found t h a t d u r i n g nerve s t i m u l a t i o n of the a d r e n a l medulla, the e n t i r e s o l u b l e c o n t e n t s of the v e s i c l e s , i n c l u d i n g DBH, were s e c r e t e d by exo-c y t o s i s , l e a v i n g the v e s i c l e membranes w i t h i n the c e l l s ( V i v e r o s , e t a l . . 1969b). R e s u l t s of a study by Weinshilboum and cowork-e r s (1971) i n sympathetic nerves were compatible with the sug-g e s t e d p r o c e s s of e x o c y t o s i s . I t was found t h a t h i g h c a l c i u m i o n c c o n c e n t r a t i o n s and the drug phenoxybenzamine enhanced the r e -l e a s e of NA and DBH i n nerves i n n e r v a t i n g g uinea p i g vas d e f -erens (Johnson ejb a_l. , 1971). P r o s t a g l a n d i n r e v e r s e d t h i s e f f e c t i n t h i s t i s s u e but not i n the a d r e n a l medulla. The au-t h o r s t h e r e f o r e suggested t h a t s i n c e p r o s t a g l a n d i n i n h i b i t e d the actions of Ca on the release process whereas phenoxybenz-amine enhanced them, phenoxybenzamine may be acting by either increasing Ca i n f l u x , decreasing Ca e f f l u x or i n h i b i t i n g prostaglandin release. A report by Thoa and coworkers (1972) indicated that the i n t e g r i t y of the microtubule and m i c r o f i l a -ment protein complex i n adrenergic nerve terminals was c r i t i c a l for depolarization-induced exocytosis of NA, implying that the rapid release of NA may involve a c o n t r a c t i l e mechanism s i m i l a r to that occurring i n muscle. Implications of the involvement of c y c l i c adenosine 3',51-monophosphate (cAMP) i n the release mechanism came about through the study of Wooten e_t al_. (1973 ). They postulated that d e p o l a r i z a t i o n by an e l e c t r i c a l stimulus may cause i n f l u x of e x t r a c e l l u l a r C a + + and a c t i v a t i o n of mem-brane-bound adenyl cyclase, producing an increase i n i n t r a c e l -l u l a r cAMP. The cAMP may then mobilize C a + + and/or act d i r e c t l y with C a + + to bring about exocytosis. K i n e t i c s studies i n r a t heart s l i c e s were performed by Bogdanski and others i n order to assess the r o l e of various i n -organic ions on the storage and release of NA from sympathetic nerves. Bogdanski and Brodie (1969) found that sodium ion (Na+_)_ i s an absolute requirement for storage of NA and proposed that Na + a f f e c t s storage by preventing spontaneous release. In 1970, Keen and Bogdanski reported a Ca + +-dependent e f f l u x of NA i n Na +-free media accompanied by an increased i n f l u x of C a + + i n -to the tissues, compatible with reports of Na + and C a + + compet-i t i o n . They suggested that i n the absence of Na +, C a + + com-bines with a receptor r e s u l t i n g i n release of NA from storage s i t e s ; a t t h e same t i m e t h e Na -dependent u p t a k e c a r r i e r mech-anism i s i n o p e r a b l e r e s u l t i n g i n r a p i d l o s s o f amine from the c e l l . F u r t h e r work i n t h i s a r e a p r o v i d e d a d d i t i o n a l e v i d e n c e + ++ t h a t Na and Ca a r e mu t u a l a n t a g o n i s t s i n t h e r e l e a s e o f NA from t i s s u e s ( B l a s z k o w s k i and B o g d a n s k i , 1971). They s u g g e s t s e v e r a l p o s s i b l e mechanisms t o e x p l a i n t h e c o m p e t i t i o n between Na and Ca . These a u t h o r s t h e n p r e s e n t e d e v i d e n c e f o r a Na -dependent, c a r r i e r - m e d i a t e d t r a n s p o r t mechanism f o r t h e r e l e a s e o f NA from the c e l l w h i c h i s a l s o i n h i b i t e d by desipr.amine. Ac-c o r d i n g t o t h e i r model, Ca e n t e r s t h e p r e s y n a p t i c t e r m i n a l i n r e s p o n s e t o d e p o l a r i z a t i o n . There, t h e f r e e C a + + p a r t i c i -p a t e s i n m o b i l i z a t i o n o f s t o r e d NA by a s a t u r a b l e p r o c e s s a n t -a g o n i z e d n o n c o m p e t i t i v e l y by N a + . When m o b i l i z e d , the NA c o u l d be t r a n s p o r t e d a c r o s s t h e membrane by t h e Na +-dependent c a r r i e r s ystem. P a t o n (1973) a l s o p r o v i d e s e v i d e n c e t h a t the e f f l u x o f NA from t h e c y t o p l a s m o f a d r e n e r g i c . n e r v e s o c c u r s by a c o c a i n e -s e n s i t i v e , c a r r i e r - m e d i a t e d p r o c e s s . Drug E f f e c t s on N o r a d r e n a l i n e R e l e a s e B e f o r e d i s c u s s i n g a c t u a l d r u g e f f e c t s on r e l e a s e , m e n t i o n s h o u l d be made o f t h e n o t i o n o f i n t r a n e u r o n a l "compartments" o r " p o o l s " o f s t o r e d NA. A l t h o u g h t h i s i s s t i l l an a r e a w h i c h i s p o o r l y d e f i n e d because o f t h e p r o f u s i o n o f complex and o f t e n c o n f l i c t i n g e v i d e n c e , many schemes have been p r o p o s e d to ex-p l a i n the d a t a . One such model has been p r e s e n t e d by I v e r s e n (1967). Ac-cording to t h i s scheme, most of the NA i n nerve terminals i s stored within p a r t i c l e s i n more than one storage form, con-s i s t i n g of a firmly-bound, tyramine-resistant complex and a tyramine-susceptible pool. This l a t t e r form can exchange f r e e l y with NA i n the extravesicular axoplasm where i t i s removed main-l y by metabolism by MAO. Release of NA by nerve impulses may occur from a small r e a d i l y a v a i l a b l e pool which seems to be r e s i s t a n t to reserpine and tyramine but i s replenished from the larger, r e s e r p i n e - s e n s i t i v e compartment. Uptake of e x t r a c e l l u -l a r NA occurs f i r s t into the axopitasm from which i t i s r a p i d l y d i s t r i b u t e d into other intraneuronal pools. Interest i n the r e l e a s i n g e f f e c t s of drugs gained promin-ence when i t became evident that sympathomimetic amines could act be d i r e c t , i n d i r e c t , or mixed e f f e c t s . Tyramine represents the prototype of the i n d i r e c t l y - a c t i n g amines which produce t h e i r sympathomimetic e f f e c t s by r e l e a s i n g NA from t i s s u e stor-age s i t e s . Evidence for a NA-releasing e f f e c t by tyramine was presented i n 1958 by Burn and Rand, who found that tyramine's action was reduced by reserpine pretreatment but restored by NA and adrenaline infusions. Stjarne (1961) showed that t y r -amine increased NA l e v e l s i n splenic blood and concluded that i n the spleen, tyramine may be acting by r e l e a s i n g NA from i n t r a -axonal storage granules or by making bound, extragranular NA a v a i l a b l e to receptors. In the same year, release of NA by tyramine i n the r a t heart was demonstrated by Hertting, Axelrod and Patrick. I n h i b i t i o n of MAO i n a t r i a produced increased re-lease bf NA by tyramine, amphetamine and mephentermine but not 24 n e r v e - s t i m u l a t e d r e l e a s e • ( S m i t h , 1966). T h i s work i n d i c a t e d t h a t s t o r a g e s i t e s f o r NA r e l e a s e d by t y r a m i n e d i f f e r from t h o s e a f f e c t e d by n e r v e s t i m u l a t i o n and t h a t s u b s t a n t i a l am-o u n t s o f NA r e l e a s e d by t h e i n d i r e c t l y - a c t i n g amines a r e oxi--d a t i v e l y d eaminated w i t h i n t h e n e r v e t e r m i n a l s . The a u t h o r s u g g e s t e d t h a t t y r a m i n e c o u l d t h e r e f o r e be p r o d u c i n g r e l e a s e from v e s i c l e s i n t o t h e c y t o p l a s m whereas n e r v e s t i m u l a t i o n a f -f e c t e d v e s i c l e s l o c a t e d a d j a c e n t t o t h e c e l l membrane. How-e v e r , C o l b u r n and K o p i n (1972), w o r k i n g w i t h r a t b r a i n synap-tosomes, i n d i c a t e d t h a t t y r a m i n e r e l e a s e s s t o r e d NA t o a s i t e where MAO i s n o t a c t i v e s i n c e i t d i d n o t r e l e a s e d e a minated m e t a b o l i t e s as r e s e r p i n e d i d . T h i s c o u l d a l t e r n a t i v e l y be ex-p l a i n e d by c o m p e t i t i o n between t y r a m i n e and NA f o r MAO s i n c e t y r a m i n e i s r a p i d l y degraded by t h i s enzyme ( w i t h a h a l f - l i f e o f about f i v e m i n u t e s ) . A l t h o u g h th e e x a c t mechanism o f t y r a m i n e - i n d u c e d r e l e a s e has n o t y e t been r e s o l v e d , i t seems a p p a r e n t t h a t t h e p r o c e s s d i f f e r s from n e r v e - s t i m u l a t e d r e l e a s e . Lindmar and c o w o r k e r s ( 1 9 6 7 ) , d e m o n s t r a t e d t h a t a c o n s t a n t i n f u s i o n o f t y r a m i n e i n r a b b i t h e a r t r e l e a s e d NA c o n t i n u o u s l y and i n d e p e n d e n t l y o f ex-t e r n a l C a + + c o n c e n t r a t i o n . A n o t h e r paper s t a t e d t h a t d r u g s such as r e s e r p i n e p r o duce a c a l c i u m - i n d e p e n d e n t , n o n - e x o c y t o t i c r e l e a s e o f NA w i t h o u t c o n c o m i t a n t r e l e a s e o f DBH (Thoa e_t a l . , 1974). Other e v i d e n c e f o r s e p a r a t e mechanisms i s g i v e n by West-f a l l and B r a s t e d (1974) who showed t h a t c o l c h i c i n e , w h i c h p r e -v e n t s r e l e a s e o f NA p r o d u c e d by n e r v e s t i m u l a t i o n , had no e f -f e c t on t h e r e l e a s e p r o d u c e d by t y r a m i n e . They a l s o d e m o n s t r a t -ed that prostaglandins E^ and E^ decreased the release of NA induced by nerve stimulation, n i c o t i n e , potassium chloride, and aminophylline, but not tyramine. Amphetamine-induced release of transmitters has also been widely studied since t h i s drug i s also classed as an i n d i r e c t -l y - a c t i n g amine. In 1965, Glowinski and Axelrod demonstrated 3 release of H-NA by both reserpine and amphetamine, although they acted i n a d i f f e r e n t manner as indicated by the d i f f e r e n t metabolic fates of NA released by the two drugs. An increase i n the concentration of normetanephrine occurred following am-phetamine treatment, suggesting that t h i s drug released NA from the neuron as unchanged amine which i s then O-methylated by COMT. This study also demonstrated an i n h i b i t i o n of NA accum-u l a t i o n by amphetamine. A s i g n i f i c a n t contribution to t h i s f i e l d of research has also been made by Rutledge and coworkers. Results of a study by Rutledge (1970) suggested that the i n h i b i t i o n of oxidative de-amination of NA by amphetamine i s p r i m a r i l y an i n d i r e c t e f f e c t r e s u l t i n g from the i n h i b i t i o n of neuronal uptake of NA, l i m i t -ing access of NA to intraneuronal MAO. Ziance and Rutledge, (1972), d i f f e r e n t i a t i n g between actual release and i n h i b i t i o n of accumulation, again showed that amphetamine releases NA pre-dominantly as unmetabolized amine because pargyline p r e t r e a t -ment had no s i g n i f i c a n t e f f e c t on t h i s release. They suggested that t h i s e f f e c t was probably not due to d i r e c t i n h i b i t i o n of uptake of NA. Complicating the picture i s evidence presented by Farnebo (1971) that amphetamine, in;low concentrations, acted p r i m a r i l y on extragranular catecholamines, having l i t t l e r e l e a s i n g e f f e c t on transmitter stored within the amine storage granules. Ziance, Azzaro and Rutledge (1972) demonstrated a marked 3 temperature-dependence of H-NA release from brain s l i c e s , chopped tis s u e , and synaptosomal homogenates produced by amphet-amine but only p a r t i a l dependence on the e x t r a c e l l u l a r calcium ion concentration. In t h i s l a t t e r respect, amphetamine-induced NA release d i f f e r s from potassium-induced release which i s 1 -strongly calcium-dependent, and also appears to d i f f e r from that produced by tyramine which occurs independently of Ca concen-t r a t i o n . This suggests that a l l i n d i r e c t l y - a c t i n g amines may not be acting i n the same manner to cause release. Amphetamine also exhibited a s e l e c t i v e release from neurons containing d i f -ferent biogenic amines (Azzaro and Rutledge, 1973) which might explain dose-dependent behavioural changes due to t h i s drug. Another experiment, employing cocaine and desipramine as in h i b -i t o r s of neuronal NA uptake, showed that amphetamine-induced e f f l u x was not due merely to i n h i b i t i o n of neuronal uptake of spontaneously-released amine (Azzaro, Ziance and Rutledge, 1974). Cocaine and desipramine also s h i f t e d the concentration-e f f e c t curve for release by amphetamine to the r i g h t but had no 3 e f f e c t on H-NA release by potassium chloride; s i m i l a r l y , cocaine and desipramine i n h i b i t e d the uptake of low concentrations of 3 H-amphetamine into synaptosomes. These findings indicate that amphetamine also acts as a substrate for the neuronal NA uptake system, thereby entering the neurone and pos s i b l y d i s p l a c i n g NA from binding s i t e s within the nerve terminal. Among the drugs which have been shown to potentiate the 3 actions of NA and to i n h i b i t the uptake of H-NA by adrenergic nerve terminals, both cocaine (Haefely e_t a_l. , 1964; Trendel-enburg, 1968; Davis and McNeill, 1973) and high doses of desip-ramine (Titus et a l . , 1966; L e i t z and Stefano, 1970) also ap-pear to possess the a b i l i t y to release endogenous NA. The effe of desipramine appears to be at the l e v e l of the NA storage granule since the catecholamine released by t h i s drug occurred p r i m a r i l y i n the form of deaminated metabolites,, (Leitz and Stefano, 1970). An i n t e r e s t i n g concept with respect to neuronal release i s the presynaptic regulation of catecholamine release, reviewed by Langer (1974). I t has been known for some time that alpha-receptor blocking agents such as phenoxybenzamine (POBZ) or phentolamine increase the nerve-stimulated overflow of NA. The hypothesis of presynaptic regulation of NA release through a ne< at i v e feedback mechanism mediated by alpha adrenergic receptors res u l t e d from studies demonstrating p o s i t i v e changes i n trans-mitter overflow by POBZ i n tissues where beta receptors pre-dominate. The theory proposes that released NA, above a c r i t i -c a l concentration i n the synaptic c l e f t , would activate presyn-apt i c alpha receptors, i n h i b i t i n g further release of the trans-mitter. In support of t h i s , alpha receptor agonists i n h i b i t , whereas the antagonists enhance nerve-stimulated release of . transmitter (Farnebo and Hamberger, 1971). According to t h i s scheme, blockade of neuronal uptake should be expected to en-28 nance presynaptic i n h i b i t i o n of NA release and thereby reduce transmitter overflow, which could also explain the r e l a t i v e i n e f f e c t i v e n e s s of drugs such as cocaine or desipramine i n i n -creasing NA overflow despite t h e i r i n h i b i t i o n of uptake of the catecholamine. Langer also proposes that since the negative feedback mechanism i s not involved i n regulation of tyramine-induced NA release (which i s also not calcium-dependent), the presynaptic feedback mechanism may act by modifying the a v a i l -a b i l i t y of calcium ions for release e l i c i t e d by nerve stimula-."Lo-t i o n . Thus, the e f f e c t s of drugs i n producing e f f l u x of NA from nerve terminals can be very complex and may be intimately con-nected with t h e i r e f f e c t s on the NA uptake system. For those drugs which have been shown to bring about transmitter release, the mechanisms are s t i l l unclear; for many other compounds which potentiate the response to NA, even less i s known and research-ers are s t i l l attempting to evaluate the r e l a t i v e contributions of the various drug actions on increased transmitter overflow. Nonspecific-Membrane E f f e c t s : L i p i d S o l u b i l i t y and i t s Rela- tionship to Potency In hiis recent review paper, membrane actions of anaesthetics brane concentrations (that i s , 1 onstrated that a l i n e a r r e l a t i o n blocking concentration of an ana fer p a r t i t i o n c o e f f i c i e n t . In a Seeman (1972) has r e l a t e d the and t r a n q u i l i z e r s to t h e i r mem-i p i d s o l u b i l i t i e s ) and has dem-" ship e x i s t s between the nerve-esthetic and i t s membrane-buf-si m i l a r manner, Carmichael and I s r a e l (1973) found that although there was no apparent r e l a t i o n -ship between narcotic analgesic potency and the i n h i b i t i o n of uptake of NA, a s i g n i f i c a n t c o r r e l a t i o n existed between the \ l i p o p h i l i c nature of these compounds and t h e i r a b i l i t y to i n -h i b i t NA uptake. Also f i t t i n g t h i s c o r r e l a t i o n were cocaine, desipramine, chlorpromazine, and benztropine, but the str u c -t u r a l analog of NA, amphetamine, did not f i t . Their r e s u l t s therefore suggested that i n h i b i t i o n of uptake of NA by many psychotropic compounds may be re l a t e d to t h e i r l i p i d s o l u b i l i t y rather than to a s p e c i f i c structure. Another study by Roach and coworkers (1973) on the e f f e c t s of ethanol on transmitter uptake by r a t brain synaptosomes also demonstrated a d i r e c t c o r r e l a t i o n between uptake i n h i b i t i o n and the oil/water par-t i t i o n c o e f f i c i e n t of the i n h i b i t o r , presenting a d d i t i o n a l e v i -dence for an i n t e r a c t i o n between the drug and membrane l i p i d . Background and Objectives of the Present Study As stated previously, pharmacologists are s t i l l t r y i n g to elucidate the precise mechanisms whereby many drugs potentiate the actions of NA and the preceeding discussion