AN INVESTIGATION INTO THE POSSIBLE RELATIONSHIP OP ADENOSINE TRIPHOSPHATE TO SENSORY SYNAPTIC TRANSMITTER SUBSTANCES by Christopher Robert Muirhead B.A., University of B r i t i s h Columbia, 1955 A Thesis Submitted i n P a r t i a l Fulfilment of The Requirements for the Degree of Master of Science in the Department of Physiology We accept th i s thesis as conforming to the required standard THE UNIVERSITY OP BRITISH COLUMBIA A p r i l , 1962 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be alloiired without my written permission. Department of Physiology The University of British Columbia, Vancouver 8, Canada. Date 26th A p r i l . 1962. ABSTRACT An attempt has been made to determine the r e l a t i o n -ship, i f any, between adenosine triphosphate and the transmitter substance responsible f o r antidromic v a s o d i l a t a t i o n . Extracts of various areas of the central nervous system have been made by d i a l y z i n g boiled, ground brain tissue against d i s t i l l e d water. These extracts were analyzed for; l a b i l e phosphate content by the method of Berenblum and Chain ( 6 6 ) , adenosine triphosphate content by paper chromatography and by the l u c i f e r i n - l u c i f e r a s e enzyme method of Strehler and Totter ( 6 9 ) . . The content of vasodilator a c t i v i t y of extracts from the same areas was determined by the method of Holton ( 3 5 ) . The extracts to be tested were injected i n t o the f a c i a l artery of a rabbit and allowed to flow Into the auricular artery and through the ear. The changes produced In the ear were detected by means of a photoelectric c e l l which measured differences i n the amount of l i g h t passing through the ear. Vasodilatation appeared as a decrease i n the amount of l i g h t transmitted through the ear and vasoconstriction as an increase. It was thought that i f there was a r e l a t i o n s h i p between adenozine triphosphate' and the transmitter material the areas containing the most ATP should also contain the most vasodilator a c t i v i t y . A comparison of the location and concentration of the two substances revealed no such c o r r e l a t i o n . The two most important questions to be answered were: 1. i s ATP the substance responsible for antidromic vasodilata-tion? and 2. i f so, is i t also a sensory synaptic transmitter substance? It was concluded that ATP was u n l i k e l y to be the substance responsible for antidromic vaso d i l a t a t i o n . I f one accepts Dale's hypothesis (48) that a neurone may employ the same transmitter substance at a l l branches of the axon, then i t would also seem to r u l e out ATP as a sensory synaptic trans-mitter agent. Hugh McLennan, Ph.D. ACKNOWLEDGMENT I wish to acknowledge with gratitude the guidance and encouragement given me throughout the course of these investigations by Dr. H, McLennan. I would l i k e to thank Dr. D.H. Copp fo r his kind assistance during my course of studies. My thanks are due also to Mr. R. Walker f o r h i 3 help with the rabbit ear prepar-ations and to Mr. K. Henze for making the i l l u s t r a t i o n s . TABLE OP CONTENTS Page I. INTRODUCTION 1 I I . MATERIALS AND METHODS 18 1. The D i s t r i b u t i o n of "Vasodilator" A c t i v i t y i n the Central Nervous System . 18 Method for Preparing Dialysates of Brain 18 Method for Preparing the Rabbit Ear . . 19 ' , Method for Measuring the Amount of Vasodilatation 21 2. D i s t r i b u t i o n of Adenosine Triphosphate In the Central Nervous System 22 Methods for Determining ATP i n Central Nervous System 22 3. Chromatography 25 4. Chemical Properties of the Vasodilator Substance and ATP 27 Acid-Base S t a b i l i t y 27 D i a l y s i s of the Vasodilator Substance and ATP 28 Treatment of Dialysates with Exchange Resins . 28 II I . RESULTS 30 1. The D i s t r i b u t i o n of Vasodilator A c t i v i t y i n the Central Nervous System 30 2. D i s t r i b u t i o n of Adenosine Triphosphate in the Central Nervous System 32 3. A Comparison of Vasodilator A c t i v i t y with Adenosine Triphosphate Content of the Central Nervous System 34 4. A Comparison of the Chemical Properties of the Vasodilator A c t i v i t y and ATP . . 35 5. Chromatography of Active Extracts for Vasodilator Material and ATP 40 Page 6. Recordings of Vasodilatation Produced in the Rabbit Ear Preparation 42 IV. DISCUSSION 44 BIBLIOGRAPHY 52 LIST OP TABLES Table Page 1. D i s t r i b u t i o n of Vasodilator A c t i v i t y i n Extracts from Various Areas of the Brain of the Cow 31 2. Adenosine Triphosphate Content of Areas of Cow Brain Expressed i n ug./gm. Fresh Brain 33 3. Comparison of the Amounts of ATP and Vasodilator Substance 36 4. The E f f e c t of Various Ion Exchange Resins on Active Dialysates and Solutions of ATP 38 5. Vasodilator A c t i v i t y , Not Due to Acetylcholine or Histamine, of Extracts From Different Regions of the Horse Brain 47 6. D i s t r i b u t i o n of Neurological Active Compounds in Areas of Central Nervous System . . . . . . . 48 - 1 -I . INTRODUCTION: A form of v a s c u l a r r e f l e x phenomenon, wh i c h has s i n c e been named " a n t i d r o m i c v a s o d i l a t a t i o n " was f i r s t d e s c r i b e d by Loveh ( l ) , who showed t h a t when the c e n t r a l end of t h e g r e a t a u r i c u l a r nerve i n t h e r a b b i t ' s ear was s t i m u l a t e d , vasocon-s t r i c t i o n 'occurred I n o t h e r organs a l t h o u g h a marked v a s o d i l a t a -t i o n was i n i t i a t e d i n the ear i t s e l f . T h i s same phenomenon was a l s o d e s c r i b e d by G o l t z ( 2 ) , who observed t h a t s t i m u l a t i o n of c e r t a i n f i b r e s of the s c i a t i c nerve would induce a p e r i p h e r a l v a s o d i l a t a t i o n i n a l o c a l i z e d a rea o f t h e s k i n . I n 1876 S t r i e k e r (3) c o n f i r m e d t h i s f i n d i n g , and f u r t h e r r e p o r t e d t h a t i t c o u l d s i m i l a r l y be brou g h t about by s t i m u l a t i o n o f t h e d i s t a l p o r t i o n o f a s e c t i o n e d p o s t e r i o r r o o t , a f i n d i n g w h i c h seemed t o be i n d i r e c t o p p o s i t i o n t o the B e l l - M a g e n d i e law. 'The B e l l - M a g e n d i e law s t a t e s t h a t p o s t e r i o r r o o t s c o n t a i n s e n s o r y f i b r e s o n l y , and t h e r e f o r e do not conduct i m p u l s e s towards t h e p e r i p h e r y . Because of the apparent c o n t r a d i c t i o n t o t h e B e l l - M a g e n d i e law, S t r i e k e r ' s work was not g e n e r a l l y accepted, u n t i l B a y l i s s (4-8) c o n f i r m e d and extended the s t u d i e s . B a y l i s s showed t h a t t h e f i b r e s con-c e r n e d seemed i n d i s t i n g u i s h a b l e from o r d i n a r y s e n s o r y f i b r e s , and he s u g g e s t e d t h a t t h e r e might be a p e r i p h e r a l nerve network around the a r t e r i o l e s common t o b o t h s e n s o r y and v a s o d i l a t o r f i b r e s . He named t h e phenomenon " a n t i d r o m i c v a s o d i l a t a t i o n " because t h e i m p u l s e s passed i n a d i r e c t i o n o p p o s i t e t o t h a t w h i c h was u s u a l l y the c a s e . Bruce (9) was a b l e t o c o n f i r m B a y l i s s ' t h e o r y w i t h some e x p e r i m e n t s i n w h i c h he p a r a l y z e d s e n s o r y f i b r e s w i t h c o c a i n e and found t h a t no v a s o d i l a t a t i o n was produced on Figure 1. Diagram of Axon Reflex - 2 -applying o i l of mustard to the area of the skin supplied by the sensory f i b r e s . However, sectioning the nerve trunk to the area without anaesthetizing i t did not prevent inflammation from occurring. He also found that when the nerve was allowed to degenerate no