shows how the study of uptake and release of catecholamines i s important pharmacologically i n helping to explain the actions of drugs on adrenergic mechanisms. McNeill and coworkers have performed re-late d experiments on the e f f e c t s of various drug pretreatments on amine uptake and amine-induced phosphorylase a c t i v a t i o n i n ra t heart, lending support to the theory that tyramine u t i l i z e s 30 the same uptake system as does NA, and that drugs which poten-t i a t e the e f f e c t s of NA but block the e f f e c t s of tyramine do so by preventing the uptake of both amines (McNeill and Brody, 1966; McNeill and Brody, 1969; Commarato, Brody and McNeill, 1969a; 1969b; McNeill and Commarato, 1969). These experiments explored the e f f e c t s of several antihistamines, t r i c y c l i c a n t i -depressants, methylphenidate and cocaine on amine uptake. A l a t e r paper by Davis and McNeill (1973) investigated further the cardiac e f f e c t s of these drugs, suggesting that a number of drugs known to potentiate the actions of noradrenaline by block-ing amine uptake i n the heart may also be exerting t h e i r p o s i -t i v e i n o t r o p i c e f f e c t s by r e l e a s i n g NA fronu asympathetic nerve endings. However, the experiments d i d not p o s i t i v e l y d i s t i n -guish between blockade of uptake off.NA and actual release of the amine. Therefore, i t was decided to pursue t h i s i n v e s t i g a t i o n of the e f f e c t s of cocaine, tyramine, and several antihistamines and t r i c y c l i c antidepressants, employing a f a i r l y simple, re-producible method involving use of catecholamine-containing nerve terminals i n r a t brain homogenate. A s i m i l a r method has been used by other in v e s t i g a t o r s to d i f f e r e n t i a t e between i n -3 hibitojon of uptake of spontaneously-released H-NA and actual release of the amine (for example, Ziance and Rutledge, 1972; Wenger and Rutledge, 1974). The s p e c i f i c objectives for the t o t a l period of research were as follows. F i r s t , i t was d e s i r -able to study the uptake of t r i t i a t e d noradrenaline by nerve terminals i n r a t brain homogenates i n the absence of t e s t drugs 31 i n order to determine the time course of uptake and the e f f e c t s of varying substrate concentrations. Secondly, the e f f e c t s of 3 both time and drug concentration on the e f f l u x of H-NA by the compounds were to be examined i n order to compare t h e i r r e l a t i v e potencies regarding amine release. A t h i r d objective was to explore the e f f e c t s of time and drug concentration on i n h i b -3 i t i o n of H-NA accumulation into nerve endings and again to det-ermine the r e l a t i v e potencies of the compounds, i n t h i s case as i n h i b i t o r s of uptake. F i n a l l y , a c o r r e l a t i o n of drug potency with l i p o p h i l i c i t y of the compounds would help to d i s t i n g u i s h between nonspecific e f f e c t s on the c e l l membrane and s p e c i f i c release or i n t e r a c t i o n with an active uptake process. MATERIALS AND METHODS Animals either sex weighing 200 to 300 grams were experiments. The animals were maintained and water ad libi t u m and were kept i n a con-u n t i l the time of s a c r i f i c e . Chemicals and Drugs The following drugs were donated: a m i t r i p t y l i n e hydro-chl o r i d e (Merch, Sharp and Dohme, Canada Ltd.), chlorphenir-amine maleate (Schering Corp. Ltd.), desipramine hydrochlor-R ide (Pertofrane , Geigy (Canada) Ltd.), diphenhydramine hydro-chloride (Benadryl , Parke-Davis and Co.), imipramine hydro-chl o r i d e (Geigy (Canada) Ltd.), n o r t r i p t y l i n e hydrochloride ( E l i L i l l y and Co. Ltd.), pargyline hydrochloride (Eutonyl , Abbott Laboratories Ltd.), phenindamine hydrochloride (Theph-o r i n , Hoffmann-LaRoche Ltd.), promethazine hydrochloride (Pou-lenc Ltd. ) , tripelennamine hydrochloride (Ciba Co. Ltd-. ) , and t r i p r o l i d i n e hydrochloride (Burroughs Wellcome and Co. (Canada) Ltd. ) . Other drugs were purchased from commercial sources. These were: cocaine hydrochloride ( B r i t i s h Drug Houses Pharmaceuti-c a l s ) , ( + )- L~7- 3H]-noradrenaline (New England Nuclear Corp.), Wistar rats of used throughout the on Purina Rat Chow t r o l l e d environment 33 (-)-noradrenaline (Sigma Chemical Co.), and tyramine hydro-chl o r i d e (Sigma Chemical Co.). A l l other chemicals were used i n the highest a v a i l a b l e p u r i t y . To prepare the sol u t i o n of l a b e l l e d noradrenaline (a solu-t i o n containing approximately 200,000 DPM as well as 200 pmoles t o t a l NA i n a volume of 10 p.1 to be added to 4 ml of incubation medium to give a f i n a l concentration of 0.05 pM NA), two other. 3 solutions were required. The f i r s t of these contained H-nor-adrenaline 0.25 mGi and 0.0045 mg i n 0.25 ml (Solution "A"). (This s p e c i f i c a c t i v i t y varies with l o t number but c a l c u l a t i o n s are analogous for each l o t received.) The second s o l u t i o n contained 3.248 pg of unlabelled noradrenaline per ml to give a concentration of 19.2 M (Solution "B"). To make the desired f i n a l s o l u t i o n , 50 pi of Solution "A" (containing 5319 pmoles 3 ft H-noradrenaline and 1.11 x 10 DPM) was added to 5269 pi of Solution B (containing 101,061 pmoles noradrenaline) g i v i n g a l a b e l l e d s o l u t i o n with a f i n a l volume of 5319 pi and contain-ing 106,380 pmoles of t o t a l noradrenaline. Tissue Preparation The method described below represents a modification of that used by Snyder and Coyle (1969). In a r e l a t e d study, these authors had pretreated rat s with reserpine i n order to i n a c t i v a t e the granular storage mechanism and deplete the en-dogenous catecholamines (Coyle and Snyder, 1969a). However, they l a t e r showed that k i n e t i c data were unaltered i n animals 34 which had received reserpine, i n d i c a t i n g that only uptake by the neuronal membrane rather than binding within storage granules was contributing to short-term experiments on NA transport (Snyder and Coyle, 1969). Therefore animals i n our study were not pretreated with reserpine. Rats were k i l l e d by a blow to the head followed by cer-v i c a l d i s l o c a t i o n . They were immediately bled by c u t t i n g the throat and the brains were r a p i d l y removed and tran s f e r r e d to a cold surface. The brains were then weighed and homogenized i n eight volumes of i c e - c o l d 0.25 M sucrose with eight to ten strokes of a motor-driven, glass homogenizing tube and t e f l o n p e s t l e (Potter-Elvehjem type, Kontes K-886000). The homogenate was then tran s f e r r e d to p l a s t i c centrifuge tubes and c e n t r i -fuged at 1000 x g (3000 rpm i n an IEC B-20 centrifuge using rotor no. 874) for ten minutes at 0° to 4° C. Following t h i s , the supernatant was aspirated from the large pink p e l l e t . This p e l l e t was discarded, the supernatant was mixed to form a uni-form suspension, and an aliq u o t of the supernatant was heated i n a t e s t tube i n a b o i l i n g water bath for ten minutes. The remaining untreated homogenate was kept i n an i c e bath u n t i l the incubation was begun (approximately t h i r t y minutes a f t e r s a c r i f i c i n g the animal). Incubation Procedure Each incubation consisted of eight tubes: one b o i l e d homogenate blank (to correct for nonspecific binding), one control (to which no drug was added), and s i x sample tubes. The t o t a l volume of incubation mixture was 4.0 ml of which, t y p i c a l l y , 0.2 ml was homogenate, 0.1 ml was drug s o l u t i o n , 3 and 10 jul was H-noradrenaline s o l u t i o n . The remaining volume consisted of modified Krebs-Henseleit bicarbonate buffer (see Table I I I ) . This buffer was prepared fresh d a i l y from stock solutions of each ingredient (except pargyline which i s un-stable i n s o l u t i o n ) . B a s i c a l l y , two procedures were used i n order to separate release experiments from studies of uptake i n h i b i t i o n . For the 3 release studies, homogenate was preincubated with H-noradren-al i n e for 25 minutes p r i o r to addition of the drug. This served 3 to load the synaptosomes with H-amine so that the e f f e c t s of the drugs on release could be measured. For studies of uptake blockade, the homogenate was instead preincubated with drug f o r 3 f i v e minutes p r i o r to addition of H-noradrenaline. This was done to enable the drug to i n t e r a c t with the c a r r i e r or mem-brane before the catecholamine was added. Before the actual incubation was begun, the tubes contain-ing buffer and drug or noradrenaline were allowed to e q u i l i -o brate to 37 C i n a Dubnoff metabolic shaker bath for approxi-mately ten minutes under 95% C-2/5% CC^ aeration. The preincu-bation was then begun with the addition of b o i l e d homogenate to the f i r s t tube. Untreated homogenate was added to each of the remaining tubes at t h i r t y second i n t e r v a l s . Tubes were mixed on a Vortex Mixer aft e r each addition. , At the end of the preincubation period, either drug s o l u t i o n or H-noradrenaline was added (depending on the experimental objective as previous-36 Table III Modified Kr-ebs-Henseleit Buffer Chemical g/1000 ml concentration (mM) NaCl* 7. 07 121 KC1 0. 35 4. 7 Ca C l 2 * * 0.183 1.4 MgS04.7H20 0. 308 1. 2 NaHC03 2.10 25 KH 2P0 4 0.16 1. 2 Glucose 2.0 11.1 Ascorbic Acid 0. 20 1.1 Disodium EDTA.2H20 0.045 0.13 Pargyline HCl 0. 030 0.16 D i s t i l l e d Water to 1000 ml since the calcium concentration was reduced (as CaCl ), extra sodium chloride was added to give the same concen-t r a t i o n of chloride that normal Krebs-Henseleit buffer contains * * calcium concentration was reduced by h a l f of the concentration found i n normal (unmodified) Krebs buffer o r i g i n a l -Henseleit 37 l y d e s c r i b e d ) a t t h i r t y second i n t e r v a l s and the i n c u b a t i o n c o n t i n u e d f o r t h e d e s i r e d l e n g t h o f t i m e . The i n c u b a t i o n was t e r m i n a t e d by i m m e d i a t e l y t r a n s f e r r i n g the i n c u b a t i o n tubes to an i c e b a t h . The m i x t u r e was p o u r e d i n t o p r e - c h i l l e d u l t r a c e n t r i f u g e t u b e s w h i c h were t h e n t i g h t l y capped and c e n t r i f u g e d a t 48 ,000 x g f o r t h i r t y m i n u t e s a t 0° to 4 ° C (27,000 rpm i n a Beckmann L2-65B u l t r a c e n t r i f u g e u s i n g r o t o r t y p e 65 ) . F o l l o w i n g c e n t r i f u g a t i o n , t u b e s were a g a i n p l a c e d i n an i c e b a t h to a w a i t f i n a l p r o c e s s i n g o f the s u p e r n a -t a n t and p e l l e t f r a c t i o n s . D e t e r m i n a t i o n o f R a d i o a c t i v i t y I m m e d i a t e l y f o l l o w i n g u l t r a c e n t r i f u g a t i o n , the s u p e r n a t -ant was d e c a n t e d from the p e l l e t f r a c t i o n s . A l i q u o t s (0 .1 ml) o f each s u p e r n a t a n t were t r a n s f e r r e d i n . d u p l i c a t e to l i q u i d s c i n t i l l a t i o n c o u n t i n g v i a l s and 10 ml o f B r a y ' s s c i n t i l l a t i o n s o l v e n t ( B r a y , 1960; see T a b l e IV) was added to each v i a l . The p e l l e t s were r i n s e d s u p e r f i c i a l l y w i t h 5 ml o f i c e -c o l d K r e b s - H e n s e l e i t b u f f e r , d r a i n e d w e l l , and suspended i n 2.0 ml o f a b s o l u t e e t h a n o l . Each p e l l e t was t h e n homogenized i n a g l a s s h o m o g e n i z i n g tube w i t h a g l a s s p e s t l e (Kontes K -885200) u n t i l a homogeneous s u s p e n s i o n was o b t a i n e d . S u s p e n -s i o n s were c e n t r i f u g e d f o r t e n minutes a t 1000 x g (3000 rpm i n an IEC B-20 c e n t r i f u g e ) and the r e s u l t i n g s u p e r n a t a n t c a r e -Table IV Bray's S c i n t i l l a t i o n Solvent Chemical Quantity PPO 4.0 g POPOP 0. 200 g Naphthalene 60 g Methanol (absolute) 100 ml Ethylene Glycol 20 ml Dioxane to 1000 ml f u l l y decanted from the p e l l e t which was discarded. Aliquots (0.5 ml) of supernatant were also transferred i n duplicate to l i q u i d s c i n t i l l a t i o n counting v i a l s and d i l u t e d with 10 ml of Bray's s c i n t i l l a t i o n f l u i d . R a d i o a c t i v i t y of each sample was determined i n a Nuclear-Chicago Isocap 300 L i q u i d S c i n t i l l a t i o n System by counting each sample for ten minutes using program number 1 for tritium-con-t a i n i n g samples. No attempt was made to separate metabolites formed i n the t i s s u e from the amine since i t had been shown i n s i m i l a r exper-iments that 85% to 95% of the r a d i o a c t i v i t y i n p a r t i c u l a t e f r a c -tions was unchanged catecholamine, with no d i f f e r e n c e between various brain areas (Snyder and Coyle, 1969; Coyle and Snyder, 1969b; Colburn e_t aJL. , 1968) since pargyline pretreatment pre-vented formation of deaminated metabolites. Calculations Employing quench curves which had been prepared exclusive-l y for the experimental system under study, counts per minute (CPM) for each sample were converted to d i s i n t e g r a t i o n s per min-ute (DPM) for both the extracted p e l l e t (P) and supernatant (S) f r a c t i o n s . Duplicate samples were averaged at t h i s point. Results were calcul a t e d f i r s t as "particulate-medium r a t i o s " (P/M r a t i o s ) of t r i t i u m , determined as d i s i n t e g r a t i o n s per min-ute per gram of p e l l e t f r a c t i o n divided by d i s i n t e g r a t i o n s per minute per m i l l i l i t r e of supernatant f l u i d . Using an analogous 40 procedure, Snyder and Coyle (1969) had determined p e l l e t weights to be between 9 and 11 mg. Therefore, a p e l l e t weight of 10 mg was assumed for the c a l c u l a t i o n s . That i s P/M r a t i o = PPM/q p e l l e t DPM/ml supernatant DPM i n extracted p e l l e t sample x 400 DPM i n supernatant sample x 10 Ratios for the blanks were subtracted from c o n t r o l and sample r a t i o s . For studies of drug e f f e c t s on the uptake and release processes, sample P/M r a t i o s were compared to control r a t i o s (with the con t r o l representing either 0% i n h i b i t i o n of uptake or 0% r e l e a s e ) . Therefore, c a l c u l a t i o n s were expressed by: % Uptake I n h i b i t i o n _ ^ , . or = Control r a t i o - Sample r a t i o % Release Control r a t i o x 1 0 0 / o for each incubation. For Time vs E f f e c t studies as well as Concentration vs E f f e c t experiments, each determination was repeated at l e a s t three times.so that the expressed values represent the mean - standard error of the mean for at le a s t three duplicate de-terminations. 41 RESULTS Uptake i n the Absence of Test Drugs The i n i t i a l experiments were conducted with the aim of e s t a b l i s h i n g the basic l e v e l s of uptake of t r i t i a t e d NA by the synaptosome-containing whole r a t brain homogehates i n the ab-sence of drug treatment. Incubations of NA and homogenate were c a r r i e d out for 2, 5, 10, 20 and 45 minutes, i n order to deter-mine the time course of NA uptake under the previously-de- ': scribed experimental conditions. This study was performed with four d i f f e r e n t concentrations of t o t a l NA (0.05, 0.27, 0.70, and 2.0 jiM) i n the incubation medium, but a constant concentra-t i o n of t r i t i a t e d NA <00?05 juM). In order to achieve these concentrations i n a t o t a l volume of 0.10 ml added to s u f f i c i e n t incubation medium to make 4.0 ml, the following scheme was used: Table V Volumes of NA Solutions Added to the Incubation Mixture ! I t ! Desired NA Volume Volume Volume 0.01 N Total cone. (p.M) "hot" sol'n "cold" t a r t a r i c acid volume 0.05 0.01 ml 0.09 ml 0.10 ml 0.27 0.01 ml 0.01 ml 0.08 ml 0.10 ml 0.70 0.01 ml 0.03 ml 0.06 ml 0.10 ml 2.0 0.01 ml 0.09 ml 0.10 ml The c o n c e n t r a t i o n o f t h e n o n - l a b e l l e d ("cold") NA s o l u t i o n was 86.67 JJM and t h a t o f t h e t r i t i a t e d ("hot") NA s o l u t i o n was 20 JUM, b o t h s o l u t i o n s h a v i n g been p r e p a r e d i n 0.01 N 1 - t a r t a r i c a c i d . From t h i s s t u d y , t h e p a r t i c l e / m e d i u m (P/M) r a t i o s were c a l c u l a t e d f o r each sample, b l a n k v a l u e s were s u b t r a c t e d , and t h e means _+ s t a n d a r d e r r o r s were d e t e r m i n e d f o r seven such d e t e r -m i n a t i o n s . The r e s u l t s a r e e x p r e s s e d i n T a b l e VI and F i g u r e 3. T a b l e VI A c c u m u l a t i o n o f N o r a d r e n a l i n e i n t h e Absence o f Drug Treatment ( V a l u e s a r e e x p r e s s e d as P a r t i c l e / M e d i u m r a t i o s . Each v a l u e r e p r e s e n t s t h e mean +_ S.E.M. o f seven d e t e r m i n a t i o n s . ) NA c o n c e n t r a - P/M r a t i o t i o n 2 min. 5 min. 10 min. 20 min. 40 min. 0.05 jiM 4.27 + 6.17 + 7.92 + 10.08 + 8.19 + 0. 38 0. 35 1.10 0. 58 0. 98 0. 27 / iM 4.32 + 5.98 + 6.80 + 8.90 + 6.43 + 0. 80 0. 74 0.47 0.81 0.85 0. 70 JJM 3.70 + 4. 76 + 5.49 + 6.75 + 4.88 + 0. 69 0.51 0. 53 0. 62 0. 68 2.0 uM 2.49 + 3.03 + 3.31 + 3.04 + 2.79 + 0. 62 0. 36 0.48 0. 38 0. 57 The graph shows t h a t t h e r e i s an i n c r e a s e i n a c c u m u l a t i o n w i t h t i m e w i t h peak upta k e o c c u r r i n g a f t e r a p p r o x i m a t e l y 20 m i n u t e s i n c u b a t i o n . f o r a l l f o u r c o n c e n t r a t i o n s of NA. F o l l o w i n g t h i s , r - 3 0 0 ^ ^ <M OIXVH wr/ a a x o a H H o o •a F i g u r e 3. A c c u m u l a t i o n o f H - n o r a d r e n a l i n e i n t h e absence o f d r u g t r e a t m e n t , e m p l o y i n g f o u r c o n c e n t r a t i o n s o f c a t e c h o l -amine: 0.05 u^M ( O ), 0.27 uM ( A ) # 0. 70 uM ( • ), and 2.0 ( A ). Each p o i n t r e p r e s e n t s t h e mean + s t a n d a r d e r r o r o f t h r e e d e t e r m i n a t i o n s . — 45 a g r a d u a l d e c l i n e i n t h e l e v e l o f u p t a k e t a k e s p l a c e . F i g u r e 3 a l s o shows t h a t t h e l o w e s t c o n c e n t r a t i o n o f NA used, 0.05 juM, p r o d u c e s the h i g h e s t P/M r a t i o s . Because d e c r e a s i n g r a t i o s w i t h i n c r e a s i n g s u b s t r a t e c o n c e n t r a t i o n s i n d i c a t e t h a t t h e u p t a k e mechanism i s becoming s a t u r a t e d , i t was d e c i d e d t o use 0.05 jaM NA f o r a l l f u r t h e r e x p e r i m e n t s s i n c e t h i s would d e m o n s t r a t e t h e g r e a t e s t changes i n a c c u m u l a t i o n w i t h v a r y i n g c o n d i t i o n s and t h u s t h e g r e a t e s t d i f f e r e n t i a t i o n between t r e a t m e n t s . An e x p e r i m e n t was a l s o p e r f o r m e d i n wh i c h c o n c e n t r a t i o n s o f NA above 2.0 ;uM were used (5, 10, and 20 ;JM), and i n c u b a ^ t i o n s were c o n t i n u e d f o r 2, 5, 10, 15, 25, and 40 m i n u t e s . The P/M r a t i o s a t t h e v a r i o u s t i m e s d i d n o t d i f f e r from one a n o t h e r f o r a l l t h r e e c o n c e n t r a t i o n s o f c a t e c h o l a m i n e and t h e s e were a l l c c o m p a r a b l e w i t h t h e r a t i o f o r t h e b l a n k , i n d i c a t i n g t h a t t h e system was s a t u r a t e d by 5.0 ;uM NA. I n o r d e r t o more p r e c i s e l y l o c a t e t h e t i m e o f maximum u p t a k e , more f r e q u e n t t i m e i n t e r v a l s were chosen i n t h e v i c i n -i t y o f the 20 m i n u t e s i n c u b a t i o n t i m e . The homogenate was i n -c u b a t e d w i t h 0.05 p.M NA and t h e r e s u l t s a r e e x p r e s s e d i n F i g -u r e 4. From t h i s c u r v e , t h e i n c u b a t i o n t i m e f o r peak accumu-l a t i o n t o o c c u r was shown t o be 25 m i n u t e s . T h e r e f o r e t h i s t i m e was chosen f o r l a t e r e x p e r i m e n t s on d r u g e f f e c t s on NA r e l e a s e , s i n c e p r e i n c u b a t i n g t h e homogenate w i t h t h e NA f o r t h i s p e r i o d o f t i m e s h o u l d a l l o w maximum l o a d i n g o f the synaptosomes b e f o r e t h e d r u g i s added. I n c l u d e d i n t h i s l a s t e x p e r i m e n t was a s t u d y o f t h e e f f e c t s o f b o t h t i m e and NA c o n c e n t r a t i o n on the P/M r a t i o f o r t h e b o i l e d homogenate b l a n k . N e i t h e r o f t h e s e p a r a m e t e r s was found t o have an e f f e c t on t h e b l a n k v a l u e s , w h i c h remained e s s e n t i a l l y c o n s t a n t . Thus, any c o n v e n i e n t i n -c u b a t i o n p e r i o d c o u l d be chosen f o r t h e b l a n k tube i n l a t e r t i m e - e f f e c t s t u d i e s . 7 E f f e c t s o f Drugs on t h e E f f l u x o f N o r a d r e n a l i n e : T i m e - E f f e c t  S t u d i e s As s t a t e d above, 25 m i n u t e s was chosen as t h e t i m e f o r p r e -3 i n c u b a t i o n o f H-NA w i t h t h e homogenate b e f o r e a d d i n g t h e d r u g so t h a t t h e synaptosomes would be l o a d e d w i t h t r i t i a t e d amine and t h e r e f o r e t h e e f f e c t s o f t h e d r u g s on r e l e a s e c o u l d be measured. The c o n c e n t r a t i o n o f d r u g s t o be used was a r b i t r a r --4 i l y chosen as 10 M and was f o u n d t o be s a t i s f a c t o r y f o r d i f -f e r e n t i a t i n g between the e f f e c t s o f t h e t w e l v e d r u g s . Once the d r u g was added t o t h e medium, i n c u b a t i o n s were c o n t i n u e d f o r 2, 5, 10, 20, and 40 m i n u t e s i n o r d e r t o show the t i m e c o u r s e o f t h e d r u g e f f e c t s . C o n t r o l t u b e s ( t o which no drugs had been added) were i n c l u d e d f o r each i n c u b a t i o n t i m e and P/M r a t i o s f o r t h e sample ( t r e a t e d ) t u b e s were compared t o r a t i o s from t h e c o n t r o l t u b e s ((a tfter f i r s t s u b t r a c t i n g the b l a n k v a l u e ) and e x p r e s s e d as "% R e l e a s e o f NA". T a b l e V I I g i v e s t h e means - s t a n d a r d e r r o r s f o r t r i p l i c a t e d e t e r m i n a t i o n s o f t h e p e r c e n t o f NA r e l e a s e d by t h e t w e l v e d r u g s a t t h e v a r -i o u s i n c u b a t i o n t i m e s . These d a t a a r e e x p r e s s e d g r a p h i c a l l y i n F i g u r e s 5a, 5b, 5c, and 5d. A l l o f t h e d r u g s which were t e s t e d p r o d u c e d an i n c r e a s e d e f f l u x o f NA w i t h t i m e o v e r t h e 47 T a b l e V I I The % R g l e a s e of N o r a d r e n a l i n e by 1 0 ~ 4 M Drugs a t V a r i o u s I n c u -b a t i o n Times (Each f i g u r e r e p r e s e n t s the mean - s t a n d a r d e r r o r o f t h r e e d e t e r m i n a t i o n s ) % Release i " ' -Of ; tNoradrenal ine D ^ u g 2 m i n . 5 m i n . 10 m i n . 20 m i n . 40 m i n . A m i t r i p t y l i n e 22.65 47 .02 65.16 79.41 92.53 + 3 .47 + i ; .40 + 3. 25 + 3. 23 + 0 .98 C h l o r p h e n i r - 13 .34 21.35 26.10 42 .29 52.46 amine + 4 .04 + 2.24 + 1.25 + 3.93 + 5.65 C o c a i n e 5.93 16 .24 22.07 36.01 42.13 + 4 .28 + 1.22 + 2.43 + 1 .91 + 0.79 D e s i p r a m i n e 17.63 40 .36 54.15 71 .79 91.73 + 2.29 + 1 .29 + 4 .53 + 3 .17 + 4 .64 D i p h e n h y d r a - 9 .11 23 .86 25.00 37.80 50.43 mine + 3.79 + 7.83 + 2.39 + 2.63 + 1.75 Imipramine 18 .00 44 .44 57.57 72.86 82 .81 + 4 .38 + 2.40 + 1 .88 + 3.39 + 2.08 N o r t r i p t y l i n e 17.05 42 .06 59.09 81.50 95.92 + 5 .46 + 3.02 + 4 .18 + 1 .09 + 1 .06 Phenindamine 6.30 24.37 39.40 58.31 72.13 + _2.12 + 4 .47 + 4 .89 + 1 .44 + 1 .92 P r o m e t h a z i n e 12 .66 44 .37 64.54 75.97 90.65 + 3.50 + 9 .49 + 7.83 + 1 .87 + 4 .68 T r i p e l e n n a - 9.78 26.20 26.18 38.19 48 .37 mine + 6.55 + 11 .56 + 2.15 + 1.77 + 1 .31 T r i p r o l i d i n e 20.44 18 .04 25.92 43 .16 48 .39 + 8 .29 + 1 .50 + 0.94 + 5.01 + 1.65 T y r a m i n e 32.75 43 .52 59.73 74.62 86 .31 + 5.95 + 2.87 + 1.23 + 2.29 + 0.85 48 100H Incubat ion T i m e (minutes) Figure 5a. The time course of e f f l u x of noradrenaline from ra brain homogenate following incubation with 10 - M amitrip t y l i n e ( • ) , imipramine ( • ) , and tripelennamine ( A ) . Each point represents the mean + standard error of t r i p l i cate determinations. 4 9 10O| Incubation Time (minutes) F i g u r e 5b. The time c o u r s e of e f f l u x o f n o r a d r e n a l i n e from r a t b r a i n homogenate f o l l o w i n g i n c u b a t i o n w i t h I O - M prometh-a z i n e ( • ) , c h l o r p h e n i r a m i n e ( • ) , and c o c a i n e ( A ) . Each p o i n t r e p r e s e n t s the mean + s t a n d a r d e r r o r of t r i p l i c a t e d e t e r m i n a t i o n s . 50 0 2 5 10 20 40 I n c u b a t i o n Time (minutes) Figure 5 c The time course of e f f l u x of noradrenaline from r a t brain homogenate following incubation with 10 - M tyramine ( • ) , phenindamine ( • ) , and t r i p r o l i d i n e ( A ) . Each point represents the mean + standard error of t r i p l i c a t e deter-minations. 51 I n c u b a t i o n Time (minutes) F i g u r e 5 d. The t i m e c o u r s e o f e f f l u x o f n o r a d r e n a l i n e from r a t b r a i n homogenate f o l l o w i n g i n c u b a t i o n w i t h 10 M n o r t r i p -t y l i n e ( • ), d e s i p r a m i n e ( • ), and d i p h e n h y d r a m i n e ( A ). Each p o i n t r e p r e s e n t s t h e mean ± s t a n d a r d e r r o r o f t r i p l i -c a t e d e t e r m i n a t i o n s . control l e v e l s . Equimolar doses of tyramine, the four t r i c y -c l i c antidepressants, and promethazine were the most e f f i c a -cious drugs and d i d not d i f f e r s i g n i f i c a n t l y from one another (P<0.05) with respect to';their e f f e c t s on release when compared a f t e r 10, 20, or 40 minutes of incubation (Figure 6). S i m i l a r -l y , at t h i s dose, cocaine, tripelennamine, t r i p r o l i d i n e , c h l o r -pheniramine and diphenhydramine appeared to be e q u i e f f e c t i v e at incubations of 10 minutes or longer but produced considerably less release than d i d the f i r s t group. Phenindamine showed an a c t i v i t y which was intermediate and s i g n i f i c a n t l y d i f f e r e n t from the other two groups of drugs at both 20 and 40 minutes. Shorter incubations did not demonstrate t h i s d i f f e r e n t i a t i o n of drug e f f e c t s on NA e f f l u x . E f f e c t s of Drugs on the E f f l u x of Noradrenaline: Concentration- E f f e c t Studies Therefore, from the above experiment, an incubation time of 20 minutes was chosen for the study of drug concentration 3 e f f e c t s on H-NA release since at t h i s time good r e s o l u t i o n between drug e f f e c t s was obtained, as well as a l e v e l l i n g of the response, approaching maximum e f f l u x . The compounds were a l l — 8 —3 tested i n s i x concentrations from 10 M to 10 M. As i n the time-response studies, the e f f e c t s of each drug were compared to a control (which represented only spontaneous e f f l u x a f t e r 45 minutes t o t a l incubation) and were expressed as "% Release of NA". Table VIII shows the means - standard errors f o r at 3 l e a s t three determinations of percent release of H-NA by the 53 F i g u r e 6. R e l a t i v e e f f i c a c y , i n d e c r e a s i n g o r d e r , f o r r e l e a s e of n o r a d r e n a l i n e from r a t b r a i n homogenate a f t e r i n c u b a -t i o n f o r 20 minutes w i t h equimolar c o n c e n t r a t i o n s of the twelve drugs. B r a c k e t s connect those compounds which do not d i f f e r s i g n i f i c a n t l y from one another. N o r t r i p t y l i n e A m i t r i p t y l i n e Promethazine Tyramine Imipramine Desipramine Phenindamine T r i p r o l i d i n e C h l o r p h e n i r a m i n e T r i p e l e n n a m i n e Diphenhydramine Cocaine T a b l e V I I I The % R e l e a s e o f N o r a d r e n a l i n e a f t e r Twenty M i n u t e s I n c u b a t i o n w i t h V a r y i n g Concen-t r a t i o n s o f t h e T e s t Drugs. (Each v a l u e r e p r e s e n t s the mean _+ s t a n d a r d e r r o r o f t h r e e d e t e r m i n a t i o n s . ) _ o _ 7 %DReareSSeC6£tNbraarenaline . Drug 10 M 10 M 1 0 - e ) M 10. M 10 M 10 M A m i t r i p t y l i n e 4.15 11.02 16.47 25.04 79.41 107.67 + 3.49 + 3.22 + 5.69 + 6.08 + 3.23 + 3.31 C h l o r p h e n i r - 2.64 14.50 19.74 31.70 42.29 72.76 amine + 0.88 + 6.97 + 5.94 + 4.70 + 3.93 + 2.44 C o c a i n e 7.20 4.20 25.34 29.38 36.01 36.01 + 3.66 + 4.48 + 5.71 + 3.36 + 1.91 + 2.06 D e s i p r a m i n e 14.72 19.95 13.27 11.41 71 79 107.18 + 4 . 0 3 + 1 . 9 5 +1.32 +5.54 +3.17 + 4 . 6 6 D i p h e n h y d r a - 12. 70 6.89 19.43 33.42 37.-80 83. 38 mine + 4.32 + 2.25 + 1.56 + 6.39 + 2.63 + 5.35 Imi p r a m i n e 2.96 9.38 5.26 8.83 72.86 108.95 + 4.19 + 2.89 + 3.58 + 4.20 + 3.39 + 3.81 N o r t r i p t y l - 13.88 12.86 22.42 15.58 81.50 108.80 i n e + 6.41 + 4.66 + 6.05 + 7.69 + 1.09 + 4.47 ( c o n t i n u e d ) T a b l e V I I I ( c o n t i n u e d ) Drug _„ % Rel'eaSenoiinNor.adipenaline 3 10 M 10 M 10 M 10 M . . . . 10. M 10 M Phenindamine -7.53 13.73 7.28 24.78 58.31 109.84 + 4.44 + 5.41 + 2.04 + 1.71 + 1.44 + 5.24 P r o m e t h a z i n e 4.51 15.37 9.09 11.24 75.97 103.08 + 2. 36 + 4. 38 + 6. 35 •+ 1. 93 + 1.87 + 4.52 T r i p e l e n n a - 14.03 25.51 32.36 34-31 38.19 62.31 mine + 4.41 + 3.99 + 4.54 + 3.07 + 1.77 + 5.08 T r i p r o l i d i n e -0.82 0.46 4.12 9.95 43.16 92.52 + 2.02 + 0.79 + 3.74 + 4.28 + 5.01 + 5.87 Tyramine 4.80 25. 65 50. 23 69.87 74.-62 72. 32 + 2. 39 + 5. 99 + 3.85 + 0. 74 + 2. 29 + 2.18 t w e l v e compounds a t s i x c o n c e n t r a t i o n s . The d a t a f o r t h e 10 M c o n c e n t r a t i o n was o b t a i n e d from t h e t i m e - r e s p o n s e s t u d i e s ( T a b l e V I I ) . F i g u r e s 7a, 7b, 7c, and 7d a r e p l o t s o f d r u g e f -f e c t (% R e l e a s e o f NA) vs d r u g c o n c e n t r a t i o n i n moles p e r l i t r e o f i n c u b a t i o n medium. From t h e s e g r a p h s i t can be seen t h a t t y r a m i n e was c l e a r l y e f f e c t i v e a t a dose much lo w e r t h a n t h a t o f any o f t h e o t h e r t e s t compounds, a l t h o u g h t h e maximum r e s p o n s e a t t a i n e d w i t h t h i s d r u g ( a p p r o x i m a t e l y 70% r e l e a s e ) was l e s s t h a n t h e peak r e s p o n s e s o f s e v e r a l of t h e o t h e r d r u g s ( t h e t r i -c y c l i c a n t i d e p r e s s a n t s , p r o m e t h a z i n e , and phenindamine) w h i c h p r o d u c e d e s s e n t i a l l y 100% r e l e a s e . The r e m a i n i n g compounds a l s o f a i l e d t o b r i n g about c o m p l e t e r e l e a s e i n the c o n c e n t r a t i o n s employed, w i t h t r i p e l e n n a m i n e and c o c a i n e p r o d u c i n g t h e l e a s t , e f f e c t . C o c a i n e , i n f a c t , f a i l e d t o a c h i e v e 50% NA r e l e a s e , even a t m i l l i m o l a r c o n c e n t r a t i o n s . These r e s u l t s were used t o compare the r e l a t i v e p o t e n c i e s o f t h e t w e l v e d r u g s , by c a l c u l a t i n g t h e c o n c e n t r a t i o n r e q u i r e d t o p r o duce a h a l f - m a x i m a l e f f e c t (EC50, o r c o n c e n t r a t i o n f o r 50% e f f e c t ) on e f f l u x o f H-NA f o r each compound. These v a l u e s , shown i n T a b l e IX, were d e r i v e d as t h e mean + s t a n d a r d e r r o r o f t h e i n d i v i d u a l EC50 v a l u e s from each of t h e t h r e e d e t e r m i n a -t i o n s . These r e s u l t s c o r r e s p o n d c l o s e l y t o t h e EC50 v a l u e s d e r i v e d from F i g u r e s i7a t o fid. The compounds i n T a b l e IX a r e l i s t e d a c c o r d i n g t o p o t e n c y i n d e s c e n d i n g o r d e r , t y r a m i n e b e i n g o u t s t a n d i n g as t h e most p o t e n t o f t h e t w e l v e d r u g s and c o c a i n e showing t h e l e a s t e f f e c t . F o r t h o s e compounds c o n n e c t e d by b r a c k e t s , t h e EC50 v a l u e s do n o t d i f f e r s i g n i f i c a n t l y from one igure 7a. The e f f e c t of varying concentrations of amitrip-t y l i n e ( • ^, imipramine ( • ) , and tripelennamine ( A ) on e f f l u x of H-noradrenaline from r a t b r a i n homogenate a f t e r incubation for 20 minutes. Data points represent the means + standard errors from three determinations. 58 8 6 T 5 4 - L o g Concentration (molar) F i g u r e 7b. The e f f e c t of v a r y i n g c o n c e n t r a t i o n s of prometha-z i n e c h l o r p h e n i r a m i n e ( • ) , and c o c a i n e ( A ) , on e f -f l u x of H - n o r a d r e n a l i n e from r a t b r a i n homogenate a f t e r i n c u b a t i o n f o r 20 minutes. Data p o i n t s r e p r e s e n t the means + s t a n d a r d e r r o r s from t h r e e d e t e r m i n a t i o n s . Figure 7c. The e f f e c t of varying concentrations of tyramine (•3*, phenindamine ( • ) , and t r i p r o l i d i n e ( • ) on e f f l u x of H-noradrenaline from r a t brain homogenate a f t e r incu-bation for 20 minutes. Data points represent the means + standard errors from three determinations. 100J 8 C H ° 60«| CD CO CO 0) 0 CC 4 C H 20« OH 60 8 4 3 ™ - L o g Concentration (molar) Figure 7d. The e f f e c t of varying concentrations of n o r t r i p -t y l i n e ( • ), desipramine ( • ), and diphenhydramine ( A ) on e f f l u x of ^-noradrenaline from r a t b r a i n homogenate aft e r incubation for 20 minutes. Data points represent the means + standard errors from three determinations. 61 Table IX The Relative Potencies, i n Decreasing Order, for Drugs Produc-ing E f f l u x of T r i t i a t e d Noradrenaline from Rat Brain Homogenate aft e r Incubation for Twenty Minutes (The concentration of each drug required to produce a h a l f -maximal e f f e c t (EC50) i s included, shown as the mean _+ standard error of three experiments. Brackets connect those compounds which do not d i f f e r s i g n i f i c a n t l y from one another.) Drug EC 50 (juM) Tyramine A m i t r i p t y l i n e N o r t r i p t y l i n e Promethazine Desipramine Imipramine Phenindamine T r i p r o l i d i n e Chlorpheniramine Diphenhydramine Tripelennamine Cocaine 1.13 + 0. 35 29. 7 + 6. 1 32. 9 + 4. 4 41.0 2. 5 44. 0 + 5. 7 44. 5 + 6 r 3 57.1 + 3. 3 146 + 31 184 + 50 192 + 28 381 + 131 >1000 another (P<0.05). A f t e r tyramine, the next s i x drugs, shown as being eguipotent, are the same as those which produced the maximum rel e a s i n g e f f e c t i n the time-response studies. The four remaining antihistamines, besides having a lower potency, were less e f f i c a c i o u s . E f f e c t s of Drugs on the I n h i b i t i o n of Noradrenaline Uptake:  Time-Effect Studies In order to investigate the i n h i b i t o r y e f f e c t s of drugs on the uptake or accumulation of NA, i t i s necessary to p r e i n -cubate the compound with the r a t brain homogenate p r i o r to ad-d i t i o n of the t r i t i a t e d amine to enable the drug to i n t e r a c t with the membrane or amine uptake mechanism. The drugs were - 4 t r i e d i n i t i a l l y at a concentration of 10 M as i n the studies of release, but t h i s gave almost t o t a l i n h i b i t i o n of uptake. Therefore 10 ^ M was chosen as the dose to be used. A preincubation time of f i v e minutes had been employed by other researchers (Coyle and Snyder, 1969a; Horn, Coyle and Snyder, 1971; Horn and Snyder, 1972; Horn, 1973) and seemed a s u i t a b l e time for t h i s experiment. In addition, a preliminary _ 7 study using 10 M cocaine preincubated with homogenate for 0, 5, and 25 minutes before adding the NA, showed no diff e r e n c e i n the i n h i b i t o r y e f f e c t of cocaine with the three separate pre incubation times. 