inflammatory reaction to the application of mustard o i l occurred. He concluded that the v a s o d i l a t a t i o n required an intact nerve supply and that bhe sensory nerve concerned contained vasodilator f i b r e s to the a r t e r i o l e s . When the skin was stimu-lated nerve impulses could pass in any d i r e c t i o n over the various branches of a neurone and therefore could pass to a vasodilator branch and give r i s e to a d i l a t a t i o n of the a r t e r i o l e s innervated. Langley (10,11,12) also studied antidromic vasodilata-t i o n and demonstrated that stimulation of posterior roots in the cat caused flushing i n the skin of the foot. In experiments in which the abdominal aorta was clamped off he allowed the feet to become quite pale and then stimulated the lumbar posterior roots on one side. He observed a combination of flushing of the pads and toes of the limb on the stimulated side, which he concluded was a r e s u l t of c a p i l l a r y d i l a t a t i o n , while there was a pallor on the opposite side. He reasoned that under the conditions of the experiment, d i l a t a t i o n of the a r t e r i e s might take blood from the c a p i l l a r i e s . As t h i s did not occur, he considered the flushing to have been due to a decrease of c a p i l l a r y tone which allowed them to be s l i g h t l y distended by the venous pressure. He sugg-ested that the vasodilatation might be the r e s u l t either of a special connection of afferent fibres with the c a p i l l a r i e s , or that the stimulated afferent fibres set free metabolites which - 3 -s e c o n d a r i l y gave r i s e t o the d i l a t a t i o n . Langley favoured the l a t t e r theory c h i e f l y because of the r e l a t i v e l y long d e l a y between s t i m u l a t i o n and the s t a r t of the d i l a t a t i o n ( 2 t o 8 seconds) which B a y l i s s had a l s o n o t i c e d . Langley c a l l e d the pathway i n v o l v e d an "axon r e f l e x " . Antidromic v a s o d i l a t a t i o n has s i n c e been shown to occur i n the f r o g (13), the r a b b i t (14) and man (15). Krogh (16,17) was able t o show that the c a l i b r e of cap-i l l a r i e s i s not a f u n c t i o n of the volume of blood coming from the a r t e r i o l e s , and t h a t i n the r e s t i n g s t a t e the c a p i l l a r i e s are i n a s t a t e of c o n t r a c t i o n and t h e r e f o r e c l o s e d to the passage of blood, but that they can be opened by s t i m u l a t i o n of p o s t e r i o r root f i b r e s . He concluded that l o c a l r e a c t i o n s of s k i n v e s s e l s t o mechanical and chemical s t i m u l i were due to axon r e f l e x e s , and that t h e r e must be some mechanism f o r r e g u l a t i n g the c a l i b r e of the c a p i l l a r i e s which would be mainly s i t u a t e d In the c a p i l l a r i e s themselves. Krogh's c o n c l u s i o n that a v a s o d i l a t o r mechanism must be found i n the immediate area of the c a p i l l a r i e s lends credance t o Langley's th e o r y that m e t a b o l i t e s are set f r e e by the a f f e r e n t f i b r e s i n the c a p i l l a r i e s , as any substance r e l e a s e d t o r e g u l a t e the c a l i b r e of the c a p i l l a r i e s would l o g i c a l l y be r e l e a s e d at the s i t e of i t s a c t i o n . G a s k e l l (18), i n 1916, f i r s t made the suggestion as t o the p o s s i b l e nature of the mediator substance causing v a s o d i l a -t i o n by p o s t u l a t i n g that a c i d m e t a b o l i t e s were r e l e a s e d from s k i n c e l l s . S p e c i a l a t t e n t i o n has been focused on humoral t r a n s m i s s i o n s i n c e Loewi's experiments i n 1921 (19) i n which he demonstrated humoral c o n t r o l of the h e a r t , s e v e r a l p i e c e s of evidence s i n c e have been o f f e r e d to strengthen the theory t h a t - 4 -humoral t r a n s m i s s i o n e l i c i t s a n t i d r o m i c v a s o d i l a t a t i o n . As men-t i o n e d above the most obvious evidence f o r humoral c o n t r o l was the long d e l a y which occurred between s t i m u l a t i o n and the onset of d i l a t a t i o n . Before any p a r t i c u l a r compound can be c o n s i d e r e d as a p o s s i b l e humoral substance f o r a n t i d r o m i c v a s o d i l a t a t i o n c e r t a i n c r i t e r i a must be met: (a) can the substance be found i n the nerves or i n the endings, (b) are the nerve f i b r e s concerned capable of syn-t h e s i z i n g the substance, (c) i s there any enzyme capable of d e s t r o y i n g the sub-stance, (d) can the presence of the substance be l o c a l i z e d t o any s p e c i a l area, such as nerve endings i n the c a p i l l a r i e s , (e) i f a s p e c i a l i z e d area can be found, can a p p l i c a -t i o n of the substance produce an e f f e c t i d e n t i c a l t o the one obtained through s t i m u l a t i o n of the nerve f i b r e s i n volved? Lewis and Marvin ( 2 0 , 2 l ) noted the d e l a y between stimu-l a t i o n and d i l a t a t i o n and went on t o do some experiments which seemed u n e q u i v o c a l l y t o show that the d i l a t a t i o n was produced by a humoral agent. They occluded the c i r c u l a t i o n t o an area of s k i n and found t h a t the f l u s h produced by a n t i d r o m i c s t i m u l a t i o n was prolonged, which they i n t e r p r e t e d t o mean th a t the "vaso-d i l a t o r substance" continued t o act d u r i n g c i r c u l a t o r y a r r e s t - 5 -and was only removed when the c i r c u l a t i o n was restored, as the subsidence of the f l u s h seemed to depend on i r r i g a t i o n of the tis s u e s . They also pointed out that while the decline of vaso-d i l a t a t i o n was delayed by c i r c u l a t o r y arrest, the time of onset of the v a s o d i l a t a t i o n was not altered; they found that the per-ipheral vessels relaxed a few seconds after the nerve had been stimulated even though the c i r c u l a t i o n was stopped. They also offered more evidence f o r humoral transmission i n experiments which related the length of stimulation with the duration of the flus h . Lewis and Marvin claimed this indicated that during stimulation a vasodilator substance was released into the tissue spaces and that i f stimulation continued, the concentration tended to r i s e . With longer occlusion of the c i r c u l a t i o n the reaction was prolonged, but only up to a point; a balance was eventually attained between l i b e r a t i o n and removal of the substance so 'that a longer reaction was not induced by lengthening the stimulation. Kibjakow (22) presented evidence which also strength-ened the humoral transmission theory when he observed a vaso-d i l a t o r a c t i v i t y in the rabbit ear preparation present i n blood col l e c t e d from a region of antidromic vasodilatation In the 'cat. It has been found that when skin i s injured by scratch-ing with a needle, by application of burning heat, by freezing or by other physical means, the vascular reaction Is threefold. The vessels d i l a t e l o c a l l y , the skin b l i s t e r s , and the surround-ing skin shows a bright red f l a r e . The f i r s t two reactions are independent of a nerve supply, and the t h i r d reaction i s known to be r e f l e x i n o r i g i n . Lewis (23) has shown that the surrounding - 6 -r e d f l a r e w h i c h can he produced by p u n c t u r e of h i s t a m i n e i n t o normal s k i n depends on an i n t a c t nerve s u p p l y ; the f l a r e was found t o d i s a p p e a r when the nerve s u p p l y t o the r e g i o n had "been a l l o w e d t o d e g e n e r a t e . I n view of t h e s e f i n d i n g s as w e l l as the o b s e r v a t i o n s of Dale (24,25) t h a t h i s t a m i n e shock was r e l a t e d t o a g e n e r a l l o s s o f tone i n t h e c a p i l l a r i e s , h i s t a m i n e was suggest-ed as the substance l i k e l y t o be r e s p o n s i b l e f o r a n t i d r o m i c vaso-d i l a t a t i o n . Lewis and Grant (26) were a b l e t o show t h a t a h i s t a -m i n e - l i k e substance was r e l e a s e d i n i n j u r e d s k i n , w h i l e K w i a t k o w s k i (27) found h i s t a m i n e i n the d i s t a l p a r t s o f s e n s o r y n e r v e s from t h e s k i n and i n t h e nerves which produce a n t i d r o m i c v a s o d i l a t a -t i o n but l i t t l e i n motor f i b r e s . He a l s o found t h a t s t i m u l a t i o n of cut p o s t e r i o r r o o t s i n t h e cat l i b e r a t e d a h i s t a m i n e - l i k e s u b s t a n c e i n t o venous b l o o d , and c l a i m e d t h a t t h i s s t r e n g t h e n e d the t h e o r y t h a t h i s t a m i n e i s the v a s o d i l a t o r s u b s t a n c e . I b r a h i m , S t e l l a , and T a l a a t (28) have a l s o c o n c l u d e d t h a t h i s t a m i n e causes the v a s o d i l a t a t i o n produced by p o s t e r i o r r o o t s t i m u l a t i o n . They found t h a t h i s t a m i n e was r e l e a s e d from the s k i n but not f r o m the muscles a f t e r a n t i d r o m i c s t i m u l a t i o n of t h e nerve f i b r e s s u p p l y i n g the a r e a , but c o n c l u d e d t h a t h i s t a -mine was r e l e a s e d from the t i s s u e s s u p p l i e d by the nerves r a t h e r t h a n f r o m the nerve f i b r e s t h e m s e l v e s . They a l s o have found t h a t a n t i h i s t a m i n e compounds reduced a n t i d r o m i c v a s o d i l a t a t i o n but d i d not a f f e c t the d i l a t a t i o n produced by i n t r a - a r t e r i a l i n j e c -t i o n of a c e t y l c h o l i n e , which t h e y c l a i m e d t o be e v i d e n c e t h a t the a n t i h i s t a m i n e compounds were p r o d u c i n g t h e i r e f f e c t by a c t i n g d i r e c t l y on h i s t a m i n e i t s e l f . E v i d e n c e has a l s o been o f f e r e d by Unger ( 2 9 , 3 0 , 3 1 ) t o - 7 -support the t h e o r y t h a t h i s t a m i n e r e l e a s e causes a n t i d r o m i c v a s o -d i l a t a t i o n . He has shown t h a t a h i s t a m i n e - l i k e s u b s t a n c e i s r e l e a s e d from p e r i p h e r a l ends of a f f e r e n t f i b r e s d u r i n g a n t i d r o m i c a c t i v i t y . He c a l l e d t h e s u b s t a n c e h i s t a m i n e - l i k e because a l -though i t has not been d e f i n i t e l y i d e n t i f i e d i t s c h e m i c a l p r o p e r -t i e s suggest i t t o be h i s t a m i n e . As a b i o l o g i c a l t e s t he has shown t h a t the substance l i b e r a t e d was c a p a b l e of augmenting g a s t r i c s e c r e t i o n , i n a manner d u p l i c a t e d o n l y by comparably s m a l l doses of h i s t a m i n e . Von E u l e r and Astrom ( 3 2 ) , s t u d y i n g segments of i s o l a t e d p e r i p h e r a l nerve from c a t t l e , have found t h a t under c e r t a i n c o n d i t i o n s a h i s t a m i n e - l i k e s u b s t a n c e was r e -l e a s e d from one end o f the segment f o l l o w i n g e l e c t r i c a l s t i m u l a -t i o n of the o t h e r , w h i c h t h e y c l a i m e d t o i n d i c a t e t h a t h i s t a m i n e was the t r a n s m i t t e r s u b s t a n c e i n v o l v e d i n a n t i d r o m i c v a s o d i l a t a -t i o n . On bhe oth e r hand, c o n s i d e r a b l e doubt has been c a s t upon the l i k e l i h o o d of h i s t a m i n e a c t i n g as a v a s o d i l a t o r sub-s t a n c e i n t h e body. U n l i k e I b r a h i m et a l . ^28), P a r r o t and L e f e b v r e ( 3 3 ) ; Prumin, % a i , and Wang (34) and H o l t o n and P e r r y (35) have a l l been unable t o show t h a t a n t i h i s t a m i n e compounds b l o c k a n t i d r o m i c v a s o d i l a t a t i o n produced by nerve s t i m u l a t i o n , w h i c h would be e x p e c t e d t o happen i f h i s t a m i n e were the t r a n s m i t t e r s u b s t a n c e . F u r t h e r , Chauchard (36) has been ab l e t o show t h a t h i s t a m i n e a c t u a l l y i n h i b i t s t r a n s m i s s i o n i n s e n s o r y neurones w h i c h would not be e x p e c t e d i f h i s t a m i n e were the compound i n q u e s t i o n . P a r r o t (37) found t h a t s t i m u l a t i o n of lumbar p o s t e r i o r r o o t s caused the r e l e a s e of a compound whose p r o p e r t i e s were s i m i l a r t o t h o s e of a d e r i v a t i v e o f a d r e n a l i n e . - 8 -He a l s o showed that a f t e r I r r i t a t i o n of the s k i n , histamine was r e l e a s e d by the i n j u r e d c e l l s , and t h i s was able t o s t i m u l a t e the p e r i p h e r a l endings of the v a s o d i l a t o r f i b r e s and e l i c i t a vaso-d i l a t o r axon r e f l e x . He suggested that histamine may not act as a mediator at the end of the axon r e f l e x but as the stimulus at i t s o r i g i n . More r e c e n t i n v e s t i g a t i o n s (38) have not supported the histamine t r a n s m i t t e r t h e o r y and the f u n c t i o n a l s i g n i f i c a n c e of histamine present i n the nervous system remains obscure. Dale and Fe l d b e r g demonstrated the r e l e a s e of a c e t y l -c h o l i n e on s t i m u l a t i o n of motor f i b r e s t o perfused v o l u n t a r y muscle (39,40,41) and i t has been suggested t h a t t h i s compound might a l s o be the mediator of antidromic v a s o d i l a t a t i o n (42). Experimental evidence has been o f f e r e d t o support the theory that a c e t y l c h o l i n e i s r e l e a s e d d u r i n g a n t i d r o m i c v a s o d i l a t a t i o n by Wybauw (43,44,45,46). In a s e r i e s of experiments on the per-f u s a t e of the hind limb of a cat c o l l e c t e d d u r i n g a n t i d r o m i c s t i m u l a t i o n of p o s t e r i o r r o o t s , he was able t o demonstrate the presence of a substance which had the same pharmacological e f f e c t s as a c e t y l c h o l i n e on the i s o l a t e d f r o g ' s h e a r t , the e s e r i n i z e d l e e c h muscle and the blood pressure of a c h l o r a l o s e d c a t . However, other observations have been made which i n d i -cate that a c e t y l c h o l i n e i s u n l i k e l y t o be the chemical mediator i n a n t i dromic v a s o d i l a t a t i o n . Holton (35) found that the vaso-d i l a t a t i o n was not i n h i b i t e d by a t r o p i n e nor was i t enhanced by e s e r i n e , so th a t although the r e l e a s e of a c e t y l c h o l i n e p e r i p h e r -a l l y on s t i m u l a t i o n of t r a n s e c t e d p o s t e r i o r r o o t s i s not denied i t seems u n l i k e l y that t h i s substance i t s e l f c o u l d be r e s p o n s i b l e f o r a n t i dromic v a s o d i l a t a t i o n (47). Dale (48) reasoned that a sensory neurone would l i k e l y release the same transmitted substance peripherally and c e n t r a l l y , and i f this were true i t would be expected that the posterior roots but not the anterior roots would contain the transmitter substance. Hellauer and Umrath (49,50) have found posterior root extracts to contain a highly active vasodilator material which was absent i n anterior root extracts. They also found that the vasodilator a c t i v i t y of the extracts was reduced by incubation with fresh brain tissue and that t h i s destruction was i n h i b i t e d by the addition of strychnine to the mixture. They suggested that the vasodilator substance was i d e n t i c a l to the central sensory transmitter and that strychnine i n h i b i t e d i t s