3 After the H-catecholamme was added to the mixture, incu-bations were continued for 2, 5, 10, 20, 40, and 60 minutes to demonstrate the e f f e c t of time on the a b i l i t y of the various d r u g s t o i n h i b i t NA a c c u m u l a t i o n . Each a s s a y measured t h e up-t a k e o f NA a t one p a r t i c u l a r i n c u b a t i o n t i m e and i n c l u d e d s i x of t h e d r u g s , a c o n t r o l and a b l a n k , as p r e v i o u s l y d e s c r i b e d . A f t e r b l a n k r a t i o s were s u b t r a c t e d , sample p a r t i c l e - m e d i u m r a -t i o s were a g a i n compared t o t h e c o n t r o l (which r e p r e s e n t e d 0% i n h i b i t i o n o f u p t a k e ) and r e s u l t s were e x p r e s s e d as "% I n h i b -i t i o n o f NA Uptake" f o r each o f t h e compounds. As i n t h e r e -l e a s e s t u d i e s , t r i p l i c a t e d e t e r m i n a t i o n s were c a r r i e d o u t and t h e r e s u l t s a r e e x p r e s s e d as mean +_ s t a n d a r d e r r o r o f t h e mean ( T a b l e X ) . These v a l u e s f o r p e r c e n t i n h i b i t i o n o f u p t a k e were p l o t t e d a g a i n s t t h e v a r i o u s i n c u b a t i o n t i m e s f o r each d r u g as d e p i c t e d i n F i g u r e s 8a, 8b, 8c, and 8d. The graphs show t h a t f o r a l l t h e d r u g s , t h e i n h i b i t i o n of NA a c c u m u l a t i o n was g r e a t e s t w i t h -i n t h e f i r s t t e n m i n u t e s of i n c u b a t i o n , a f t e r which t h e e f f e c t d e c r e a s e d g r a d u a l l y and a t t a i n e d a r e l a t i v e l y s t e a d y s t a t e a f t e r f o r t y m i n u t e s . Comparing r e s p o n s e s a t f o r t y m i n u t e s , ( F i g u r e 9) t y r a m i n e was d e m o n s t r a t e d t o have the g r e a t e s t i n h i b i t i n g e f -f e c t , b u t was n o t s i g n i f i c a n t l y d i f f e r e n t from t r i p e l e n n a m i n e w h i c h , i n t u r n , d i d n o t d i f f e r from c o c a i n e (P<0.05). A s e c -ond group o f compounds, c o n s i s t i n g o f t h e f o u r t r i c y c l i c a n t i -d e p r e s s a n t s and c h l o r p h e n i r a m i n e , appeared t o be e q u i e f f e c t i v e when t e s t e d a t the same dose, b u t were s i g n i f i c a n t l y l e s s e f f i -c a c i o u s t h a n c o c a i n e . Diphenhydramine, phenindamine, t r i p r o l i -d i n e , and p r o m e t h a z i n e had t h e l e a s t i n h i b i t o r y e f f e c t o f t h e t w e l v e d r u g s . Because an i n c u b a t i o n t i m e o f f o r t y m i n u t e s showed a l e v e l l i n g o f t h e r e s p o n s e and a l s o because i t gave a good sep-T a b l e X The % I n h i b i t i o n o f T r i t i a t e d N o r a d r e n a l i n e Uptake by 10 M Drugs at V a r i o u s Incu-b a t i o n T i m e s . (Each f i g u r e r e p r e s e n t s the mean + s t a n d a r d e r r o r of t h r e e d e t e r m i n -a t i o n s . ) Drug % InMbi- t ioho 'ofTNo.radren 'a l ' ine Uptake 2 m i n . 5 m i n . 10 m i n . . 20. m i n . . . 40 min . 60- min. A m i t r i p t y l i n e 48 .22 29.75 41.83 32.71 28.80 28.62 + 4 .25 + 3.70 + 2.86 + 2.45 + 4 .28 + 1 .74 C h l o r p h e n i r - 72 .19 50.74 56.84 41.00 31.05 30.94 amine + 2 .79 + 2.89 + 2.36 + 3.91 + 2.68 + 3.97 C o c a i n e 94.71 81 .09 , 77.91 68.73 56.55 46 .05 + 6.29 + 1 .66 + 0.90 + 2.31 + 4 .01 + 1.34 D e s i p r a m i n e 48 .25 42.32 37.18 35.74 25.60 37.98 + 3.22 + 5 .97 + 4 .93 + 2.65 + 2.44 + 3.23 D i p h e n h y d r a - 45 .32 37 .49 33.74 31.79 12.83 23.69 mine + 4 .10 + 6.33 + 2.69 + 3.01 + 1 .80 + 4 .07 Imipramine 45 .58 41.42 48.50 33.40 25.18 25.57 + 8. 70 +-:5.46 + 4. 93 + 2.31 + 6. 36 + 5. 36 N o r t r i p t y l - . 57 .07 37.80 40. 66 42 .19 29.02 39. 66 i n e + 5. 55 + 5.80 + 9.02 + 1. 56 + 2.82 + 0. 70 ( c o n t i n u e d ) T a b l e X ( c o n t i n u e d ) Drug % I n h i b i - t i b n t 6 - f i ' . N © 2 m i n . 5 m i n . 10 m i n . 20 m i n . 40. m i n . 60 m i n . • - = ~-"' -' - -= -•Wn t- a K e Phenindamine 37.63 24 .69 33.05 27.82 8.90 13 .59 ± 4 .66 + 1.14 + 0.85 + 1.46 + 3.14 + 1 .47 P r o m e t h a z i n e 3 .89 19 .58 15.22 18.10 1.17 6.79 + 9.73 + 3.18 + 3.59 + 3.80 + 4 .59 + 5.71 T r i p e l e n n a - -110.25 88 .65 89.59 82.68 67.98 64.85 mine + 5 .67 + 3.41 + 1 .10 + 0.64 + 2.45 + 2.86 T r i p r o l i d i n e 20.62 12 .27 16.22 4 .18 4 .56 12 .34 + 2.02 + 5.04 + 6.04 + 5.11 + 3.89 + 5.18 T y r a m i n e 100.90 84 .52 84.62 77.22 74.62 73.86 + 3.52 + 5:71 + 2.38 + 1.96 + 1.57 + 1 .09 66 Figure 8a . The time course of i n h i b i t i o n o f , H-noradrenaline uptake in t o r a t brain homogenate by 10 M tripelennamine ( • ), a m i t r i p t y l i n e ( • ), and imipramine ( A ) . Each point represents the mean +_ standard error of t r i p l i c a t e deter-minations . 67 Figure 8b. The time course of i n h i b i t i o n o f , H-noradrenaline uptake in t o r a t brain homogenate by 10 M cocaine ( • ), chlorpheniramine ( • ) , and promethazine ( A ) . Each point represents the mean + standard error of t r i p l i c a t e deter-minations. 68 F i g u r e 8c. The t i m e c o u r s e o f i n h i b i t i o n o f , H - n o r a d r e n a l i n e u p t a k e i n t o r a t b r a i n homogenate by 10 M t y r a m i n e ( • ), phenindamine ( • ), and t r i p r o l i d i n e ( A ) „ Each p o i n t r e p -r e s e n t s t h e mean +_ s t a n d a r d e r r o r o f t r i p l i c a t e d e t e r m i n -a t i o n s . 69 savidn NOiuaiHNi % Figure 8 d . The time course of i n h i b i t i o n o f , H-noradrenaline uptake into r a t brain homogenate by 1 0 - M n o r t r i p t y l i n e ( • ) , desipramine ( • ) , and diphenhydramine ( A ) . Each point represents the mean _+ standard error of t r i p l i c a t e determinations. 70 F i g u r e 9. R e l a t i v e e f f e c t i v e n e s s , i n d e c r e a s i n g o r d e r , o f e q u i m o l a r c o n c e n t r a t i o n s o f t h e t e s t compounds f o r i n h i b -i t i o n o f u p t a k e o f t r i t i a t e d n o r a d r e n a l i n e a f t e r f o r t y m i n u t e s i n c u b a t i o n . ( B r a c k e t s c o n n e c t t h o s e compounds w h i c h do n o t d i f f e r s i g n i f i c a n t l y from one a n o t h e r . ) Tyramine T r i p e l e n n a m i n e C o c a i n e C h l o r p h e n i r a m i n e N o r t r i p t y l i n e A m i t r i p t y l i n e I m i p ramine D e s i p r a m i n e Diphenhydramine Phenindamine T r i p r o l i d i n e P r o m e t h a z i n e 7 1 a r a t i o n of groups of drugs which produced e q u i v a l e n t e f f e c t s , t h i s time was chosen f o r the i n v e s t i g a t i o n of the i n f l u e n c e of drug c o n c e n t r a t i o n on uptake i n h i b i t i o n . E f f e c t s of Drugs on the I n h i b i t i o n of N o r a d r e n a l i n e Uptake:  C o n c e n t r a t i o n - E f f e c t S t u d i e s — 8 The twelve drugs were t e s t e d i n c o n c e n t r a t i o n s from 10 -3 M to 10 M as i n the r e l e a s e experiments. With the f i v e min-ute p r e i n c u b a t i o n p e r i o d , P/M r a t i o s f o r the c o n t r o l tube r e p -3 r e s e n t accumulation of H-NA by the synaptosomes a f t e r 45 min-utes i n the absence of an i n h i b i t o r y i n f l u e n c e . Responses to the drugs were t h e r e f o r e expressed r e l a t i v e to t h i s 0% c o n t r o l as "% I n h i b i t i o n of NA Uptake". A minimum of t h r e e determina-t i o n s a t each dose f o r each drug was made and these r e s u l t s were averaged and expressed i n Table XI as the mean +_ s t a n d a r d — 6 e r r o r . Data for..the 10 M c o n c e n t r a t i o n was d e r i v e d , i n p a r t , from the time-response s t u d i e s of i n h i b i t i o n of NA uptake. The r e s u l t s are a l s o r e p r e s e n t e d g r a p h i c a l l y i n F i g u r e s 10a, 10b, 10c, and lOd by p l o t t i n g % I n h i b i t i o n of NA Uptake a g a i n s t Log Drug C o n c e n t r a t i o n ( i n moles per l i t r e ) . The graphs show t h a t a l l the drugs produced e s s e n t i a l l y 100% i n h i b -i t i o n a t the h i g h e s t doses, and demonstrate t h a t tyramine, .1 which was p r e v i o u s l y shown to be a p o t e n t r e l e a s i n g agent, i s a l s o an e f f e c t i v e i n h i b i t o r y agent a t v e r y low doses. In comparison, both c o c a i n e and t r i p e l e n n a m i n e , which had the l e a s t e f f e c t s i n 3 p r o d u c i n g r e l e a s e of H-NA, showed i n h i b i t i o n o f NA uptake a t doses which were comparable to t h a t of tyramine. In o r d e r to T a b l e XI The % I n h i b i t i o n o f T r i t i a t e d N o r a d r e n a l i n e A c c u m u l a t i o n a f t e r F o r t y M i n u t e s I n c u b -a t i o n w i t h V a r y i n g Doses of t h e T e s t Drugs. (Each p o i n t r e p r e s e n t s t h e mean + st a n -d a r d e r r o r o f a minimum o f t h r e e d e t e r m i n a t i o n s . ) Drug 10 8 M 7 % I n h i b i t i o n o f N o r a d r e n a l i n e . U p t a k e 10 M 10 M 10 M 10 M 10 M A m i t r i p t y l -i n e 6. 67 + 4.17 7. 09 + 6. 21 28. 95 + 3.03 59.04 + 1.94 107.07 + 2.58 103.62 + 2.91 C h l o r p h e n i r -amine 5. 88 + 5.83 0. 97 + 2.18 34. 03 + 3.53 91. 65 + 2.01 108.27 + 2.32 108.38 + 2. 78 C o c a i n e 1.88 + 8. 66 13. 73 + 3. 76 58. 98 + 3.74 102.23 + 3.74 108.20 + 2.63 104.59 + 2.66 D e s i p r a m i n e 11.01 23.87 23.21 42.84 102.60 104.47 + 1.48 +4.75 + 2.95 + 4.74 + 3.19 + 1.93 Diphenhydra-mine 1.46 + 4.48 14. 01 + 5.88 16. 65 + 4.03 78. 50 + 0.19 102.16 + 1.80 106.91 + 3.36 Imip r a m i n e 3. 35 + 6.60 9. 75 + 5.46 24. 51 + 4.54 43.48 + 7. 25 106.47 + 1. 29 105.92 + 3.01 N o r t r i p t y l -i n e 0. 09 + 4.11 18.86 + 2.94 26. 82 + 2.97 66. 71 + 2.57 106.25 + 3.02 104.99 + 0.83 ( c o n t i n u e d ) T a b l e XI ( c o n t i n u e d ) Drug o % I n h i b i t i o n of N o r a d r e n a l i n e .Uptake 10 M 10" M 10 M 10 M 10 M 10 M Phenindamine - 7 . 3 4 - 3 . 4 3 12.10 75.15 104.20 107.04 + 6.39 + 2.21 + 3.90 + 2.37 + 2 . 3 3 + 1.75 P r o m e t h a z i n e - 6 . 9 9 - 2 . 3 6 2.96 32.29 99.64 102.13 + 1 .22 + 1 .26 + 3.71 + 2.45 + 4 .21 + 0.32 Tripelennar- : " .. '• 4. 31 11 .12 65.07 98. 28 105.23 106.41 mine + 5 .80 + 4 .75 + 3.38 + 1.95 + 1 .96 + 3 . 5 7 T r i p r o l i d i n e 3.35 - 1 . 3 7 4 .56 40.45 89.94 106.06 + 3.11 + 7.73 + 2.75 + 1 .86 + 2.33 + 2.54 T y r a m i n e 9.56 10 .17 74.85 105.55 109.12 107.11 + 5.63 + 4 .56 + 1.13 + 4 .09 + 2.81 + 2.60 74 - L o g Drug Concentration (M) F i g u r e 10a. The e f f e c t o f v a r y i n g c o n c e n t r a t i o n s o f t r i p e l e n -namine ( • ), a m i t r i p t y l i n e ( • ), and i m i p r a m i n e ( A ) on i n h i b i t i o n o f u p t a k e o f H - n o r a d r e n a l i n e i n t o r a t b r a i n homogenate a f t e r 40 m i n u t e s i n c u b a t i o n . Each p o i n t r e p -r e s e n t s t h e mean +_ s t a n d a r d e r r o r o f a t l e a s t t h r e e d e t -e r m i n a t i o n s . 75 Figure 10 b. The e f f e c t of varying concentrations of cocaine (• ), chlorpheniramine^( • ), and promethazine ( A ) on i n -h i b i t i o n of uptake of H-noradrenaline into r a t brai n hom-ogenate a f t e r 40 minutes incubation. Each point represents the mean + standard error of at l e a s t three determinations. 7 6 o « 80 o fl o ' .fl 60H 4CH 20H o-l i 8 T 7 T 5 I 4 T - L o g D r u g C o n c e n t r a t i o n ( M ) F i g u r e 10c. The e f f e c t o f v a r y i n g c o n c e n t r a t i o n s o f t y r a m i n e ( • ), p h e n i n d a m i n e ^ • ), and t r i p r o l i d i n e ( A ) on i n h i b -i t i o n o f u p t a k e o f H - n o r a d r e n a l i n e i n t o r a t b r a i n homo-genate a f t e r 40 m i n u t e s i n c u b a t i o n . Each p o i n t r e p r e s e n t s the mean + s t a n d a r d e r r o r o f a t l e a s t t h r e e d e t e r m i n a t i o n s . - L o g D r u g C o n c e n t r a t i o n ( M ) gure lOd. The e f f e c t of varying concentrations of diphen-hydramine ( • ), n o r t r i p t y l i n e ( • ), and desipramine ( A ) on i n h i b i t i o n of uptake of H-noradrenaline in t o r a t bra i n homogenate a f t e r 40 minutes incubation. Each point rep-resents the mean + standard error of at l e a s t three deter-minations. 78 compare the r e l a t i v e potencies of the twelve drugs, a s t a t i s -t i c a l analysis (using the t - t e s t for unpaired data) was made of the concentrations necessary to produce 50% i n h i b i t i o n of uptake (IC50), derived from the i n d i v i d u a l experiments. The compounds, l i s t e d i n decreasing order according to r e l a t i v e potency, as well as the i r respective IC50 values are given i n Table XII. As i n the comparison of potencies derived from the time-effect study of uptake i n h i b i t i o n , the comparison of IC50 values shows tyramine to be the most potent i n h i b i t o r but not d i f f e r i n g s i g n i f i c a n t l y from tripelennamine (P<0.05). Simi l a r -l y , the potencies of tripelennamine and cocaine were not s i g -n i f i c a n t l y d i f f e r e n t . Promethazine appeared to be the l e a s t potent of a l l drugs tested i n i n h i b i t i n g the uptake of the t r i t i a t e d amine. However, the compounds did not seem to f a l l i n to any d e f i n i t e groups according to therapeutic c l a s s i f i c a t i o n with respect to t h e i r r e l a t i v e i n h i b i t o r y potencies. F i n a l l y , a comparison of potencies of the twelve drugs with regard to both NA e f f l u x as well as uptake i n h i b i t i o n i s given i n Table XIII i n order to more e a s i l y contrast the two e f f e c t s for each drug. In a l l cases, i n h i b i t i o n of accumulation i s produced at a lower dose than i s release of NA, although tyramine shows the l e a s t d i s p a r i t y i n t h i s respect since i t s IC50 i s only approximately one-half of the EC50. For t r i p e l -ennamine and cocaine, the differences between potencies for NA release and i n h i b i t i o n of uptake are the greatest. 79 Table XII Relative Potencies, i n Decreasing Order, for Drugs Producing I n h i b i t i o n of Uptake of T r i t i a t e d Noradrenaline aft e r Forty Minutes Incubation (The concentration of each drug required to produce a half-max-imal i n h i b i t i o n (IC50) i s also given, representing the mean +_ standard error of three experiments. Brackets connect those drugs which do not d i f f e r s i g n i f i c a n t l y from one another.) Drug EC50 (juM) Tyramine Tripelennamine Cocaine Chlorpheniramine^ Diphenhydramine N o r t r i p t y l i n e Phenindamine A m i t r i p t y l i n e j Desipramine Imipramine T r i p r o l i d i n e Promethazine 0.405 + 0.016 0. 546 + 0. 070 0. 571 + 0.038 1. 75 + 0.18 3.29 + 0. 28 3. 62 + 0. 26 3.85 ± 0. 37 4. 93 + 0.04 11. 3 + 1. 9 15. 6 + 1.0 15. 7 + 1. 3 18. 3 + 0. 5 80 T a b l e X I I I A Comparison o f Drug P o t e n c i e s i n D e c r e a s i n g Order f o r E f f e c t s on B o t h E f f l u x o f N o r a d r e n a l i n e and I n h i b i t i o n o f Uptake o f the C a t e c h o l a m i n e (a c o m p o s i t e o f T a b l e s IX and X I I ) R e l e a s e Uptake I n h i b i t i o n EC50 (uM) Drug Drug IC50 (uM) 1.13 ± 0 . 35 Tyramine Tyramine 0.405 + 0. 016 29. 7 + 6.1 A m i t r i p t y l i n e T r i p e l e n n a m i n e 0. 546 + 0.070 32. 9 + 4.4 N o r t r i p t y l i n e C o c a i n e 0. 571 + 0.038 41.0 + 2. 5 P r o m e t h a z i n e C h l o r p h e n i r a m i n e 1.75 + 0.18 44. 0 + 5. 7 D e s i p r a m i n e Diphenhydramine 3. 29 + 0. 28 44. 5 + 6. 3 Imipramine N o r t r i p t y l i n e 3. 62 + 0. 26 57.1 + 3.3 Phenindamine Phenindamine 3.85 + 0. 37 146 + 31 T r i p r o l i d i n e A m i t r i p t y l i n e 4. 93 + 0.04 184 + 50 C h l o r p h e n i r a m i n e D e s i p r a m i n e 11. 3 + 1. 9 192 + 28 Diphenhydramine Imipramine 15.6 + 1.0 381 + 131 T r i p e l e n n a m i n e T r i p f o l i d i n e 15. 7 + 1.3 >1000 C o c a i n e P r o m e t h a z i n e 18. 3 + 0. 5 81 A C o r r e l a t i o n of Drug E f f e c t s with L i p i d S o l u b i l i t i e s of the  Compounds In order to determine whether a c o r r e l a t i o n e x i s t s be-tween the i n h i b i t i o n of NA uptake and/or release of t r i t i a t e d amine and the l i p i d s o l u b i l i t y of the drug molecule, octanol-water p a r t i t i o n c o e f f i c i e n t s for each compound were ca l c u l a t e d f i r s t , according to the methods of Leo e_t _al. (1971) and are summarized i n Table XIV. Graphs were then p l o t t e d with e i t h e r the logarithm of the EC50 (Figure, 11) or the logarithm of the IC50 (Figure 12) on the ordinate against the logarithm of the octanol/water p a r t i t i o n c o e f f i c i e n t on the abscissa. ZIn Figure H , nine of the twelve compounds gave points which yielded a l i n e a r r e l a t i o n s h i p between the two parameters (r = -0.936) and the slope of the r e s u l t i n g l i n e was c a l c u l a t e d to be -0.531. The value for cocaine could not be p l o t t e d since t h i s drug did not achieve 50% NA release; the value for phenin-damine f e l l to the r i g h t of the l i n e , i n d i c a t i n g that t h i s com-pound had a lower potency than would be expected based on l i p i d s o l u b i l i t y alone; and tyramine showed the opposite e f f e c t since t h i s point f e l l f ar to the l e f t of the l i n e , suggesting that tyramine's pronounced potency was i n no way connected to i t s l i p i d s o l u b i l i t y . With respect to the i n h i b i t i o n of NA uptake (Figure 12), the l i n e a r c o r r e l a t i o n between potency and l i p i d s o l u b i l i t y was not as good (r = -0.624) and more of the points were scat-tered at a distance from the computed l i n e , although the slope (-0.535) was very close to that of Figure l l . Points for only 82 T a b l e XIV L o g a r i t h m s o f t h e O c t a n o l / W a t e r P a r t i t i o n C o e f f i c i e n t s f o r t h e Twelve Drugs (The p a r t i t i o n c o e f f i c i e n t s , d e r i v e d a c c o r d i n g t o methods o u t -l i n e d by Leo ejb al_. (1971), a r e shown +_ " u n c e r t a i n t y u n i t s " w h i c h a r e an a l o g o u s t o s t a n d a r d d e v i a t i o n s . S u p e r s c r i p t s a r e e x p l a i n e d f o l l o w i n g t h e t a b l e , g i v i n g t h e d e r i v a t i o n s o f i n -d i v i d u a l l o g P v a l u e s . ) Drug Log P A m i t r i p t y l i n e 4. 