normal enzymic breakdown, the presence of a destroying enzyme being one of the c r i t e r i a for a transmitter substance. I f t h i s were so, i t would be expected that strychnine should potentiate antidromic vasodilatation, by analogy with the action of eserine in cholin-ergic nerve transmission, i f the enzyme i s present at the periph-e r a l ends of the neurone. This has not been found to be the case (35). In order to c l a r i f y the problem further Holton (35) has attempted to measure the effects of s p e c i f i c antagonists and synergists of acetylcholine and histamine, as well as the effect of strychnine on antidromic vasodilatation. The e f f e c t of a c e t y l -choline i t s e l f was measured and was fcund to produce a vasodila-t a t i o n of less than a minute's duration, while antidromic stimu-l a t i o n produced a d i l a t a t i o n l a s t i n g from 3 to 4 minutes. Injec-t i o n of 1Q>^ histamine produced a vasoconstriction, while smaller do3es tended to give a mild d i l a t a t i o n . Atropine, which i n h i b i t s - 10 -most peripheral actions of acetylcholine, was found to abolish the effects of injected acetylcholine but not those of antidromic nerve stimulation. Eserine, an anticholinesterase, was found to diminish the effects of antidromic stimulation rather than poten-t i a t e them, while the action of injected acetylcholine was en-hanced. Mepyramine, an antihistamine, was found to abolish the effects of injected histamine completely but not the e f f e c t s of stimulation. From these findings Holt on suggested that antidromic vasodilatation i s brought about by the l i b e r a t i o n of a chemical transmitter but the long latency and duration as w e l l as the pharmacological evidence, make i t u n l i k e l y that the transmitter is either acetylcholine or histamine. Two other facts make i t unl i k e l y that histamine is the transmitter, (a) histamine does not disappear from sensory nerves of the rabbit's ear on degener-ation and (b) histamine appears to i n h i b i t transmission i n sensory neurones (36). As strychnine did not potentiate the response t o antidromic stimulation i t seems u n l i k e l y that the transmitter l i b e r a t e d is destroyed by a strychnine-sensitive enzyme. Holton suggested, however, that t h i s l a s t observation did not exclude the p o s s i b i l i t y that the transmitter substance is i d e n t i c a l with the central synaptic transmitter, and that the enzyme responsible for i t s destruction may be present in the spinal cord but not in the peripheral nerve endings. In another paper Holton (51) found that boiled saline extracts of both posterior and anterior roots contained a sub-stance which causes vasodilatation very s i m i l a r to antidromic vasodilatation when injected a r t e r i a l l y into the rabbit's ear. - 11 -A v a s o d i l a t o r e f f e c t could a l s o be obtained from e x t r a c t s of acetone d r i e d powders of both r o o t s and there appeared t o be no d i f f e r e n c e i n the content of the a c t i v e substance when e x t r a c t s from the two s e t s of r o o t s were compared. The a c t i v e substance was heat s t a b l e at pH7 and d i a l y s a b l e , but was d e s t r o y e d by b o i l -i n g m i n e r a l a c i d although not by a l k a l i . The chemical p r o p e r t i e s of t h i s v a s o d i l a t o r substance r u l e out v a r i o u s compounds which upon I n j e c t i o n cause a d i l a t a t i o n and which t h e r e f o r e might be the t r a n s m i t t e r i n v o l v e d . Thus substance P i s s o l u b l e i n acetone (52), k a l l i k r e i n i s heat l a b i l e (53), n e c r o s i n i s not d i a l y s a b l e (54), and b r a d y k i n i n i s r e s i s t a n t t o b o i l i n g a c i d (55). The acetone d r i e d powders of both p o s t e r i o r and a n t e r i o r r o o t s l o s t t h e i r a c t i v i t y e n z y m i c a l l y when incubated i n s a l i n e . Fresh p o s t e r i o r r o o t s were found t o r e t a i n t h e i r a c t i v i t y when mashed and incubated w i t h s a l i n e w h ile f r e s h a n t e r i o r r o o t s l o s t t h e i r a c t i v i t y as r a p i d l y as a c e t o n e - d r i e d powders. Holt on (56) has t r i e d v a r i o u s v a s o d i l a t o r compounds t o determine whether any c o u l d produce a d i l a t a t i o n resembling that r e s u l t i n g from a n t i d r o m i c s t i m u l a t i o n . She found that i n j e c t i o n of s o l u t i o n s of adenosine t r i p h o s p h a t e (ATP) s a t i s f i e d t h i s c r i t e r i o n and f u l f i l l e d the chemical s p e c i f i c a t i o n s g i v e n above. These p r o p e r t i e s were shown t o be shared by adenosine diphosphate (ADP), but not adenosine monophosphate (AMP) or adenosine. In experiments on the perfused, i s o l a t e d r a b b i t ' s ear Holt on was able t o show an i n c r e a s e i n a b s o r p t i o n of u l t r a - v i o l e t l i g h t i n the 2 5 5 - 2 6 5 ^ range, c h a r a c t e r i s t i c of purine compounds, on s t i m u l a t i o n of the a u r i c u l a r nerve. She suggested these r e s u l t s - 12 -i n d i c a t e d t hat ATP or a s i m i l a r compound was r e l e a s e d on stimu-l a t i o n of the sensory f i b r e s . H o lton (57) ha3 made an e f f o r t t o determine the content of v a s o d i l a t o r m a t e r i a l i n v a r i o u s areas of the c e n t r a l nervous system, but has not compared these w i t h the ATP contents of these areas. She found t h a t although the absolute v a s o d i l a t o r a c t i v i t y v a r i e d from b r a i n to b r a i n , t h e r e was a d e f i n i t e p a t t e r n of a c t i v i t y w i t h i n each b r a i n , w i t h h i g h e s t c o n c e n t r a t i o n s i n the caudate nucleus and nucleus cuneatus. However, she admits that i t was u n l i k e l y that a l l the v a s o d i l a t o r a c t i v i t y i n any one e x t r a c t could be due t o a s i n g l e chemical substance s i n c e the methods used could not d i s c r i m i n a t e between substances other than a c e t y l c h o l i n e and h i s t a m i n e . I t was maintained that the r e s u l t s showed no c l e a r r e l a t i o n s h i p i n v e r s e or d i r e c t between the vaso-d i l a t o r a c t i v i t y of incubated e x t r a c t s from d i f f e r e n t r e g i o n s of the c e n t r a l nervous system and the d i s t r i b u t i o n of c h o l i n e r g i c neurones. Holton thought t h a t the type of response of the vaso-d i l a t a t i o n t o i n j e c t i o n of e x t r a c t s c o u l d give some i n f o r m a t i o n t o the s i t e of a c t i o n (58). For example, a substance which d i l a t e s the l a r g e v e s s e l s , the c a p i l l a r i e s as w e l l as opening the a r t e r i o - v e n o u s anastomoses, should g i v e a quick response as the m a t e r i a l would be r a p i d l y swept away by the b l o o d . I f the substance d i l a t e d only the c a p i l l a r i e s a prolonged response should r e s u l t as the i n j e c t e d d i l a t o r substance would have to flow through the whole c a p i l l a r y bed before the response would be over. A l s o , one would expect a d e l a y while the substance reaches the c a p i l l a r i e s . I f t h e l a r g e r v e s s e l s were a l r e a d y d i l a t e d and - 1 3 -the arterio-venous anastomoses were open before the i n j e c t i o n the response should be short l i v e d . Holton's experiments demonstrated that the c a p i l l a r i e s were the only vessels to respond t o antidromic stimulation, pro-vided the stimulation was short enough. In t e s t i n g possible trans-mitters she found that adenosine, which d i l a t e s the large vessels, produced a quick response whether the rabbit's ear vessels were d i l a t e d or not. Spinal root extracts, however, were found to give a prolonged response as well as a degree of latency in pre-parations with constricted vessels and closed arterio-venous anastomoses, i f the larger vessels were d i l a t e d and the a r t e r i o -venous anastomoses were open the response was over quickly. Holt on concluded that the vasodilator substance may be l i b e r a t e d in the c a p i l l a r y bed at some distance from the larger vessels and that the c a p i l l a r i e s may be more sensitive to the substance. Furthermore, the vasodilator substance of the s p i n a l root ex-tracts acted primarily on the c a p i l l a r i e s i n the same way as does antidromic stimulation. The prolonged nature of the vasodilata-t i o n indicates that the d i l a t o r substance is not quickly destroyed in the blood. Holton has done various biochemical analyses i n an e f f o r t to Identify more closely the substance causing antidromic vasodilatation ( 5 9 ) . Extracts of acetone powders of posterior and anterior roots were chromatograpbed, intense u l t r a v i o l e t absorption occurred in positions corresponding to ATP and ADP. These spots were eluted and assayed for vasodilator a c t i v i t y , 1 6 $ of the a c t i v i t y was recovered from the ATP portion and 4 $ of the a c t i v i t y was recovered from the ADP portion with respect to the - 14 -o r i g i n a l e x t r a c t . A s e r i e s of determinations were done i n which the a c t i v e substance was compared ag a i n s t pure ATP, as w e l l as enzymic determinations of l a b i l e phosphate content of the e x t r a c t s . U l t r a v i o l e t a b s o r p t i o n was a l s o done t o o b t a i n a rough e s t i m a t i o n of the adenine n u c l e o t i d e present i n the e x t r a c t s . Holton con-cluded that t h e t h r e e separate d e t e r m i n a t i o n s agreed c l o s e l y enough t o be able t o say t h a t the v a s o d i l a t o r a c t i v i t y i n the e x t r a c t s was due t o a mixture of ATP and ADP and small amounts of f u r t h e r breakdown pr o d u c t s . Holton has used the f i r e f l y luminescence method, which Is h i g h l y s p e c i f i c f o r ATP, on p e r f u s a t e s from the s t i m u l a t e d r a b b i t ' s ear (60,61). She observed that ATP was r e l e a s e d i n t o the p e r f u s a t e on a n t i d r o m i c s t i m u l a t i o n of the sensory nerve only a f t e r the nerve had been s e c t i o n e d but not a f t e r i t had degener-ated; as the amount l i b e r a t e d could not be r e l a t e d t o the amount of hemolyzed e r y t h r o c y t e s present she concluded the source of ATP t o be i n t r a c e l l u l a r and from the nerve. However, the amount r e -covered was o n l y 0.16$ of the amount of ATP which had to be i n j e c t e d a r t e r i a l l y t o g i v e a comparable d i l a t a t i o n . T h i s i n t r o -duces one of the most p u z z l i n g aspects of the problem, how t o account f o r the l a r g e d i s c r e p a n c y of 10^ order, between the amount of ATP which must be i n j e c t e d t o d u p l i c a t e the v a s o d i l a t a -t i o n e f f e c t s of s t i m u l a t i o n and the maximum amount of ATP r e c o v e r -ed from the p e r f u s a t e a f t e r s t i m u l a t i o n . Brown, Dale and Peldberg (62) have been able t o r e c o v e r l/lOO of the amount of a c e t y l c h o l -ine i n j e c t e d necessary t o e l i c i t a response comparable t o stimu-l a t i o n of c h o l i n e r g i c nerves. In l i g h t of t h i s r e s e a r c h a d i s -crepancy of 10^ order i n r e c o v e r y of i n j e c t e d ATP seems too l a r g e - 15 -t o i n d i c a t e that i t i s the substance r e s p o n s i b l e f o r the vaso-d i l a t a t i o n . Holton (61) has suggested an e x p l a n a t i o n f o r the d i s -crepancy. In p e r f u s i o n experiments the ATP c o l l e c t e d a f t e r s t i m u l a t i o n had to pass through at l e a s t one l a y e r of c e l l s which would c o n t a i n adenosine t r i p h o s p h a t a s e before r e a c h i n g the lumen of the b l o o d v e s s e l s , and that there i s no convenienb method of p r o t e c t i n g ATP a g a i n s t enzymic breakdown. In a d d i t i o n to the d i s c r e p a n c y mentioned, one a l s o cannot ignore the w e l l known r o l e ATP p l a y s In metabolism through-out the body. Since ATP i s present i n a l l nerve c e l l s and i s so c l o s e l y a s s o c i a t e d w i t h the metabolism of the neurone i t i s not s u r p r i s i n g t hat ATP would f i t some of the c r i t e r i a f o r a t r a n s -m i t t e r substance. Thus ATP i s present i n a l l c e l l s , i n c l u d i n g neurones. Secondly, as the neurones must have some c o n t r o l over t h e i r metabolism, they must be capable of s y n t h e s i z i n g and des-t r o y i n g ATP. I t seems u n l i k e l y , t h e r e f o r e , that such a compound would a l s o be a t r a n s m i t t e r substance. T h i s becomes more appar-ent when one c o n s i d e r s the d i s t r i b u t i o n of a c e t y l c h o l i n e through-out the c e n t r a l nervous system. I t has been shown t h a t not a l l neurones are capable of s y n t h e s i z i n g a c e t y l c h o l i n e , and t h a t t h e r e i s a tendency f o r neurones w i t h h i g h c h o l i n e a c e t y l a s e (the enzyme which s y n t h e s i z e s a c e t y l c h o l i n e ) content t o a l t e r n a t e with c e l l s In which the enzyme i s absent. Sensory impulses reach the b r a i n by a three neurone pathway, the f i r s t and t h i r d of which are d e f i c i e n t i n c h o l i n e a c e t y l a s e while the second has a h i g h a c e t y l c h o l i n e s y n t h e s i z i n g power. It seems reasonable t o - 16 -b e l i e v e t h a t t h i s a l t e r a t i o n of neurones with d i f f e r e n t enzyme a c t i v i t i e s corresponds t o an a l t e r a t i o n of c h o l i n e r g i c and non-c h o l i n e r g i c elements which suggests that a c e t y l c h o l i n e i s prob-a b l y the mediator of t r a n s m i s s i o n across a l a r g e number of synapses i n the c e n t r a l nervous system (63). J u s t such a d i s t r i -b u t i o n of ATP, i n c o n t r a s t , has not been shown to e x i s t , as s t a t e d above, ATP i s present i n a l l body c e l l s . Although ATP does cause v a s o d i l a t a t i o n , there i s s t i l l doubt t h a t i t i s the t r a n s m i t t e r substance i n q u e s t i o n because no s u i t a b l e b l o c k i n g agents are known f o r ATP so i t s e f f e c t s cannot be r u l e d out as are the e f f e c t s of a c e t y l c h o l i n e and histamine f o r which b l o c k i n g agents are known. P l o r e y and McLennan (64) have produced some evidence which i n d i c a t e s t h a t ATP i s not the t r a n s m i t t e r substance. It i s known that when sensory f i b r e s have degenerated the area f o r m e r l y s u p p l i e d by them becomes s e n s i t i z e d t o t h e i r t r a n s m i t t e r substance. Basing t h e i r work on t h i s phenomenon, P l o r e y and McLennan cut segments out of the great a u r i c u l a r nerves of r a b b i t s and allowed the nerves t o degenerate. I f ATP were the t r a n s m i t t e r substance r e s p o n s i b l e then the r e a c t i o n to ATP i n the denervated prepara-t i o n s should be p o t e n t i a t e d . However, t h i s was not found to be the case. When e x t r a c t s of p o s t e r i o r s p i n a l r o o t s were i n j e c t e d i n t o the denervated area the v a s o d i l a t a t i o n was v e r y marked while the responses t o v e n t r a l r o o t s e x t r a c t and s o l u t i o n s of ATP were decreased. The q u e s