92 + 0. 0 2 a C h l o r p h e n i r a m i n e 3. 88 +_ 0. 1 3 b C o c a i n e 2. 55 + 0. 0 9 C D e s i p r a m i n e 4. 28 + 0. 0 2 a Diphenhydramine 3. 34 + 0. 0 2 a I m i p r a m i n e 4. 62 + 0. 0 2 a N o r t r i p t y l i n e 4. 60 + 0. 0 6 d Phenindamine 6. 12 + 0. 4 7 e P r o m e t h a z i n e 4. 35 + 0. 0 4 f T r i p e l e n n a m i n e 2. 59 + 0. 0 8 g T r i p r o l i d i n e 3. 92 + 0. 0 2 a Tyramine 0. 74 + 0.06 h a. e x p e r i m e n t a l v a l u e , l i s t e d i n T a b l e X V I I o f Leo e t a l . (1971) ( c o n t i n u e d ) 83 Table XIV (continued) b. log P (pyridine) + log P (6-phenylpropyldimethylamine) + [log P (chlorobenzene) - log P (benzene)J + i r(branch) = 0. 65 + 2. 73 + 2.84 - 2.14 + (-0. 20) = 3.88 c. log P (atropine) - [log P (C 6H 5CH 2CH OH) + TT (branch)] + log P (benzene) + log P (acetic acia) = 1.81 - (1.36 -0.20) + 2.14 -0.24 = 2.55 d. log P (ami t r i p t y l i n e ) - \jt (methyl) + TT (branch )} = 4.92 -(0. 52 - 0. 20) = 4. 60 e. log P (carbazole) - *rr(2 double bonds) + log P (benzene) + [log P (N-methylpiperidine) - log P (piperidine)] = 3.29 - 2(-0.30) + 2.14 + (0.94 - 0.85) = 6.12 f. log P (promazine) +TT(branch) = 4.55 + (-0. 20) = 4. 35 g. log P (N,N,N' ,N'-tetramethylethylenediamine) - Tr(methyl) + log P (benzene) + log P (pyridine) = 0.30 - 0.50 + 2.14 + 0.65 = 2.59 h. log P (2-phenylethylamine) + ["log P (phenol) - log P (benz-ene )J = 1.41 + 1.47 - 2.14 = 0.74 igure 11. The r e l a t i o n s h i p between drug potency with respect to e f f e c t s on e f f l u x of H-noradrenaline (EC50) and the l i p i d s o l u b i l i t y of the compound (calcula t e d as the loga-rithm of the octanol/water p a r t i t i o n c o e f f i c i e n t ) . The c o r r e l a t i o n c o e f f i c i e n t for the curve was -0.936. Figure 12. The r e l a t i o n s h i p between drug potency with respect to i n h i b i t o r y e f f e c t s on H-noradrenaline uptake (IC50) and the l i p i d s o l u b i l i t y of the compound (calculated as the logarithm of the octanol/water p a r t i t i o n c o e f f i c i e n t ) . The c o r r e l a t i o n c o e f f i c i e n t f o r t h i s curve was -0.624. 86 s i x of the drugs (promethazine, t r i p r o l i d i n e , and the four t r i c y c l i c antidepressants) were used i n the c a l c u l a t i o n s . Again, phenindamine seemed to exhi b i t a lower potency than i t s p a r t i t i o n c o e f f i c i e n t would pr e d i c t whereas the potent e f f e c t of tyramine did not r e f l e c t i t s low l i p i d s o l u b i l i t y . In con-t r a s t t o - t h e i r e f f e c t s i n r e l e a s i n g NA, both tripelennamine and cocaine gave points which f e l l f ar to the l e f t of the c a l c u l -ated l i n e , i n d i c a t i n g that these compounds were acting to i n -h i b i t NA uptake through a mechanism (possibly the same as for tyramine) which did not depend on l i p i d s o l u b i l i t y . C o r r e l -ations between i n h i b i t o r y potency and l i p i d s o l u b i l i t y for d i -phenhydramine and chlorpheniramine also appeared to deviate from the ca l c u l a t e d l i n e a r r e l a t i o n s h i p ; however the discrep-ancy was not as great as with cocaine, tyramine, or t r i p e l e n -namine, po s s i b l y due to a complex mode of action. 87 DISCUSSION In an attempt to d i f f e r e n t i a t e between drug e f f e c t s on blockade of NA uptake and release of the catecholamine, the r e s u l t s of t h i s study have shown that although many substances appear to a f f e c t both processes there are s i g n i f i c a n t dose-de-pendent differences i n t h e i r a c t i v i t i e s . This observation, along with the c o r r e l a t i o n of potency with p a r t i t i o n c o e f f i c i e n t for some of the drugs, would imply that separation of drug ef-fe c t s on NA uptake i n h i b i t i o n and NA release can be achieved but that t h e i r actions cannot be explained by only one mechan-ism. These r e s u l t s also confirmed the observations of many other researchers that tyramine i s extremely e f f e c t i v e both i n i n h i b i t i n g neuronal uptake and i n producing release of NA from nerve endings. 3 The i n i t i a l studies of H-NA uptake i n the absence of drugs show that the time course of t h i s uptake compares well with that demonstrated by Snyder and Coyle (1969) i n which the rate of uptake declined r a p i d l y a f t e r ten minutes incubation time. However, i n t h e i r experiment, the P/M r a t i o s d i d not reach a maximum after twenty minutes but instead showed an i n -crease up to f o r t y minutes. Our r e s u l t s could be explained by the observation that control r a t i o s i n the early experiments also declined as the incubation time increased beyond twenty minutes, c o r r e l a t i n g with the age of the prepared homogenate. This occurred because the assay techniques had not yet been per-fected; when i t was discovered that the a c t i v i t y of the homo-88 genate decreased during the day even though i t was stored i n an ice-bath, the procedure was changed so that a fresh homo-genate was prepared immediately before each incubation. When t h i s was done, the P/M r a t i o s d i d not reach a maximum but i n -stead continued to increase beyond the twenty minute incuba-t i o n time. The actual P/M r a t i o s which we obtained were also compar-able to those of Snyder and Coyle (1969) although these authors showed that there were marked regional differences i n t h i s res-pect, with r a t i o s i n s t r i a t a l t i s s u e being approximately ten-f o l d higher than those i n other brain areas. Since whole brain homogenate was used i n our study, only a general comparison can be made. As i n the k i n e t i c study, we also showed that P/M 3 . r a t i o s for H-NA uptake decreased with increasing amine concen-t r a t i o n , i n d i c a t i n g a saturation of the uptake process. I n i t -i a l l y , only 0°G-zero-time blanks and zero-tissue blanks were used. As i n the study by Snyder and Coyle, at zero time the P/M r a t i o s always exceeded unity, i n d i c a t i n g a s i g n i f i c a n t up-take of amine i n the cold. Later, these two blanks were sub-s t i t u t e d by a si n g l e b o i l e d homogenate blank since t h i s would better represent non-specific binding than using a zero-tissue blank. As well, i t would replace the 0°-zero-time blank since the b o i l e d homogenate blank gave c o n s i s t e n t l y higher P/M r a t i o s . 3 Once the time course for H-NA uptake had been established for the present system, the e f f e c t s of the various drugs on 3 H-NA e f f l u x showed that a l l drugs tested produced an increased release with time as compared to the respective control l e v e l s . 89 That the apparent release produced by these drugs i s due simp-l y to i n h i b i t i o n of uptake i s u n l i k e l y since a drug such as co-caine, which i s regarded as a potent i n h i b i t o r of neuronal up-take, showed the l e a s t release. However, i t i s obvious that both release of NA and i n h i b i t i o n of amine uptake may be af-fected simultaneously by some of the drugs (since there i s no known way of i n a c t i v a t i n g one process completely without i n -t e r f e r i n g with the other) and therefore i t i s impossible to assess the exact contribution of uptake blockade to the meas-ured e f f e c t on NA release. Concentration-effect studies on release i l l u s t r a t e d that tyramine was e f f e c t i v e at producing NA release at a much lower dose than for any of the other drugs. The apparent l e v e l l i n g of t h i s curve with high doses of tyramine could be explained by conversion of tyramine to octopamine which could replace some of the noradrenaline normally released or, a l t e r n a t i v e l y , 3 by competition between tyramine with the H-NA for postulated c a r r i e r s i t e s f o r e f f l u x , causing a decline i n e f f l u x of the catecholamine as suggested by Paton (1973). On the other hand, I n h i b i t i o n of NA uptake alone could not explain t h i s decline 3 since a reduction i n H-NA accumulation by t h i s process would be manifested as an increase rather than a decrease i n apparent e f f l u x . Tyramine was the only drug studied which displayed a very potent r e l e a s i n g a b i l i t y but an extremely low octanol/water p a r t i t i o n c o e f f i c i e n t (Figure 11). The lack of r e l a t i o n s h i p between these two properties of tyramine suggests that t h i s substance acts s p e c i f i c a l l y on the amine uptake process of the 90 neuronal membrane through i t s s t r u c t u r a l resemblance to NA, rather than depending on i t s l i p i d s o l u b i l i t y . This i s consis-tent with r e s u l t s from other experiments which have shown that tyramine, as well as being a potent i n h i b i t o r of the NA uptake system, i s also accumulated by a mechanism which i s s e n s i t i v e to both cocaine and ouabain (Iversen, 1971b). In addition, tyramine appears to be taken up by i n t r a c e l l u l a r storage par-t i c l e s , from which i t s t o i c h i o m e t r i c a l l y displaces endogenous catecholamine. This has been demonstrated i n the adrenal med-u l l a (Schumann and Philippu, 1962) and i t i s believed that par-t i c l e s i n adrenergic neurons behave s i m i l a r l y . Tyramine also i n h i b i t s NA uptake into the medullary storage p a r t i c l e s (Carls-son e_t a^l. , 1963). Neither cocaine nor tripelennamine demonstrated any apprec-3 i a b l e release of H-NA, even at the highest doses. Similar re-s u l t s with cocaine were observed by Paton (1973) who i n t e r p r e t -ed them as being pos s i b l y due to a concomitant blockade of both the neuronal uptake process as well as the e f f l u x system, so that no appreciable net release would occur. However, Paton does not o f f e r an explanation of how cocaine (an i n h i b i t o r of NA uptake which does not appear to act as a substrate for the uptake process and therefore should not be accumulated i n the nerve ending) exerts i t s e f f e c t on e f f l u x , although he rules out a non-specific l o c a l anesthetic e f f e c t . In support of Paton's findings, Azarro and coworkers (1974) also provided evidence that cocaine did not i n h i b i t spontaneous release of 3 H-NA although i t i n h i b i t e d amphetamine-induced release. They 91 s u g g e s t e d t h a t c o c a i n e may be i n h i b i t i n g amphetamine-induced r e l e a s e by co m p e t i n g w i t h amphetamine f o r i n t r a - n e u r o n a l b i n d -3 i n g s i t e s o f H-NA. T h i s h y p o t h e s i s was based on t h e i r e v i d e n c e t h a t c o c a i n e was e q u i p o t e n t w i t h amphetamine i n e f f e c t i n g H-NA r e l e a s e ; a c a r e f u l e x a m i n a t i o n o f t h e i r data,. however, r e v e a l s t h a t t h e two drugs a r e n o t ~ e q u i p o t e n t , b u t r a t h e r appear t o have t h e same a f f i n i t i e s f o r s i t e s o f e f f l u x ( s i n c e e q u a l con-c e n t r a t i o n s o f t h e two d r u g s p r o d u c e d h a l f o f t h e i r r e s p e c t i v e maximum e f f e c t s ) . I n 1961, H e r t t i n g , A x e l r o d and P a t r i c k showed t h a t c o c a i n e 3 b l o c k e d t h e upt a k e o f H-NA b u t d i d n o t r e l e a s e NA i n t h e r a t h e a r t . C o n v e r s e l y , e v i d e n c e t h a t p a r t of t h e a b i l i t y o f c o c a i n e t o p o t e n t i a t e NA o c c u r s t h r o u g h r e l e a s e o f the t r a n s m i t t e r has been p r e s e n t e d by H a e f e l y , H u r l i m a n n , and Thoenen (1964) and T r e n d e l e n b u r g (1968). I n t h e forme r c a s e , t h i s c o n c l u s i o n was th e r e s u l t o f t h e d e m o n s t r a t i o n t h a t p r e v i o u s t r e a t m e n t w i t h r e s e r p i n e g r e a t l y r e d u c e d t h e sympathomimetic e f f e c t o f c o c a i n e . However t h i s r e s u l t c o u l d j u s t as e a s i l y be e x p l a i n e d by t h e a b i l i t y o f c o c a i n e t o i n h i b i t NA u p t a k e . T r e n d e l e n b u r g (1968) e x p r e s s e d s i m i l a r f i n d i n g s and a l s o d e m o n s t r a t e d an augmented r e l e a s i n g e f f e c t by c o c a i n e when p a r g y l i n e was employed t o i n -h i b i t MAO. D a v i s and M c N e i l l (1973) showed an i n c r e a s e d e f f l u x 3 o f H-NA from g u i n e a - p i g a t r i a by c o c a i n e w h i c h was a s s o c i a t e d w i t h a p o s i t i v e i n o t r o p i c e f f e c t , a l t h o u g h t h i s e f f l u x was con-s i d e r a b l y l e s s t h a n t h a t p r o d u c e d by t y r a m i n e . They t h e r e f o r e s u g g e s t e d t h a t r e l e a s e o f t h e c a t e c h o l a m i n e may be c o n t r i b u t i n g t o t h e p h a r m a c o l o g i c a l e f f e c t s of c o c a i n e . 92 Cocaine was the only drug of the twelve tested i n our study which could not be included i n Figure 11 since the maximum re-_ 3 lease achieved by t h i s compound, at a concentration of 10 M (the highest dose studied), did not reach 50% release of NA and therefore an EC50 value could not be calculated. Nevertheless, i t appears that cocaine would not deviate g r e a t l y from the de-termined r e l a t i o n s h i p between potency and l i p o p h i l i c i t y because t h i s drug possesses both a r e l a t i v e l y low l i p i d s o l u b i l i t y (only tyramine had a lower value) and a weak capacity to pro-duce release. In contrast, a nonspecific depression of neuro-transmitter release was deemed u n l i k e l y by Westfall and Brasted (1974) because cocaine and four other agents d i d not uniformly depress NA release by four agonists. The f a i l u r e of high doses of cocaine to show an apprec-3 i a b l e release of H-NA i s i n t e r e s t i n g . If cocaine were inh i b -i t i n g uptake of spontaneously-released catecholamine i n a com-p e t i t i v e manner, i t might be expected to displace NA from up-take s i t e s and reduce i n t r a c e l l u l a r accumulation of the amine. Because of the experimental design used, t h i s might be i n t e r -preted as increased NA e f f l u x , which was observed to a l i m i t e d degree. This i s possible since the competitive nature of co-caine's i n h i b i t i o n of uptake has been shown (Iversen, 1963). An a l t e r n a t i v e explanation i s that i n h i b i t i o n of NA-uptake- by cocaine might r e s u l t i n presynaptic regulation of release i n the manner proposed by Langer (1974), thus l i m i t i n g the extent of apparent release. If t h i s were so, i t may explain some d i s -crepancies i n the r e s u l t s of our experiments and studies employ-93 ing a constant perfusion technique. In t h i s l a t t e r case, i f a neuronal uptake blocker were present, released NA would be washed away from the synaptic c l e f t , preventing, i t s action on presynaptic i n h i b i t o r y receptors, r e s u l t i n g i n an increased ef-f l u x . Desipramine has also been purported to have the a b i l i t y to release NA when present i n high concentrations, and, as Table IX shows, the four t r i p t y l i n e compounds tested i n our ex-3 periments also produced release of H-NA but, although equipo-tent, displayed a lower potency for t h i s action than did t y r -amine. Figure 11 indicates that t h i s r e l e a s i n g e f f e c t may be p a r t i a l l y the r e s u l t of a nonspecific action of these l i p i d -soluble compounds on c e l l membranes. Brodie and coworkers (1968) provided evidence that desip-ramine, i n r e l a t i v e l y large doses, does not block cardiac ac-cumulation of high doses of tyramine or of amphetamine, yet an-tagonizes the depletion of heart NA e l i c i t e d by the two amines. The authors i n f e r that the antagonism of the r e l e a s i n g e f f e c t on sympathomimetic amines by desipramine i s due to an action within the nerve terminal, p o s s i b l y on the synaptic v e s i c l e s . However, the f a i l u r e to block tyramine uptake may be a dose-dependent, competitive phenomenon. The a b i l i t y of desipramine to prevent the amine-induced release may be a r e s u l t of i t s blocking e f f e c t on the granule uptake process which also accum-ulates tyramine (Iversen, 1971b). Iversen has suggested that the actions of i n d i r e c t l y - a c t i n g amines seem to be r e l a t e d to displacement of NA from storage p a r t i c l e s . L e i t z and Stefano 94 3 (1970) demonstrated that the released H-NA was mainly i n the form of deaminated metabolites i n d i c a t i n g that desipramine exerts an e f f e c t on the amine storage granule to deplete the NA stores. At low doses of the drug, only i n h i b i t i o n of uptake 3 of H-NA was seen. Again, t h i s could be explained by the i n -h i b i t i o n by desipramine of the granule amine uptake process (rather than true release), which would prevent the NA from being protected against intraneuronal metabolism. This seems to c o r r e l a t e well with the observation that higher doses of desip-ramine are required to i n h i b i t the p a r t i c l e uptake system as compared to the neuronal uptake process (Iversen, 1971b). Titus and coworkers (1966) had also observed that very high 3 l e v e l s of desipramine produced an appreciable release of H-NA. They commented that t h i s drug may have an e f f e c t on the storage granules, but also suggested that because many of the compounds which blocked catecholamine uptake were of diverse structures, were l i p i d soluble, and showed a m u l t i p l i c i t y of e f f e c t s on membranes, i t i s u n l i k e l y that these drugs would act on a spec-i f i c c a r r i e r for NA. Thus the r e l e a s i n g e f f e c t s of such drugs' i n high doses may likewise be p a r t l y due to nonspecific mem-brane e f f e c t s . Except for promethazine and possibly phenindamine, which seemed to have a NA r e l e a s i n g action equivalent to the t r i c y -c l i c antidepressants, the remaining antihistamine compounds 3 displayed a low potency for causing e f f l u x of H-NA. In addi-t i o n , the antihistaminic compounds showed a close c o r r e l a t i o n between potency and l i p o p h i l i c i t y with respect to release of H-NA, again suggesting that a nonspecific mechanism of action may be responsible for the r e l e a s i n g e f f e c t s of high doses of these drugs. The only exception i s phenindamine, whose high p a r t i t i o n c o e f f i c i e n t did not correspond to i t s only moderate e f f e c t on e f f l u x . This may be the r e s u l t of an excessive af-f i n i t y for the l i p i d portions of the membrane, sequestering the molecule and thus preventing i t s m o b i l i z a t i o n to possible s i t e s of action. Similar observations were made by Isaac and Goth (1965) i n an in. vivo study i n r a t hearts which indicated that none of the antihistamines tested (chlorpheniramine, phenindamine, prometh-3 azine, pyrilamine, and tripelennamine) caused release of H-NA. However, Davis and McNeill (1973) found that chlorpheniramine, t r i p r o l i d i n e , and tripelennamine produced an increased e f f l u x 3 of H-NA from i s o l a t e d guinea p i g a t r i a i n conjunction with a p o s i t i v e i n o t r o p i c e f f e c t whereas promethazine did not. These r e s u l t s do not agree with those of the present study. 