t i o n which n a t u r a l l y a r i s e s i s what substance i s r e s p o n s i b l e f o r antidromic v a s o d i l a t a t i o n ? T h i s question assumes great importance when one c o n s i d e r s Dale's (48) remark - 17 -that since the bipolar afferent neurones involved in cutaneous f l a r e do not synapse u n t i l they are within the ce n t r a l nervous system, the vasodilator substance released i n the periphery dur-ing antidromic vasodilatation may also be the transmitter at the central synapse of the afferent neurones, which would further suggest that sensory neurones only and not motor neurones, would contain the humoral agent. Peldberg (65) also expresses the same idea when he says that d i f f e r e n t branches of the same axon are u n l i k e l y to have d i f f e r e n t transmitter substances. In t h i s case any Information made available on the substance responsible for antidromic vasodilatation could also perhaps give a valuable insight into the function of the central nervous system i t s e l f . It has been proposed that ATP is the substance respon-si b l e for antidromic vasodilatation (56) and evidence has been produced to support t h i s suggestion. However, by the same token, evidence has also come to l i g h t which appears to contradict t h i s proposal (61,64), consequently the problem remains unsolved. S p e c i f i c a l l y i t was in an e f f o r t to resolve these apparent contradictions and to determine just what role ATP had, i f any, in antidromic vasodilatation that the following research was undertaken. - 18 -I I . MATERIALS AND METHODS: 1. The D i s t r i b u t i o n of "vasodilator" a c t i v i t y In the Central Nervous System. (a) Method for Preparing Dlalysates of Brain Beef brain tissue was obtained from fr e s h l y k i l l e d c a t t l e , known weights of tissue were taken from the areas of cerebral cortex, caudate nucleaus, thalamus, sub-co r t i c a l white matter, r e t i c u l a r formation, f l o o r of the fourth v e n t r i c l e , cerebellar cortex, optic chiasma, lentiform nucleus, nuclei cuneatus et g r a c i l i s , superior corpora quadrigemina, i n f e r i o r corpora quadrigemina, dorsal roots, ventral roots, dorsal columns of s p i n a l cord, and deep cerebellar structures. As quickly as possible the fresh tissue was b o i l e d for f i v e minutes i n a volume of d i s t i l l e d water equal to two times the weight of the fresh tissue. This was necessary i n order to destroy any i n a c t i v a t i n g enzymes present i n the t i s s u e . The boiled material was then homogenized by grinding i n a mortar with fine sand or i n a Waring blendor, depending on the volume involved. The homogenate was placed i n cellophane d i a l y s i s tubing and dialyzed against d i s t i l l e d water twenty times the volume of the homogenate for forty-eight hours at 5°C, The dialysates were evaporated to dryness i n a f l a s h evaporator and redissolved in physiological saline which was one-third the volume of the fresh t i s s u e . These extracts were stored at -5°C. u n t i l used. Certain extracts were treated to remove the majority of potassium present, and t h i s was done by adding one-tenth the extract volume of 11.6 N. perchloric acid, f i l t e r i n g out the resultant p r e c i p i t a t e , and n e u t r a l i z i n g to - 19 -pH7 by adding 1 N. sodium hydroxide. These solutions were stored at -5°C. also. (b) Method for Preparing the Rabbit Ear The vasodilator a c t i v i t y of the extracts was deter-mined by i n j e c t i n g 0.125 cc. of the material to be tested into the rabbit's ear and measuring the amount of vasodilatation produced.in the ear. The rabbit was f i r s t anaesthetized with nembutal (50 mg./ml.). A tracheal cannula was inserted f i r s t to ensure adequate v e n t i l a t i o n , a cannula was then inserted into the femoral vein of one of the hind legs so more anaes-t h e t i c could be given i f necessary. A t h i r d cannula (a small polyethylene tube) was inserted i n t o the f a c i a l artery, point-ing towards the heart, on the same side of the rabbit as the ear to be tested. A l l the major branches of the carotid artery including the i n t e r n a l carotid artery were t i e d off, with the exception of the auricular artery. This allowed free passage to the ear of the Injected extract. On i n j e c t i o n the extract was forced to flow backward.down the f a c i a l artery into the external carotid artery where i t was then swept into the auricu-l a r artery by the blood flow and thereby c a r r i e d to the ear i t s e l f . To avoid interference from extraneous nerve Impulses the auricular nerve of the ear to be tested was sectioned before the experiments began. The rabbit was also given an i n j e c t i o n of atropine ( l mg./Kg.), a blocking agent for acetylcholine, and benadryl (10 mg./Kg.), a blocking agent for histamine, before experimen-ta t i o n in order to rule out any effects of acetylcholine and histamine. - 20 -Key A. Polyethylene Cannula B. Auricular Artery C. F a c i a l Artery Cannulated D. Superior Thyroid Artery Tied Off E. Direction of Injection Flow F. Internal Carotid Tied Off G. Direction of Blood Flow H. Common Carotid I. Tracheal Cannula J. Trachea Figure 2. Cannulation for Rabbit's Ear Preparation - 21 -(c) Method for Measuring the Amount of Vasodilatation Vasodilatation i n the rabbit's ear was measured elect rophotometrically; the white rabbit's ear was shaved and made more translucent by spreading p a r a f f i n oil over " i t . A l i g h t with a green f i l t e r over i t was shone through the ear, which was taped to a p l a s t i c box; beneath the ear was a clear window i n the box which allowed the l i g h t passing through the ear to f a l l onto a photoelectric c e l l . Changes in the i n t e n s i t y of l i g h t passing through the ear were detected as changes in i n t e n s i t y of a current produced by the photocell. These changes were amplified and recorded on a moving s t r i p of f i l m which passed In front of an oscilloscope screen, A vasodilatation, therefore, was shown as a curve r i s i n g from a predetermined base l i n e on the f i l m s t r i p and s i m i l a r l y , a vasoconstriction was shown as a f a l l below this base l i n e (see Figure 6.). Before each i n -ject i o n of extract, a photograph of the s t a b i l i z e d base l i n e was taken to enable any change i n base l i n e to be measured. Green F i l t e r Rabbit Ear C l e a r Window Light-tight Box To Amplifier and Oscilloscope Figure 3. System for Measuring Vasodilatation in Rabbit's Ear - 22 -2. D i s t r i b u t i o n of Adenosine Triphosphate i n the Central Nervous System. (a) Methods for Determining; ATP in Central Nervous System (i) Phosphomolybdic acid method (66). This meth-od i s based on the s o l u b i l i t y of reducible phosphomolybdic acid i n isobutyl alcohol. It is e s s e n t i a l l y the reduction of colour-less phosphomolybdic acid to the blue reduced form by shaking the alcoholic extract with an a c i d i f i e d aqueous s o l u t i o n of stannous chloride. The amount of inorganic phosphate present i n the d i a -lysates was determined f i r s t , then the dialysates were hydro-lyzed in hot 1 N. hydrochloride acid for seven minutes and the phosphate again determined. The difference between the two measurements was taken to be the acid l a b i l e phosphate which was assumed to be adenosine triphosphate. Five ml. of the dialysate to be determined for phos-phate content was put into a separatory funnel and the following were added: 0.5 ml. of 10 N. sulphuric acid, 2 ml. d i s t i l l e d water, 2.5 ml. of an 85% solution of ammonium molybdate, and 10 ml, of isobutyl alcohol. This mixture was shaken for two minutes and the aqueous layer discarded. The alcoholic solution was washed with two portions of 5 ml. each of 1 N. sulphuric acid and then shaken with 15 ml. of a solution of 0.4 gm. stannous chl o r i d e / ml. concentrated hydrochloric acid which had been d i l u t e d two hundred times with 1 N. sulphuric acid for t h i r t y seconds, and the aqueous layer discarded. The r e s u l t i n g blue solution was poured into a 10 ml. volumetric fl a s k , the - 23 -separately funnel washed with ethyl alcohol and the solution made up to 10 ml. with the washings. The solutions were read i n a K l e t t colorimeter and the amount of phosphate determined from a standard curve which was prepared with each experiment. ( i i ) The f i r e f l y luminescence enzyme method for determining ATP in dialysates of areas of the CNS. Strehler and Totter (68,69) have reported that l i g h t emission by l i v i n g organisms was a product of a series of chemical reactions and that i t was possible under c e r t a i n conditions to extract from luminous organs a heat stable substance, l u c i f e r i n , and a heat l a b i l e substance, l u c i f e r a s e , which when mixed together would emit l i g h t . The l i g h t emitting step depended upon the oxidation of the l u c i f e r i n molecule i n the presence of oxygen and the enzyme, l u c i f e r a s e . It was shown that a p a r t i a l l y p u r i f i e d Cypridlna l u c i f e r i n (from a species of luminous fish) contained l a b i l e phosphate groups which were removed during the l i g h t emitting reaction and i t was postulated that the energy derived from the breakdown of the sidechain was conserved as phosphate energy bonds. Support for t h i s suggestion that l a b i l e phosphate groups are concerned in the luminescent reaction was obtained with extracts from f i r e f l y material. It was possible to restore l i g h t emission i n extracts which had ceased to luminesce by add-ing ATP and a divalent ion Mg+-r. Implicit in the finding that f i r e f l y luminous organ extracts which had become dark would respond to added ATP by the production of l i g h t was the p o s s i b i l i t y of using t h i s phenom enon for the assay of ATP i n l i v i n g material under various - 24(a) -j Standard Curve of Luciferine - Luciferote Enzyme Method 3000 n ^jg /ml Adenosine Triphosphate - 24 -c o n d i t i o n s . T h i s has been found t o be the case u s i n g both standard and h i g h l y r e f i n e d l i g h t - m e a s u r i n g d e v i c e s . ATP can be determined d i r e c t l y i n mixture with other compounds by mak-ing use of a l i n e a r r e l a t i o n between l i g h t output and ATP added ,to an e x t r a c t of f i r e f l y l a n t e r n s . The method used i n t h i s r e s e a r c h was based on the method developed by S t r e h l e r and T o t t e r w i t h seme m o d i f i c a t i o n s by H o l t o n . F i f t y mg. of d r i e d f i r e f l y l a n t e r n s were e x t r a c t e d w i t h 5 ml. of 0.1 M. s r s e n a t e b u f f e r at pH 7.4, the mixture was c e n t r i f u g e d and 20 mg./ml. of magnesium sulphate was added t o the supernatant l i q u i d . For each d e t e r m i n a t i o n 1.0 ml. of t h i s enzyme mixture was pla c e d i n a tube, 1.0 ml. of d i a l y s a t e was added, the s o l u t i o n mixed, a stopwatch s t a r t e d , and e x a c t l y 15 seconds l a t e r the luminescence was measured. Standard s o l u t i o n s of known c o n c e n t r a t i o n s of ATP were run with each s e t of d i l u -t i o n s of the unknown d i a l y s a t e s . The l i g h t e m i t t e d by the luminescent r e a c t i o n was measured by means of a commercial s c i n t i l l a t i o n c ounter from which the phosphor had been removed and r e p l a c e d by a c l e a r window of perspex •§• inch t h i c k . A h o l e the s i z e of the r e a c t i o n tubes was d r i l l e d i n t o the t h i c k n e s s of the window and a m i r r o r , s i l v e r e d s i d e f a c i n g i n , was attached t o the window on the s i d e opposite the p h o t o m u l t i p l l e r tube so that most of the l i g h t emitted would be r e f l e c t e d back i n t o the counter. The whole device was enclosed i n a l i g h t - p r o o f e n c l o s u r e . - 25 -L i g h t T i g h t Box E l e c t r o n i c Counter P h o t o m n l t l n i 1ftr Tube C l e a r Window with Hole f o r Reaction Tubes M i r r o r F i g u r e 5. ' System f o r Measuring F i r e f l y Luminescence 3. Chromatography. The b a s i c technique was the same i n a l l ca s e s ; v a r y -i n g amounts of ATP were a p p l i e d t o the papers and g e n e r a l l y the papers were developed by the two di m e n s i o n a l descending method, the d i f f e r e n c e s being i n the s o l v e n t s used and the methods of d e t e c t i n g the ATP i n the developed chromatograms. (a) 5, 10, 20 and 30 lambda amounts (0.005, 0.010, 0.020, 0.030 ml.) of a 1 mg./ml. s o l u t i o n of ATP were s p o t t e d on four f u l l s i z e sheets of Whatman #4 chromatography paper. The papers were developed u s i n g the two dimensional descending s o l v e n t run t e c h n i q u e . The f i r s t dimension s o l v e n t system was n. propanol, ammonium hydroxide, water (60:30:10) and was run f o r 8 hours. The second dimension s o l v e n t system was t e r t : b u t a n o l , p i c r i c a c i d , water (80 ml., 4 gm., 20 ml.) which was run f o r 5 hours. The papers were then sprayed with a c o l o u r i z -ing s o l u t i o n which contained 5 ml. of 60$ p e r c h l o r i c a c i d , 25 ml. of 4$ ammonium molybdate s o l u t i o n , 10 ml. of 1 N. hydro-c h l o r i c a c i d , and 60 ml. of water. The papers were then heated f o r 15 minutes at 85°C. - 26 -(b) 25, 50, 75 and 100 lambda amounts of 1 mg./ml. solution of ATP were spotted on sheets of Whatman #4 chromato-gram paper as in (a), the solvent systems and techniques were the same as i n (a) except the second dimension solvent system was allowed t o run for 20 hours instead of 5. The colourizing solution used was the same as (a) except the sprayed papers were heated after being allowed t o dry completely f i r s t . (c) 20, 50, 75 and 100 lambdas of 1 mg./ml. solution of ATP were spotted on Whatman #4 papers, and the papers were developed as above. The colourizing solution used th i s time contained 1 gm. of ammonium molybdate In 8 ml. of water, 3 ml. of concentrated hydrochloric acid, 3 ml. of 70$ perchloric acid, and thi s mixture then di l u t e d to 100 ml. with acetone. The developed papers were dipped in this solution, allowed to dry, and exposed to u l t r a v i o l e t l i g h t f o r 30 minutes to bring out the colour. (d) Four papers of Whatman #4 grade, each having the equivalent of 100 mg. of ATP spotted on them, were run i n two dimensions using the descending solvent run technique. The two solvent systems were n-propanol, ammonium hydroxide, water (60:30:10) and isopropanol, saturated ammonium sulphate solu-ti o n , and water (2:79:19); the f i r s t system was run for 18 hours, the second for 4-g- hours (70) . The same colourizing solution was used as above. (e) Four papers were spotted with 500 lambdas (0.5 ml.) of a water solution of acetone powders made from the caudate nucleus, 50 mg. of ATP were also spotted to serve as a marker - 27 -compound; a second 50 mg. portion was also put on aft e r the f i r s t solvent system was run, and t h i s served as a marker compound during the second solvent run. The developed papers were scanned f i r s t with u l t r a v i o l e t l i g h t and then dipped i n the colourizing solution used i n (c). (f) Five hundred lambdas (0.5 ml.) of the dialysate from the thalamus region was spotted on each of 4 papers, and 50 mg. of ATP was also spotted as