3 In experiments on the i n h i b i t i o n of H-NA uptake produced by the drugs, the time course of i n h i b i t i o n showed an i n i t i a l peak i n the f i r s t ten minutes of incubation (with s l i g h t f l u c -tuations) followed by a gradual decline i n the degree of block-ade to an approximate steady-state l e v e l a f t e r f o r t y minutes i n -cubation time. The decrease i n e f f e c t with time i s probably due to competition between the NA and the drug, the extent de-pending on the r e l a t i v e a f f i n i t i e s of each f o r the membrane c a r r i e r . The catecholamine would displace compounds with lower a f f i n i t i e s u n t i l an equilibrium s i t u a t i o n i s reached. There-96 fore, the i n i t i a l degree of i n h i b i t i o n attained by each agent should represent the r e l a t i v e a f f i n i t y of that compound for the uptake s i t e i n the absence of a s i g n i f i c a n t interference by NA, whereas the l e v e l portion of the curve r e f l e c t s the e q u i l i b -rium between the drug and the t r i t i a t e d amine. The concentration-effect studies of NA uptake blockade afte r f o r t y minutes incubation showed that a l l drugs studied 3 produced an i n h i b i t i o n of accumulation of H-NA, with essen-_ 3 t i a l l y 100% blockade occurring when a maximum dose of 10 M was used. In contrast, the experiments of Isaac and Goth (1965, 1967) found that promethazine d i d not i n h i b i t uptake s i g n i f i -c a n tly as compared to s a l i n e although diphenhydramine, t r i p e l -ennamine, chlorpheniramine and phenindamine were e f f e c t i v e i n -h i b i t o r s . However, i n these studies, only a single dose of drug was used, c o n s i s t i n g of 10 mg/kg promethazine administered i n -t r a p e r i t o n e a l l y i n in_ vivo studies (Isaac and Goth, 1965) and 1 x 10 -^ M promethazine i n the in_ v i t r o experiments (Isaac and Goth, 1967). Because i t was the l e a s t potent of the twelve drugs, and t h i s l a t t e r dose of promethazine would have shown less than 5% uptake i n h i b i t i o n i n our i n v e s t i g a t i o n , i t i n d i -cates that the absence of an i n h i b i t o r y e f f e c t as well as a po t e n t i a t i n g e f f e c t by promethazine may be due to an i n s u f f i c -i e nt concentration of the drug. The remaining antihistamines showed a considerable i n h i b i t i o n of uptake i n our experiments i n the doses employed by Isaac and Goth (1967), corresponding to t h e i r observations. The findings of Davis and McNeill (1973) show a greater contrast with those of the present study. These authors found that desipramine appeared to be the most potent i n h i b i t o r of 3 H-NA uptake i n i s o l a t e d guinea-pig a t r i a , producing 83% i n -h i b i t i o n at a dose.of 1 x 10 M. In comparison, desipramine showed only about 25% blockade of uptake at t h i s concentration _5 according to Figure lOd. S i m i l a r l y , 3 x 10 M promethazine and tripelennamine, which produced 31% and 62% i n h i b i t i o n of uptake r e s p e c t i v e l y according to Davis and McNeill, appear to produce a much greater response i n the present study as re-f l e c t e d i n Figures 10b and 10a. These c o n f l i c t i n g r e s u l t s could be explained by a number of d i f f e r e n t f a c t o r s such as species and/or tissu e v a r i a t i o n s (which w i l l be discussed i n more d e t a i l l a t e r ) , as well as differences between perfusion and incubation techniques. Another contributing factor may be the presence of an MAO i n h i b i t o r i n the incubation medium at a concentration of 1.6 x 10~ 4 M. Iversen (1965b) observed that of seven MAO i n h i b i t o r s tested, three possessed the a b i l i t y to also i n h i b i t NA uptake i n r a t heart. Pargyline, which was used i n our experiments, _5 displayed no blockade of uptake at a dose of 10 M; however no other concentrations were tested to examine the e f f e c t s of higher doses of the drug. Hendley and Snyder (1968) demonstra-ted that, i n r a t cortex, pargyline was moderately e f f e c t i v e as an i n h i b i t o r of uptake of metaraminol, a phenethylamine deriv-a t i v e which i s r e s i s t a n t to MAO and COMT but has a high a f f i n -i t y for the neuronal amine uptake system. The ID50 value for -4 uptake i n h i b i t i o n by pargyline was found to be 1.2 x 10 M, 98 a c o n c e n t r a t i o n l e s s t h a n t h a t used i n t h i s s t u d y , s u g g e s t i n g t h a t t h i s MAO i n h i b i t o r may i n f l u e n c e t h e measured e f f e c t s o f o t h e r d r u g s on t h e u p t a k e mechanism. A g a i n , t h e r e l e a s i n g e f f e c t s o f t h e v a r i o u s d r u g s c a n n o t be d i s r e g a r d e d when comparing t h e compounds as i n h i b i t o r s o f u p t a k e . Because of t h e methods employed, any s u b s t a n c e w h i c h p r o d u c e s c o n s i d e r a b l e e f f l u x o f NA would a l s o appear t o have a g r e a t e r b l o c k i n g a c t i o n t h a n a c t u a l l y o c c u r s , s i n c e b o t h e f r 3 f e c t s a r e m a n i f e s t e d by d e c r e a s e d a c c u m u l a t i o n o f H-NA. How-e v e r , t h e i n f l u e n c e o f r e l e a s e does n o t appear t o be i m p o r t a n t s i n c e b o t h c o c a i n e and t r i p e l e n n a m i n e , n e i t h e r o f w h i c h p r o d u c e d any a p p r e c i a b l e e f f l u x , were h i g h l y e f f e c t i v e i n h i b i t o r s of NA u p t a k e . I n c o n t r a s t , t h e t r i p t y l i n e compounds were f a i r l y p o t e n t r e l e a s i n g a g e n t s b u t were c o n s i d e r a b l y l e s s e f f e c t i v e t h a n c o c a i n e , t y r a m i n e or t r i p e l e n n a m i n e w i t h r e s p e c t t o u p t a k e i n h i b i t i o n . I n a d d i t i o n , a l l t h e compounds were more p o t e n t as i n h i b i t o r s o f u p t a k e t h a n as r e l e a s i n g a g e n t s , a f a c t w h i c h a l s o t e n d s t o d i s s o c i a t e e f f e c t s on e f f l u x f r om e f f e c t s on up-t a k e b l o c k a d e . T a b l e XV shows IC50 v a l u e s f o r some o f t h e t e s t d r u g s as i n h i b i t o r s o f c a t e c h o l a m i n e u p t a k e , o b t a i n e d by o t h e r i n v e s -t i g a t o r s . I n g e n e r a l , t h e v a l u e s o b t a i n e d i n our s t u d y a g r e e v e r y w e l l w i t h t h o s e r e p o r t e d i n the l i t e r a t u r e ( a l t h o u g h t r i -p e l e n n a m i n e and t r i p r o l i d i n e c o u l d n o t be compared s i n c e c o r r e s -p o n d i n g f i g u r e s f o r c a t e c h o l a m i n e u p t a k e i n h i b i t i o n i n t h e l i t -e r a t u r e c o u l d n o t be f o u n d ) . O n l y i n t h e case o f t h e f o u r t r i p t y l i n e compounds d i d our r e s u l t s show some d e v i a t i o n from 99 T a b l e XV I n h i b i t i o n o f C a t e c h o l a m i n e Uptake by t h e T e s t Drugs i n V a r -i o u s T i s s u e s and S p e c i e s ( C o n c e n t r a t i o n s f o r h a l f - m a x i m a l i n h i b i t i o n (IC50) a r e g i v e n . ) Drug IC50 (p.M) T r a n s - S p e c i e s T i s s u e R e f e r -m i t t e r ence A m i t r i p t y l i n e 0.11 0. 055 4. 0 NA NA DA Rat Rat R a t H e a r t a,b H y p o t h a l - c amus S t r i a t u m c C h l o r p h e n i r a m i n e 2.0 1. 2 1. 6 2. 5 NA NA DA DA Rat R a t R a t R a t H y p o t h a l - d amus H y p o t h a l - c amus S t r i a t u m c S t r i a t u m d 0. 38 NA Rat H e a r t e,f 0. 8 NA Rat C o r t e x g 2. 0 NA R a b b i t H e a r t b 2. 0 NA R a b b i t S t r i a t u m h 1.0 NA Mouse C o r t e x h 0.47 NA Mouse Whole i b r a i n D e s i p r a m i n e 0. 007 NA Ra t H e a r t a 0. 013 NA Rat H e a r t e,f 0. 050 NA Rat H y p o t h a l -amus c,d : 0.4 NA Ra t C o r t e x g 50 DA Ra t S t r i a t u m c, 0. 03 a, j NA R a b b i t H e a r t b 50 NA R a b b i t S t r i a t u m h 0.03 NA Mouse C o r t e x h 0. 006 NA Mouse Whole b r a i n i ( c o n t i n u e d ) 100 Table XV (continued) Drug IC50 (uM) Trans-mitter Species Tissue Refer ence Diphenhydramine 2. 70 NA Rat Hypothal- c amus 4. 2 NA Rat Hypothal- d amus 4. 6 DA Rat Striatum d 3.49 DA Rat Striatum c Imipramine 0.04 NA Rat Heart a,b 0. 09 NA Rat Heart e,f 1. 00 NA Rat Hypothal- c amus 8.00 DA Rat Striatum c 20. 0 NA Rabbit Striatum h 0. 20 NA Mouse Cortex h N o r t r i p t y l i n e 0. 02 NA Rat Heart a,b 1. 300 NA Rat Hypothal- c amus 5.49 DA Rat Striatum c Phenindamine 8.0 NA Rat Hypothal- c amus 4.0 NA Rat Hypothal- d amus 4. 5 NA Rat Hypothal- j amus 4. 8 DA Rat Striatum d,j Promethazine 5.01 NA Rat Hypothal- d amus 17.86 NA Rat Hypothal- c amus 26. 32 DA Rat Striatum c 15.0 DA Rat Striatum d Tyramine 0.45 NA Rat Heart k 1.0 NA Rat Hypothal- 1 0. 54 DA Rat Striatum 1 (continued) 101 Table XV (continued) References: a. Callingham, 1966 b. B e r t i and Shore, 1967 c. Horn, Coyle and Snyder, 1971 d. Snyder, 1970 e. Iversen, 1965b f. Iversen, 1967 g. Azzaro et. _al. , 1974 h. Ross and Renyi, 1967 i . Carmichael and I s r a e l , 1973 j . Coyle and Snyder, 1969a k. Burgen and Iversen, 1965 1. Horn, 1973 reported IC50 values, e s p e c i a l l y those obtained i n heart, cor-t i c a l , and hypothalamic t i s s u e s . However, these t r i c y c l i c an-tidepressants appear to be the only compounds studied which show a discrepancy i n uptake i n h i b i t o r y potencies between s t r i a t a l and heart or hypothalamic tiss u e s , with i n h i b i t i o n of s t r i a t a l uptake r e q u i r i n g a considerably higher concentration than up-take blockade i n the other t i s s u e s . This i s most pronounced for desipramine, which shows a thousand-fold d i f f e r e n c e between the two doses, i n d i c a t i n g s i g n i f i c a n t tissue d i f ferences between the two brain regions. The IC50 values for these drugs ob-= tained i n the present study c o r r e l a t e more c l o s e l y with those reported f o r i n h i b i t i o n of catecholamine uptake i n the s t r i a -tum. This may have been due to the use of whole brain homogen-ates i n our study, so that the measured e f f e c t would be a com-posite of the various regions. Since the IC50 values for t r i p t y l i n e compounds obtained i n our whole brain homogenate approximated uptake i n h i b i t o r y concentrations for s t r i a t a l t i s s u e although t h i s brain region 102 represents only a small proportion of the t o t a l t i s s u e , i t may be that there are f a r more uptake binding s i t e s per u n i t weight i n the striatum. This could also be a possible explanation for r e s u l t s obtained by Snyder and Coyle (1969) who observed 3 that P/M r a t i o s for H-NA uptake i n the striatum were approx-imately tenfold higher than those i n the hypothalamus and other brain regions, even though H-NA demonstrated a higher a f f i n i t y for the catecholamine uptake system i n n o n s t r i a t a l areas. How-ever i t may also be possible that there i s a high degree of nonspecific binding of desipramine and r e l a t e d compounds i n s t r i a t a l tissue, to explain the greater concentration of drug required for uptake i n h i b i t i o n . In support of t h i s , Horn, Coyle and Snyder (1971) showed through k i n e t i c analyses that i n h i b -i t i o n by the t r i c y c l i c compounds was competitive i n the hypo-thalamus but noncompetitive i n the corpus striatum. F a i l u r e to show competitive i n h i b i t i o n indicates that the i n h i b i t o r y sub-stance i s probably acting at a s i t e separate from that occupied by the substrate, suggesting that nonspecific binding could be involved. In accordance with t h i s , Mundo et_ al. (1974) demon-strated that the accumulation of t r i t i a t e d t r i p t y l i n e compounds into r a t a t r i a l t i s s u e appeared to be a passive and unsaturable process for a l l the tested drugs. They also observed that drug 3 t i s s u e concentrations c o r r e l a t e d with H-NA uptake i n h i b i t i o n but not with p o t e n t i a t i o n of the chronotropic response to NA. Another factor to be considered i s that i n a l l cases but one (Ross and Renyi, 1967) uptake into the' striatum was meas-3 3 ured with H-dopamine rather than H-NA. Since the two cate-103 cholamines d i f f e r i n t h e i r r e l a t i v e a f f i n i t i e s for the amine uptake mechanism (Snyder and Coyle, 1969) with dopamine accum-u l a t i o n exceeding that of NA i n a l l brain areas, care must be taken i n making a comparison of i n h i b i t o r y potencies of various drugs i n the two brain regions. One would expect a higher con-centration of drug to be required to i n h i b i t accumulation of a transmitter d i s p l a y i n g a greater a f f i n i t y . I t i s now apparent that there are marked tissu e d i f f e r -ences i n the neuronal amine uptake systems, e s p e c i a l l y i n the various brain regions (Snyder and Coyle, 1969). However, the properties of the system i n NA-containing neurones of the cen-t r a l nervous system (primarily the hypothalamus) seem to be i n -di s t i n g u i s h a b l e from those i n the peripheral sympathetic neur-one (Iversen, 1971b). Squires (1974) has observed the i n h i b -i t i o n of uptake i n several regions of r a t brain by t r i c y c l i c compounds and has presented evidence that even the cerebral cortex does not appear to be uniform but rather, exhibits areas such as the f r o n t a l cortex with a high density of dopamine t e r -minals (see Table XVI). As mentioned before, Horn, Coyle and Snyder (1971) found that a large number of c e n t r a l l y - a c t i n g drugs were competitive i n h i b i t o r s of catecholamine uptake into hypothalamic synaptosomes but were noncompetitive i n h i b i t o r s i n the corpus striatum. In addition to regional or tissue v a r i a t i o n s i n properties of the uptake system, there also seems to be a quantitative species v a r i a t i o n as shown i n Tables XVII and XVIII (Iversen, 1971b) although the general properties appear to be s i m i l a r i n most species. However, Iversen states that the NA uptake sys-104 T a b l e XVI I n h i b i t i o n o f N o r a d r e n a l i n e Uptake i n t o Synaptosomes P r e p a r e d from S e v e r a l B r a i n R e g i o n s ( C o n c e n t r a t i o n s f o r h a l f - m a x i m a l i n h i b i t i o n (IC50) a r e g i v e n . The t a b l e i s a d a p t e d from S q u i r e s , 1974. v) I n h i b i t o r IC50 (uM) T i s s u e D e s i p r a m i n e 0.001 O c c i p i t a l C o r t e x 10 F r o n t a l C o r t e x 0. 001 Hippocampus 10 Whole F o r e b r a i n I m i p r a m i n e 0. 03 O c c i p i t a l C o r t e x 2. 5 F r o n t a l C o r t e x 0.006 Hippocampus 10 Whole F o r e b r a i n A m i t r i p t y l i n e 0. 05 O c c i p i t a l C o r t e x 2.5 F r o n t a l C o r t e x 0.03 Hippocampus 3.0 Whole F o r e b r a i n tern i n g u i n e a p i g and r a b b i t seems t o l a c k s t e r e o c h e m i c a l s p e c -i f i c i t y w h i c h s u g g e s t s t h a t some q u a l i t a t i v e e s p e e i e s ^ d i f f e r -ences may e x i s t . To e x p l a i n t h e d i s c r e p a n c i e s between the r e s u l t s o f t h i s s t u d y and t h o s e o f o t h e r a u t h o r s , more t h a n j u s t t i s s u e and s p e c i e s v a r i a t i o n may be i n v o l v e d . The measurement o f r e c e p -t o r r e s p o n s e i n e v a l u a t i n g t h e r e l a t i v e p o t e n c i e s o f v a r i o u s i n h i b i t o r s c o u l d be m i s l e a d i n g i f t h e d r u g s a l s o p r o d u c e d p o s t -s y n a p t i c e f f e c t s . F o r example, c o c a i n e has been shown t o po-105 Table XVII Species Differences i n Catecholamine Uptake i n the Perfused Hearts of Various Vertebrates (Iversen, 1971b) Species ID50 (;uM) (-)-Adrenaline (-)-NA Rat 0. 28 1.02 Mouse 0. 65 1. 08 Guinea Pig 0. 98 2. 72 Pigeon 1.15 2. 96 Toad (Bufo marinus) 2. 34 0. 96 (The ID50 i s the concentration required to produce 50% i n h i b -i t i o n of uptake of (+)- H-NA and i s approximately equal to the K. . ) l Table XVIII A f f i n i t y Constants for (+)-NA Uptake by Cerebral Cortex of Various Species (Iversen, 1971b) Species Km ( + )-NA (jaM) Mouse 0.40 Rat 0. 40 Monkey 0.40 Cat 0. 75 Guinea Pig 1.10 106 t e n t i a t e t h e r e s p o n s e s t o NA i n systems (such as d e n e r v a t e d t i s s u e s ) where amine up t a k e c a n n o t o c c u r , i m p l y i n g t h a t c o -c a i n e has some d i r e c t e f f e c t s as w e l l as p r o d u c i n g p o t e n t i a -t i o n t h r o u g h i n h i b i t i o n o f u p t a k e ( R e i f f e n s t e i n , 1968; N a k a t s u and R e i f f e n s t e i n , 1968; M a x w e l l e_t al_. , 1966; R e i f f e n s t e i n and T r i g g l e , 1974). I t has been s u g g e s t e d t h a t t h e p o s t - s y n a p t i c a c t i o n s o f c o c a i n e may be due t o an a l l o s t e r i c c o n f o r m a t i o n a l change near t h e r e c e p t o r t o p o s s i b l y a l t e r t h e i n t r i n s i c a c -t i v i t y o f t h e r e c e p t o r o r t h e a g o n i s t - r e c e p t o r b i n d i n g ( R e i f ^ f e n s t e i n and T r i g g l e , 1974). T h e r e f o r e t h i s a c t i o n c o u l d ac-c o u n t f o r p a r t o f t h e p o t e n t i a t i o n p r o d u c e d by d r u g s i n p o o r -l y o r n o n - i n n e r v a t e d t i s s u e s . C o n v e r s e l y , i n more d e n s e l y i n -n e r v a t e d t i s s u e s , where the u p t a k e system has been shown t o p l a y an i m p o r t a n t r o l e i n t e r m i n a t i n g the r e s p o n s e s t o NA, i n -h i b i t i o n o f t h i s u p t a k e and t h e degree o f p o t e n t i a t i o n may be more c l o s e l y c o r r e l a t e d . As n o t e d i n t h e R e s u l t s s e c t i o n , t h e c o r r e l a t i o n between i n h i b i t o r y p o t e n c y and l i p i d s o l u b i l i t i e s o f t h e t e s t e d com-pounds was n o t as w e l l - d e f i n e d as f o r t h e r e l e a s i n g e f f e c t s . T h i s may r e f l e c t m u l t i p l e s i t e s o r modes o f a c t i o n o f many o f t h e s e d r u g s . Drugs such as c o c a i n e , t y r a m i n e , o r t r i p e l e n n a -mine, w h i c h do n o t show a c o r r e l a t i o n between p o t e n c y and s o l -u b i l i t y , may i n h i b i t u p t a k e t h r o u g h a s p e c i f i c i n t e r a c t i o n w i t h t h e t r a n s p o r t p r o c e s s . I n f a c t , c o c a i n e and t y r a m i n e have been shown t o a c t as c o m p e t i t i v e u p t a k e i n h i b i t o r s ( I v e r s e n , 1963; I v e r s e n , 1971b). The i n h i b i t o r y a c t i v i t y o f t h e t r i c y c l i c a n t i d e p r e s s a n t s 1 0 7 r e l a t e s more c l o s e l y to t h e i r l i p i d s o l u b i l i t i e s , which may imply a nonspecific rather than a competitive e f f e c t , i n con-t r a s t to the mechanism described by Iversen,(1971b). But since the NA uptake i n h i b i t i o n produced by these compounds i n our study resembles t h e i r action i n the striatum where they have been shown to act noncompetitively (Horn, Coyle, and Snyder, 1971), t h i s could explain t h e i r apparent nonspecific e f f e c t . The antihistamines which, l i k e cocaine, were included by Seeman (1972) i n h i s general c l a s s i f i c a t i o n of anesthetics, d i d not seem to f i t the r e l a t i o n s h i p shown i n Figure 12. Only promethazine and t r i p r o l i d i n e f e l l close to the computed curve. Thus other f a c t o r s besides nonspecific membrane e f f e c t s may con-t r i b u t e to the measured i n h i b i t o r y response. S t r u c t u r a l l y nonspecific drugs may exert t h e i r action as a r e s u l t of-such physicochemical properties as degree of ion-i z a t i o n , s o l u b i l i t y , thermodynamic a c t i v i t y and surface tension e f f e c t s rather than through an i n t e r a c t i o n with a s p e c i f i c re-ceptor (Korolkovas, 1970). For most drugs which lack a struc-t u r a l resemblance to a c t i v e l y transported compounds, t h e i r rate of passage across l i p o p r o t e i n c e l l u l a r membranes w i l l depend to a great extent on t h e i r l i p o s o l u b i l i t y , the l i p o p h i l i c com-pounds crossing membrane b a r r i e r s r a p i d l y . S i m i l a r l y , such drugs could accumulate at some point of v i t a l importance to a c e l l and thereby disrupt c e r t a i n metabolic processes. For example, Seeman (1972) summarizes the membrane actions of the l i p i d - s o l u b l e anesthetics and t r a n q u i l i z e r s as e l e c t r i -c a l s t a b i l i z a t i o n of the membrane along with f l u i d i z a t i o n and 108 disordering of the membrane components. As a r e s u l t of these • conformational changes and membrane expansion, either stimu-l a t i o n or i n h i b i t i o n of the associated enzymes and proteins may occur, depending also on the e l e c t r i c a l charge of the drug, by modifying the permeability of membranes to ions or solutes. Seeman also postulates that enhanced neurosecretion of bound substances may occur during membrane f l u i d i z a t i o n through ex-pansion and subsequent fusion of two membranes. However, since l i p i d - s o l u b l e drugs such as the anesthetics displace Ca?"*, which i s required for stimulus-secretion coupling, i n h i b i t i o n of secretion may poss i b l y mask any enhanced neurosecretion. In the same way, such drugs could t h e o r e t i c a l l y diminish the up-take of neurotransmitters i n d i r e c t l y through interference with membrane permeability to ions, whether these fluxes be passive,, active or f a c i l i t a t e d . Therefore, the r e s u l t s of these experiments have provided evidence that the actions of c e r t a i n drugs i n causing release of NA from nerve endings may be d i f f e r e n t i a t e d from the inh i b -i t o r y e f f e c t s of these drugs on NA uptake using the methods employed. However, the mechanisms whereby these drugs act may be complicated and i n most cases can only be sp e c u l a t i v e since, fo r most of the processes involved, the knowledge i s s t i l l i n -complete. SUMMARY AND CONCLUSIONS 109 In t h i s i n v i t r o study, a homogenate of r a t whole brain t i s s u e was employed to examine the e f f e c t s of twelve drugs on 3 the uptake and release of H-noradrenaline from nerve terminals since t h i s preparation has been previously used by other inves-t i g a t o r s and has been shown to contain synaptosomes, i s o l a t e d nerve endings which were pinched o f f from t h e i r axons during the homogenization process. To d i f f e r e n t i a t e between r e l e a s i n g e f f e c t s of the drugs and i n h i b i t i o n of NA uptake, the incub-ation procedure was varied. That i s , to investigate NA release, 3 the homogenate was f i r s t preincubated with H-NA to load the synaptosome p r i o r to addition of the drug; to study uptake i n -h i b i t i o n , the homogenate was instead preincubated with drug and 3 then H-NA was added and the incubation continued* The r e s u l t s of t h i s study may be summarized as follows: 3 1. In the absence of drug treatment, the accumulation of H-NA increased with time (showing a peak accumulation a f t e r 25 min-utes incubation) and diminished with increasing concentrations of NA ( i n d i c a t i n g saturation by 5.0 jaM NA). -4 2. Employed i n a concentration of 10 M, a l l twelve drugs studied demonstrated a r e l e a s i n g action which increased with time. Although simultaneous i n h i b i t i o n of uptake could have contributed to the measured e f f e c t , i t was f e l t that t h i s would be minimal or u n l i k e l y since cocaine, a potent i n h i b i t o r of neuronal NA uptake, showed the least- release. 110 3. Following incubation for 20 minutes, a l l the drugs showed increasing r e l e a s i n g e f f e c t with increasing dose although t y r -amine was e f f e c t i v e at a much lower dose than for any of the remaining compounds. From these data, concentrations required to produce half-maximal e f f e c t s on release (EC50) were deter-mined, demonstrating tyramine to be by f a r the most potent drug i n t h i s respect, whereas cocaine and tripelennamine showed the le a s t release of NA. 4. A p l o t of the logarithm of the EC50 value _vs the logarithm of the octanol/water p a r t i t i o n c o e f f i c i e n t showed a l i n e a r r e -l a t i o n s h i p for nine of the twelve drugs i n d i c a t i n g that a non-s p e c i f i c mechanism of action based on l i p i d s o l u b i l i t y may be responsible for the re l e a s i n g e f f e c t s of high doses of these drugs. The f a i l u r e of tyramine to f i t t h i s c o r r e l a t i o n suggests that t h i s drug may be acting by competing for the neuronal cat-echolamine uptake process to gain entry into the nerve terminal where i t can displace bound NA and thus produce release of the amine. This evidence supports that of many other i n v e s t i g a t o r s who have implied a s i m i l a r mechanism of action for tyramine. The f a i l u r e of cocaine to produce any appreciable release of NA despite i t s reported competitive i n h i b i t i o n of uptake may be due to postulated presynaptic i n h i b i t i o n of release; or i t may instead be i n d i c a t i v e that cocaine i s not a substrate f o r the up-take process so that, although i t might block the uptake system, i t could not gain entry to the nerve terminal to displace the bound catecholamine. I l l — 6 5. A t a c o n c e n t r a t i o n o f 10" Mf a l l t w e l v e d r u g s d e m o n s t r a t e d an i n h i b i t o r y e f f e c t on NA u p t a k e w h i c h showed an i n i t i a l de-c r e a s e w i t h t i m e and th e n l e v e l l e d a f t e r a p p r o x i m a t e l y 40 min-u t e s i n c u b a t i o n . A g a i n , t h e e f f e c t s w h i c h t h e s e d rugs may have on e f f l u x d i d n o t seem t o i n f l u e n c e t h e measured r e s p o n s e on up-t a k e i n h i b i t i o n s i n c e c o c a i n e and t r i p e l e n n a m i n e w h i c h seemed t o p o s s e s s a m i n i m a l r e l e a s i n g a c t i o n were h i g h l y e f f e c t i v e i n -h i b i t o r s of NA u p t a k e . 3 6. When th e d r u g s were i n c u b a t e d f o r 40 m i n u t e s w i t h t h e H-NA, i n c r e a s i n g c o n c e n t r a t i o n s o f t h e compounds were o b s e r v e d t o p r o d u c e i n c r e a s i n g i n h i b i t i o n o f u p t a k e r e a c h i n g 100% w i t h maximum c o n c e n t r a t i o n s o f a l l t w e l v e d r u g s . From t h e s e d a t a , c o n c e n t r a t i o n s r e q u i r e d t o p r o d u c e 50% i n h i b i t i o n (IC50) were c a l c u l a t e d , a l l o w i n g r e l a t i v e p o t e n c i e s o f t h e drugs t o be com-p a r e d . As i n s t u d i e s o f r e l e a s e , t y r a m i n e was five 1 most p o t e n t compound, w i t h i t s IC50 and EC50 v a l u e s b e i n g comparable. I n c o n t r a s t , t r i p e l e n n a m i n e and c o c a i n e were a p p r o x i m a t e l y e q u i -p o t e n t w i t h t y r a m i n e as i n h i b i t o r s o f NA u p t a k e a l t h o u g h t h e i r p o t e n c i e s w i t h r e s p e c t t o r e l e a s e were f a r l o w e r . T h i s i n d i -c a t e s t h a t c o c a i n e and t r i p e l e n n a m i n e , l i k e t y r a m i n e , a r e p r o b -a b l y a c t i n g s p e c i f i c a l l y on t h e NA u p t a k e mechanism t o produce t h e i r b l o c k a d e . The r e m a i n i n g d r u g s , a l t h o u g h l e s s p o t e n t t h a n t y r a m i n e , a l s o showed a g r e a t e r p o t e n c y as i n h i b i t o r s o f NA up-t a k e t h a n as r e l e a s i n g a g e n t s , and t h e i r IC50 v a l u e s compare w e l l w i t h r e p o r t e d v a l u e s i n t h e l i t e r a t u r e . These compounds c o u l d be a c t i n g by a n o n c o m p e t i t i v e mechanism t o produce t h e i r i n h i b i t i o n . 7. Because t h e c o r r e l a t i o n between i n h i b i t o r y p o t e n c y and l i p i d s o l u b i l i t y was not v e r y g o o d , i t may be t h a t the t e s t e d compounds are a c t i n g t h r o u g h s e v e r a l p o s s i b l e mechanisms or w i t h mixed e f f e c t s . Those p d f u g s which f a l l on or c l o s e to t h e l i n e are p r o b a b l y a c t i n g i n a n o n s p e c i f i c manner which i s r e -l a t e d to t h e i r l i p i d s o l u b i l i t i e s ; compounds which d e v i a t e g r e a t l y f rom the e s t a b l i s h e d r e l a t i o n s h i p p r o b a b l y are i n h i b i t -i n g t h r o u g h a s p e c i f i c i n t e r a c t i o n w i t h the u p t a k e p r o c e s s ; and t h o s e d r u g s whiich f a l l between may be a c t i n g t h r o u g h a c o m b i n -a t i o n o f t h e s e e f f e c t s or a t y e t a n o t h e r s i t e . BIBLIOGRAPHY 113 Axelrod, J., Gordon, E., Heftting, G., Kopin, I.J., and Potter, L.T. On the Mechanism of Tachyphylaxis to Tyramine i n the Isolated Rat Heart. Br. J. Pharmacol. , JL9: 56-63 (1962) Axelrod, J., Hertting, G., and potter, L. E f f e c t of Drugs on the Uptake and Release of H-Norepinephrine i n the Rat Heart. Nature, 194: 297 (1962) Axelrod, J., Weil-Malherbe, H., and Tomchick, R. The P h y s i o l -o g i c a l D i s p o s i t i o n of H -Epinephrine and i t s Metabolite Metanephrine. J. Pharmacol., 127: 251-256 (1959) Azzaro, A.J. and Rutledge, CO. S e l e c t i v i t y of Release of Nor-epinephrine, Dopamine, and 5-Hydroxytryptamine by Amphet-amine i n Various Regions of Rat Brain. Biochem. Pharmacol., 22: 2801-2813 (1973) Azzaro, A.J., Ziance, R.J., and Rutledge, CO. The Importance of Neuronal Uptake of Amines for Amphetamine-Induced Re-lease of H-Norepinephrine from Isolated Brain Tissue. J. Pharmacol. Exp. Ther., 189: 110-118 (1974) B e r t i , F. and Shore, P.A. A K i n e t i c Analysis of Drugs that I n h i b i t the Adrenergic Neuronal Membrane Amine Pump. Biochem. Pharmacol., 16-:. 2091-2094 (1967) Blaszkowski, T.P. and Bogdanski, D.F. Possible Role of Sodium and Calcium Ions i n Retention and P h y s i o l o g i c a l Release of Norepinephrine by Adrenergic Nerve Endings. Biochem. Pharmacol., _20: 3281-3294 (1971) Bogdanski, D.F., Blaszkowski, T.P., and T i s s a r i , A.H. Mechan-isms of Biogenic Amine Transport and Storage. IV. Rela-ti o n s h i p Between K and the Na Requirement for Transport and Storage of 5-Hydroxytryptamine and Norepinephrine i n Synaptosomes. Biochim. Biophys. Acta, 211: 521-532 (1970) Bogdanski, D.F. and Brodie, B.B. The E f f e c t s of Inorganic Ions on the Storage and Uptake of H-Norepinephrine by Rat Heart S l i c e s . J. Pharmacol. Exp. Ther., 165: 181-189 (1969) Bogdanski, D.F., T i s s a r i , A. and Brodie, B.B. Role of Sodium, Potassium, Ouabain, and Reserpine i n Uptake, Storage, and Metabolism of Biogenic Amines i n Synaptosomes. L i f e S c i . , 2: 419-428 (1968) Bray, G.A. A Simple E f f i c i e n t L i q u i d S c i n t i l l a t i o n Method for Counting Aqueous Solutions i n a L i q u i d S c i n t i l l a t i o n Coun-ter. Anal. Biochem., !_: 279 (1960) 114 Brodie, B.B., Costa, E., Groppetti, A., and Matsumoto, C. Interaction Between Desipramine, Tyramine, and Amphetamine at Adrenergic Neurones. B r i t . J. Pharmacol. , . 3_4: 648-658 (1968) Burgen, A.S.V. and Iversen, L.L. The I n h i b i t i o n of Noradren-a l i n e Uptake by Sympathomimetic Amines i n the Rat Isolated Heart. B r i t . J. Pharmacol., 25} 34-49 (1965) Burn, J.H. The Action of Tyramine and Ephedrine. J. Pharma-c o l . Exp. Ther., 46: 75-95 (1932) Burn, J.H. and Rand, M.J. The Action of Sympathomimetic Amines i n Animals Treated with Reserpine.. J. Physiol. (Lond.), 144: 314-336 (1958) Burnstock, G., and Holman, M.E. Junction Potentials at Adren-ergic Synapses. Pharmacol. Rev., 18: 481-493 (1966) Callingham, B.A. The E f f e c t s of Imipramine and Related Com-pounds on the Uptake of Noradrenaline into Sympathetic Nerve Endings, i n : Antidepressant Drugs, Proceedings of the F i r s t International Symposium, International Congress Series No. 122, ed. S. G a r a t t i n i and M.N.G. Dukes, Amster-dam, Exerpta Medica Foundation, 1966, pp. 35-43. Carlsson A., H i l l a r p , N-A., and Waldeck, B. Analysis of the Mg -ATP Dependent Storage Mechanism i n the Amine Granules of the Adrenal Medulla. Acta Physiol. Scand. , _59: Suppl. 215 (1963) Carmichael, F.J. and I s r e a l , Y. In V i t r o I n h i b i t o r y E f f e c t s of Narcotic Analgesics and Other Psychotropic Drugs on the Active Uptake of Norepinephrine i n Mouse Brain Tissue. J. Pharmacol. Exp. Ther., 186: 253-260 (1973) Clout, D.H. and Williams, N. The E f f e c t of Narcotic Analgesic Drugs on the Uptake and Release of Neurotransmitters i n Isolated Synaptosomes. J. Pharmacol. Exp. Ther., 188: 419-428 (1974) Colburn, R.W., Goodwin, F.K., Murphy, D.L., Bunney, W.E., and Davis, J.M. Quantitative Studies of Norepinephrine Up-take by Synaptosomes. Biochem. Pharmacol. , JL7: 957-964 (1968) Colburn, R.W.,and Kopin, I.J. E f f e c t s of Reserpine and Tyr-amine on Release of Norepinephrine from Synaptosomes. Biochem. Pharmacol., 2 1 : 733-736 (1972) Commarato, M.A., Brody, T.M., and McNeill, J.H. The E f f e c t o| Various Drugs on the Uptake and Metabolism of Tyramine-H i n the Rat Heart. J. Pharmacol. Exp. Ther. , 1 6 7 1 5 1 -158 (1969a) 115 Commarato, M.A., Brody, T.M., and M c N e i l l , J.H. F u r t h e r S t u d -i e s on ^he E f f e c t o f V a r i o u s Drugs on :the Uptake o f T y r -amine-H and F o r m a t i o n o f Octopamine-H i n t h e R a t H e a r t . Can. J . P h y s i o l . P h a r m a c o l . , 47: 511-514 (1969b) C o y l e , J.T. and Snyder, S.H. A n t i p a r k i n s o n i a n Drugs; I n h i b -i t i o n o f Dopamine Uptake i n the Corpus S t r i a t u m as a Pos-s i b l e Mechanism of A c t i o n . S c i e n c e , 166: 899-901 (1969a) C o y l e , J.T. and Snyder, S.H. C a t e c h o l a m i n e Uptake by S y n a p t o -somes i n Homogenates o f Ra t B r a i n : S t e r e o s p e c i f i c i t y i n D i f f e r e n t A r e a s . J . P h a r m a c o l . Exp. Ther. 170: 221-231 (1969b) D a v i s , R.S. and M c N e i l l , J.H. The C a r d i a c E f f e c t s o f C o c a i n e and C e r t a i n A n t i h i s t a m i n e s and A n t i d e p r e s s a n t s . A r c h . I n t . Pharmacodyn. Ther., 201: 262-279 (1973) d e B e l l e r o c h e , J.S. and B r a d f o r , H.F. The Synaptosome: An I s o -l a t e d , W o r k i n g , N e u r o n a l Compartment. P r o g . N e u r o b i o l . , 1: 275-298 (1973) D e n g l e r , H.J., M i c h a e l s o n , I.A., S p i e g e l , H.E., and T i t u s , E. The Uptake of L a b e l l e d N o r e p i n e p h r i n e by I s o l a t e d B r a i n and Other T i s s u e s o f t h e C a t . I n t . J . N e u r o p h a r m a c o l . , 1: 23-38 (1962) D e n g l e r , H.J., S p i e g e l , H.E., and T i t u s , E.O. E f f e c t o f Drugs on ..Uptake o f I s o t o p i c N o r e p i n e p h r i n e by C a t T i s s u e s . N a t u r e , 191: 816-817 (1961a) D e n g l e r , H.J., S p i e g e l , H.E., and T i t u s , E.O. Uptake o f T r i t i u m L a b e l l e d N o r e p i n e p h r i n e i n B r a i n and Other T i s s u e s o f C a t i n v i t r o . S c i e n c e , 133: 1072-1073 (1961b) De R o b e r t i s , E., De I r a l d i , A.P., De L o r e s A r n a i s , G.R., and S a l g a n i c o f f , L. C h o l i n e r g i c and N o n - C h o l i n e r g i c Nerve E n d i n g s i n R a t B r a i n . J . Neurochem. _9: 23-35 (1962) D o u g l a s , W.W. The Mechanism o f R e l e a s e o f C a t e c h o l a m i n e s from t h e A d r e n a l M e d u l l a . P h a r m a c o l . Rev., 1_8: 471-480 (1966) E u l e r , U.S. von, and L i s h a j k o , F. C a t e c h o l a m i n e R e l e a s e and Uptake i n I s o l a t e d A d r e n e r g i c Nerve G r a n u l e s . A c t a , P h y s -i o l . Scand. , J57: 468-480 (1963) F a r n e b o , L-O. E f f e c t o f d-Amphetamine on Spontaneous and S t i m -u l a t i o n - I n d u c e d R e l e a s e o f C a t e c h o l a m i n e s . A c t a P h y s i o l . Scand., S u p p l . 371: 45-52 (1971) Far n e b o , L-0., and Hamberger, B. Drug-Induced Changes i n t h e R e l e a s e o f H-Monoamines from F i e l d S t i m u l a t e d R a t B r a i n S l i c e s . A c t a P h y s i o l . Scand., S u p p l . 371: 35-44 (1971) 116 Furchgott, R.F., Kirpekar, S.M., Rieker, M. , and Schwab, A. Actions and Interactions of Norepinephrine, Tyramine and Cocaine on A o r t i c S t r i p s of Rabbit and L e f t A t r i a of Guinea Pig and Cat. J. Pharmacol. Exp. Ther.. 142: 39-58 (1963) G i l l e s p i e , J.S. Uptake of Noradrenaline by Smooth Muscle. B r i t . Med. B u l l . , _29: 136-141 (1973) Glowinski, • J. and Axelrod, J. E f f e c t of Drugs on the Uptake, Release, and Metabolism of H -Norepinephrine i n the Rat Brain. J. Pharmacol. Exp. Ther., 149: 43-49 (1965) Gray, E.G. and Whittaker, V.P. The I s o l a t i o n of Nerve Endings from Brain: An Electron-Microscopic Study of C e l l Frag-ments Derived by Homogenization and Centrifugation. J. Anat., Lond. , _96: 79-88 (1962) Haefely, W., Hurlimann, A., and Thoenen, H. A Quantitative Study of the E f f e c t of Cocaine on the Response of the Cat N i c t i t a t i n g Membrane to Nerve Stimulation and to In-jected Noradrenaline. B r i t . J. Pharmacol., 2_2: 5-21 (1964) Hendley, E.D. and Snyder, S.H. Relationship Between the Action of Monoamine Oxidase I n h i b i t o r s on the Noradrenaline Up-take System and t h e i r Antidepressant E f f i c a c y . Nature, 220: 1330-1331 (1968) Herblin, W.F. Amantadine and Catecholamine Uptake. Biochem. Pharmacol., 2 1 : 1993-1995 (1972) Hertting, G. , Axelrod,. J. , and Patrick, R.W. Actions cjf Co-caine and Tyramine on the Uptake and Release of H -Nor-epinephrine i n the Heart. Biochem. Pharmacol. , _8: 246-248 (1961) Hertting, G., Axelrod, J., and Whitby, 3 L» G- E f f e c t of Drugs on the Uptake and Metabolism of H -Norepinephrine. J. Pharmacol. Exp. Ther., 134: 146-153 (1961) Horn, A.S. S t r u c t u r e - A c t i v i t y Relations for the I n h i b i t i o n of Catecholamine Uptake into Synaptosomes from Noradren-a l i n e and Dopaminergic Neurones i n Rat Brain Homogenates. B r i t . J. Pharmacol., 47: 332-338 (1973) Horn, A.S., Coyle, J.T., and Snyder, S.H. Catecholamine Uptake by Synaptosomes from Rat Brain. S t r u c t u r e - A c t i v i t y Rela-tionships of Drugs with D i f f e r e n t i a l E f f e c t s on Dopamine and Norepinephrine Neurons. Mol. Pharmacol. , _7: 66-80 (1971) 117 Horn, A.S. and Snyder, S.H. S t e r i c R e q u i r e m e n t s f o r C a t e c h o l -amine Uptake by R a t B r a i n Synaptosomes: S t u d i e s w i t h R i g i d A n a l o g s o f Amphetamine. J . P h a r m a c o l . Exp. Ther., 180: 523-530 (1972) I s a a c , L. and Goth, A. I n t e r a c t i o n o f A n t i h i s t a m i n i c s w i t h N o r e p i n e p h r i n e Uptake: A C o c a i n e - L i k e E f f e c t . L i f e S c i . , 4: 1899-1904 (1965) I s a a c , L. and Goth, A. The Mechanism o f t h e P o t e n t i a t i o n o f N o r e p i n e p h r i n e by A n t i h i s t a m i n i c s . J . P h a r m a c o l . Exp. Ther., 156: 463-468 (1967) I v e r s e n , L.L. The Uptake o f N o r a d r e n a l i n e by t h e I s o l a t e d P e r f u s e d R a t H e a r t . B r i t . J . P h a r m a c o l . , 21: 523-537 (1963) I v e r s e n , L.L. The I n h i b i t i o n o f N o r a d r e n a l i n e Uptake by Drugs, i n : Advances i n Drug R e s e a r c h , V o l . 2, ed. N.J. H a r p e r and A.B. Simmonds, London, Academic P r e s s , 1965a, pp. 1-46. I v e r s e n , L.L. I n h i b i t i o n o f N o r a d r e n a l i n e Uptake by Drugs. J . Pharm. P h a r m a c o l . , r 7 : 62-64 (1965b) I v e r s e n , L.L. The Uptake o f C a t e c h o l Amines a t H i g h P e r f u s i o n C o n c e n t r a t i o n s i n t h e R a t I s o l a t e d H e a r t : A N o v e l C a t -e c h o l Amine Uptake P r o c e s s . B r i t . J . P h a r m a c o l . , 2.5: 18-33 (1965c) I v e r s e n , L.L. The Uptake o f A d r e n a l i n e by the I s o l a t e d R a t H e a r t . B r i t . J . P h a r m a c o l . , 24: 387^394 (1965d) I v e r s e n , L.L. The Uptake and S t o r a g e o f N o r a d r e n a l i n e i n Sym-p a t h e t i c N e r v e s . Cambridge, Cambridge U n i v e r s i t y P r e s s , 1967. I v e r s e n , L.L. R o l e o f T r a n s m i t t e r Uptake Mechanisms i n Syn-a p t i c N e u r o t r a n s m i s s i o n . B r i t . J . P h a r m a c o l . , 41.: 571-591 (1971a) I v e r s e n , L.L. The Uptake o f B i o g e n i c Amines, i n : B i o g e n i c Amines and P h y s i o l o g i c a l Membranes i n Drug Therapy, P a r t B, ed. J.H. B i e l and L.G. Abood, New York, M a r c e l Dekker, 1971b, pp. 259-327. I v e r s e n , L.L. C a t e c h o l a m i n e Uptake P r o c e s s e s . B r i t . Med. B u l l . , 29.: 130-135 (1973 ) I v e r s e n , L.L. and K r a v i t z , E.A. Sodium Dependence o f T r a n s -m i t t e r Uptake a t A d r e n e r g i c Nerve T e r m i n a l s . M o l . Pharm-a c o l . , 2 : 360 (1966) 118 Johnson, D.G., Thoa, N.B., Weinshilboum, R., Axelrod, J ., and Kopin, I.J. Enhanced Release of Dopamine-($-Hydroxylase from Sympathetic Nerves by Calcium and Phenoxybenzamine and i t s Reversal by Prostaglandins. Proc. Nat. Acad. S c i . , U.S.A., _68: 2227-2230 (1971 ) Johnson, G.L., Kahn, J.B., and Mansfield, J. Cocaine-Like Ef-fec t s of Some Antihistaminics. Fed. P r o c , _24: 515 (1965) Katz, B. The Transmission of Impulses from Nerve to Muscle, and the Subcellular Unit of Synaptic Action. Proc. Roy. Soc. Lond., 155: 455-477 (1962) Keen, P.M. and Bogdanski, D.F. Sodium and Calcium Ions i n Up-take and Release of Norepinephrine by Nerve Endings. Amer. J. Physiol., 219: 677-682 (1970) Kirpekar, S.M. and Misu, Y. Release of Noradrenaline by Splen-i c Nerve Stimulation and i t s Dependence on Calcium. J. Physiol. (Lond.), 188: 219-234 (1967) Korolkovas, A. E s s e n t i a l s of Molecular Pharmacology, Back-ground for Drug Design. New York, John Wiley and Sons, 1970, pp. 312-320. Langer, S.Z. Presynaptic Regulation of Catecholamine Release. Biochem. Pharmacol., _23: 1793-1800 (1974) L e i t z , F.H. and Stefano, F.J.E. Desipramine-Induced Release of Norepinephrine from Heart. Biochem. Pharmacol., 19: 1797-1801 (1970) Leo, A., Hansch, C., and E l k i n s , D. P a r t i t i o n C o e f f i c i e n t s and Their Uses. Chem Rev., 71: 525-616 (1971) Lindmar, R., L o f f e l h o l z , K., and Muscholl, E. Unterschiede + +_ swischen Tyramin und Dimethylphenylpiperazin i n der Ca -Abhangigkeit und im z e i t l i c h e n Verlauf der Noradrenalin-Freisetzung am i s o l i e r t e n Kaninchenherzen. Experientia, 23: 933-934 (1967) McNeill, J.H. and Brody, T.M. The E f f e c t of Antihistaminics, Cocaine and Reserpine on Amine-Induced Rat Cardiac Phos-phorylase A c t i v a t i o n . J. Pharmacol. Exp. Ther., 152: 478-487 (1966) McNeill, J.H. and Brody, T.M. The E f f e c t of Various Drug Pre-treatments on Amine-Induced Phosphorylase A c t i v a t i o n and Amine Uptake. J. Pharmacol. Exp. Ther., 162: 121-133 (1968) McNeill, J.H. and Commarato, M.A. Blockade by T r i p t y l i n e Com-pounds of Tyramine Uptake and of Tyramine-Induced Phos-phorylase A c t i v a t i o n i n Cardiac Tissue. Can. J. Physiol. Pharmacol., £7: 735-738 (1969) 119 Maxwell, R.A., Wastila, W.B., and Eckhardt, S.B. Some Factors Determining the Response of Rabbit A o r t i c S t r i p s to d l -Norepinephrine -7-H Hydrochloride and the Influence of Co-caine, Guanethidine, and Methylphenidate on these Factors. J. Pharmacol. Exp. Ther., 151: 253-261 (1966) Montel, H. and Starke, K. E f f e c t s of Narcotic Analgesics and Their Antagonists on the Rabbit Isolated Heart and i t s Adrenergic Nerves. B r i t . J. Pharmacol. , 4_9: 628-641 (1973) Mundo, A.S., Bonaccorsi, A., Bareggi, S.R., Franco, R., Morsel-l i , P.L., Riva, E., and G a r a t t i n i , S. Relationships Be-tween T r i c y l c i c Antidepressant Concentrations and 1-H -Noradrenaline Uptake and Chronotropic E f f e c t i n Isolated A t r i a of Rats. Eur. J. Pharmacol., i n press. Nakatsu, K. and R e i f f e n s t e i n , R.J. Increased Receptor U t i l i z a -t i o n : Mechanism of Cocaine Potentiation. Nature, 217: 1276-1277 (1968) Paton, D.M. Mechanism of E f f l u x of Noradrenaline from Adren-erg i c Nerves i n Rabbit A t r i a . B r i t . J. Pharmacol., 49: 614-627 (1973) R e i f f e n s t e i n , R.J. E f f e c t s of Cocaine on the Rate of Contrac-t i o n to Noradrenaline i n the Cat Spleen S t r i p : Mode of Action of Cocaine. B r i t . J. Pharmacol. , 3_2: 591-597 (1968) R e i f f e n s t e i n , R.J. and Tr i g g l e , C.R. Cocaine-Induced Super-s e n s i t i v i t y i n the Human Umbilical Artery. Can. J. Phys-i o l . Pharmacol., 52: 687-698 (1974) Roach, M.K., Davis, D.L., Pennington, W., and Nordyke, E. E f f e c t of Ethanol on the Uptake by Rat Brain Synaptosomes of ( H-)-dl-Norepinephrine, ( H)-5-Hydroxytryptamine, ( H) GABA, and ( H) Glutamate- :Life Sci,., JL2: 433-441 (1973) Ross, S.B. and Renyi, A.L. I n h i b i t i o n of the Uptake of T r i t i a t e d Catecholamines by Antidepressant and Related Agents. Eur. J. Pharmacol., 2: 181-186 (1967) Rubin, R.P.?. The Role of Calcium i n the Release of Neurotrans-mitter Substances and Hormones. Pharmacol. Rev., 22: 389-428 (1970) Rutledge, CO. The Mechanisms by Which Amphetamine I n h i b i t s Oxidative Deamination of Norepinephrine i n Brain. J. Pharmacol. Exp. Ther. ., 171: 188-195 (1970) 120 Schumann, H.J., and Philippu, A. Release of Catechol Amines from Isolated Medullary Granules by Sympathomimetic Amines. Nature, 193: 890-891 (1962) Seeman, P. The Membrane Actions of Anesthetics and Tranq u i l -i z e r s . Pharmacol. Rev., 24: 583-655 (1972) Shore, P.A. Transport and Storage of Biogenic Amines. Ann. Rev. Pharmacol., 12:: 209-226 (1972) Smith, C.B. The Role of Monoamine Oxidase i n the Intraneuronal Metabolism of Norepinephrine Released by I n d i r e c t l y - A c t i n g Sympathomimetic Amines or by Adrenergic Nerve Stimulation. J. Pharmacol. Exp. Ther., 151: 207-220 (1966) Snyder, S.H. Putative Neurotransmitters i n the Brain: Selec-t i v e Neuronal Uptake, Subcellular L o c a l i z a t i o n , and Inter-actions with C e n t r a l l y Acting Drugs. B i o l o g i c a l Psychiat. ~2:' 367-389 (.1970) 3 Snyder, S.H. and Coyl<|, J.T. Regional Differences i n H-Nor-epinephrine and H-Dopamine Uptake into Rat Brain Homogen-ates. J. Pharmacol. Exp. Ther., 165: 78-86 (1969) Squires, R.F. E f f e c t s of Noradrenaline Pump Blockers on i t s Uptake by Synaptosomes from Several Brain Regions; Addit-i o n a l Evidence for Dopamine Terminals i n the Fr o n t a l Cor-tex. J. Pharm. Pharmacol., 26: 364-367 (1974) Stjarne, L. Tyramine E f f e c t s on Catechol Amine Release from Spleen and Adrenals i n the Cat. Acta Physiol. Scand., 51: 224-2.29 (1961) Thoa, N.B., Costa, J.L., Moss, J., and Kopin, I.J. Mechanism of Release of Norepinephrine from Peripheral Adrenergic Neurones by the Calcium Ionophores X 537A and A 23187. L i f e S c i . , 14: 1705-1719 (1974) Thoa, N.B., Wooten, G.F., Axelrod, J., and Kopin, I.J. Inhib-i t i o n of Release of Dopamine -(^-Hydroxylase and Norepi-nephrine from Sympathetic Nerves by Colchicine, Vinblas-tin e , or Cytochalasin-B. Proc. Nat. Acad. S c i . U.S.A., 69: 520-522 (1972) Thoenen, H. , Hiirlimann, A. , and Haefely, W. Mechanism of Am-phetamine Accumulation i n the Isolated Perfused Heart of the Rat. J. Pharm. Pharmacol., 20.'- 1 - 1 1 (1968) T i t u s , E.O., Matussek, N., Spiegel, H.E., and Brodie, B.B. The E f f e c t s of Desmethylimipramine on Uptake of dl-Norepi-nephrine-7-H i n the Heart. J. Pharmacol. Exp. Ther., 152:: 469-477 (1966) 121 Trendelenburg, U. The E f f e c t of Cocaine on the Pacemaker of Isolated Guinea-Pig A t r i a . J. Pharmacol. Exp. Ther., 161: 222-231 (1968) Viveros, O.H., Arqueros, L. , Connett, R.J., and Kirshner, N. Mechanism of Secretion from the Adrenal Medulla. I I I . Studies of Dopamine-^-Hydroxylase as a Marker for Cate-cholamine Storage V e s i c l e Membranes i n Rabbit Adrenal Glands. Mol. Pharmacol., 5_: 60-68 (1969a) Viveros, O.H., Arqueros, L., Connett, R.J., and Kirshner, N. Mechanism of Secretion from the Adrenal Medulla. IV. The Fate of the Storage Vesicles Following I n s u l i n and Reserpine Administration. Mol. Pharmacol. , _5: 69-82 (1969b) Wakade, A.R. and Furchgott, R.F. Metabolic Requirements for the Uptake and Storage of Norepinephrine by the Isolated L e f t Atrium of the Guinea Pig. J. Pharmacol. Exp. Ther., 163: 123-135 (1968) Weinshilboum, R.M., Thoa, N.B., Johnson, D.G., Kopin, I.J., and Axelrod, J. Proportional Release of Norepinephrine and Dopamine-fi-Hydroxylase from Sympathetic Nerves. Science, 174: 1349-1351 (1971) Wenger, G.R. and Rutledge, CO. A Comparison of the E f f e c t s of Amphetamine and i t s Metabolites, p-Hydroxyamphetamine and p-Hydrc^xynorephedrine, on Uptake, Release, and Cata-bolism of H-Norepinephrine i n Cerebral Cortex of Rat Brain. J. Pharmacol. Exp. Ther., 189::-. 725-732 (1974) Westfall, T.C and Brasted, M. S p e c i f i c i t y of Blockade of the Nicotine-Induced Release of H-Norepinephrine from Adren-ergic Neurons of the Guinea-Pig Heart by Various Pharma-c o l o g i c a l Agents. J. Pharmacol. Exp. Ther., 189: 659-664 (1974) Whitby 3 L.G., Axelrod, J., and Wei1-Malherbe, H. The Fate of H -Norepinephrine i n Animals. J. Pharmacol. Exp. Ther., 132: 193-201 (1961) White, T.D. and Keen, P. The Role_of Internal and External Na + and K on the Uptake of ( H-)- Noradrenaline by Synap-tosomes Prepared from Rat Brain. Biochim. Biophys. Acta, 196: 285-295 (1970) White, T.D. and Keen, P. E f f e c t s of I n h i b i t o r s of (Na + K )-Dependent Adenosine Triphosphatase on the Uptake of Nor-epinephrine by Synaptosomes. Mol. Pharmacol. , 7_: 40-45 (1971) 122 White, T.D., and Paton, D.M. E f f e c t s of E x t e r n a l N a T and K' on the I n i t i a l Rates of N o r a d r e n a l i n e Uptake by Synapto-somes Prepared from Rat B r a i n . Biochim. Biophys. A c t a , 266: 116-127 (1972) Wooten, G.F., Thoa, N.B., Kopin, I . J . , and A x e l r o d , J . ; En-hanced Rele a s e of D o p a m i n e s-Hydroxylase and N o r e p i -nephrine from Sympathetic Nerves by D i b u t y r y l C y c l i c Aden-o s i n e 3 1,5'-Monophosphate and T h e o p h y l l i n e . Mol. Pharm-a c o l . , 9_: 178-183 (1973) Z i a n c e , R.J., A z z a r o , A . J . , and Rutledge, C O . C h a r a c t e r i s t i c s of Amphetamine-Induced Relea s e of N o r e p i n e p h r i n e from Rat C e r e b r a l C o r t e x iri v i t r o . J . Pharmacol. Exp. Ther., 182: 284-294 (1972a) Z i a n c e , R.J. and Rutledge, C O . A Comparison of the E f f e c t s of F e n f l u r a m i n e and Amphetamine on Uptake, Release, and C a t a b o l i s m of N o r e p i n e p h r i n e i n Rat B r a i n . J . Pharmacol. Exp. Ther., 180: 118-126 (1972b) 

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