a marker compound. The chrom-atograms were developed and the spots coinciding with the marker spots were eluted with d i s t i l l e d water and analyzed for acid l a b i l e phosphate as mentioned above by the method of Berenblum and Chain. (g) One hundred lambda amounts (0.1 ml.) of the dia-lysates of the caudate nucleus and the cerebral cortex were spotted on 3 MM. chromatogram paper, the papers were developed by the descending technique i n n-propanol, ammonium hydroxide, water (60:30:10) for 16 hours. The length of the developed chromatogram from o r i g i n to solvent front was divided into ten sections, each of these sections was eluted with d i s t i l l e d water, evaporated to dryness, and redissolved i n physiological saline of a volume approximately equal to the o r i g i n a l dialysate sample volume. These samples, representing the ten areas on the chrom-atogram, were then tested on the rabbit ear preparation described above for vasodilator a c t i v i t y , 4. Chemical properties of the vasodilator substance and ATP, (a) Acid-base stab i l i t y . -One m i l l i l i t e r portion of the dialysate from the caud-ate nucleus was boiled with an equal volume of 2 N. hydrochloric - 28 -acid and neutralized on cooling, another portion was boiled with an equal amount of 1 N. sodium hydroxide and also neutral-ized on cooling, a t h i r d portion was boiled for the same time but no acid or base was added to i t . The same procedure was followed with portions of the dialysate from the crebral cortex area. These samples were then tested on the rabbit ear prepara-t i o n for vasodilator a c t i v i t y . The same thing was also done with a 1 mg./ml. solution of ATP. (b) D i a l y s i s of the vasodilator substance and ATP Experiments were done t o t r y and determine how much ATP was dialysed by the method used for d i a l y z i n g the areas of brain for vasodilator material. Five m i l l i l i t e r s of a 1 mg./ml. solution of ATP were placed In a d i a l y s i s bag and dialyzed against d i s t i l l e d water in the cold for 48 hours. At the end of thi s time the dialysate and the contents of the bag were both analyzed for ATP content by the f i r e f l y luminescence enzyme method mentioned above. (c) Treatment of dialysates with exchange resins Two active dialysates were treated with ion exchange resins in an e f f o r t to determine some basic chemical properties of the substance responsible for vasodilatation. Two m i l l i l i t e r portions of the dialysates from the caudate nucleus and the cerebral cortex were treated with por-tions of Dowex-50 exchange r e s i n , separate portions were simi-l a r l y treated with Dowex-1 exchange r e s i n . After f i l t e r i n g the mixtures to separate out the resins, the dialysates were then tested on the rabbit ear preparation for vasodilator a c t i v i t y . - 29 -Further. 1 m i l l i l i t e r portions of the same dialysates were treated with re s i n IR-45, a weak anion exchanger, and IRC-50, a weak cation exchanger. Before treatment both resins were washed thoroughly; IR-45 in 1 N. hydrochloric acid to ob-ta i n the chloride form, and then twice i n d i s t i l l e d water, IRC-50 with 1 N. sodium hydroxide to obtsin the hydroxide form, and then twice i n d i s t i l l e d water. After shaking the dialysate with the resins, the l i q u i d was saved. The remaining r e s i n IR-45, was eluted with 1 N. and 3 N. ammonium hydroxide, the eluate was evaporated to dryness and redissolved i n phy s i o l o g i c a l saline and neutralized. S i m i l a r l y , IRC-50 was eluted with 3 N. acetic acid, t h i s eluate being also evaporated to dryness and redissolved i n physiological saline and neutralized. These samples were a l l also tested for vasodilator a c t i v i t y . - 30 -I I I . RESULTS: Holton (57) has tested various areas of the central nervous system for vasodilator a c t i v i t y and has reported that a d e f i n i t e pattern of d i s t r i b u t i o n existed, the most a c t i v i t y being found i n caudate nucleus and nuclear cuneatus. The following groups of experiments were undertaken i n an e f f o r t to determine the d i s t r i b u t i o n of the vasodilator a c t i v i t y and to see i f the d i s t r i b u t i o n p a r a l l e l e d that claimed by Holton. Also, i t was f e l t that i f ATP were the transmitter substance or related to the substance i n question, the d i s t r i b u t i o n of ATP in the central nervous system should be the same as, or clo s e l y s i m i l a r to, the d i s t r i b u t i o n of vasodilator a c t i v i t y , therefore the d i s t r i b u t i o n and concentration of ATP in the brain was also determined. 1. The d i s t r i b u t i o n of vasodilator a c t i v i t y i n the central nervous system Table I shows the r e l a t i v e amounts of the substance responsible f o r producing vasodilatation i n the rabbit's ear found i n the areas of the central nervous system mentioned. The degree of vasodilatation produced by dialysates of the areas are graded from the strongest effect to the weakest by a number of +'s; ++++being the strongest effect and + being the weakest. Where no vasodilatation was obtained an 0 is indicated. Holton found caudate nucleus and nucleus cuneatus to contain the most vasodilator a c t i v i t y followed by thalamus and hypothalamus. The results here agree generally with those of Holton i n that caudate nucleus contains the greatest amount of vasodilator material, while the thalamus also contains much - 31 -TABLE I: DISTRIBUTION OF VASODILATOR ACTIVITY IN EXTRACTS FROM VARIOUS AREAS OF THE BRAIN OF THE COW Area of CNS. Tested Expt. 1 Expt. 2 Expt. 3 Expt. In f e r i o r c o l l i c u l u s - - ++-++ -IVth v e n t r i c l e f l o o r (mixed) 0 - - 0 Lenticular nucleus ( c e l l u l a r ) -++- + 0 -Thalamus ( c e l l u l a r ) - n - ++ ++ Caudate nucleus ( c e l l u l a r ) +++ -hi-Reticular formation (mixed) 0 - + + + -Cerebral cortex (precentral areas) +++ •++-»- + -f-n-Superior c o l l i c u l u s - 0 0 -Subcortical white matter +- -t-+ 0 -Hypothalamus ( c e l l u l a r ) - 0 0 Cerebellar cortex ( c e l l u l a r ) -t--t-+-t- - -f-+"t -i Anterior roots (white) + + - -Nucleic cuneatus et g r a c i l i o ( c e l l u l a r ) 0 - -Optic chiasma (white) 0 - - -Deep cerebellum (mixed) 0 0 - -Dorsal roots - - -Ventral roots - - - -+ "t-+ Posterior columns (white) +++ -n-t- mt - 32 -a c t i v i t y . There the agreement ends. Holton claims hypothalamus has much a c t i v i t y while these results indicate i t has no a c t i v -i t y ; these results also show that cerebral cortex contains a great deal of a c t i v i t y but Holton reported l i t t l e a c t i v i t y i n the same region. 2. D i s t r i b u t i o n of adenosine triphosphate i n the central nervous system An e f f o r t was made to discover a c o r r e l a t i o n between the d i s t r i b u t i o n of vasodilator a c t i v i t y and the d i s t r i b u t i o n of ATP i n the central nervous system. I f ATP were in some way connected with antidromic vasodilatation, the d i s t r i b u t i o n of vasodilator a c t i v i t y should be similar to that of ATP. The method employed was the f i r e f l y luminescence enzyme method which i s highly s p e c i f i c for ATP and was described i n the methods and materials above. The determinations were carried out on water dialysates as well as acetone powders of areas of the central nervous system In order to determine whether the methods of i s o l a t i o n might be a f f e c t i n g the amount of ATP recov-ered. Table II shows the amounts of ATP expressed in^g./gm. of fresh brain tissue i n water dialysates as well as acetone powders from the areas indicated. The water dialysate of the f l o o r of the fourth vent-r i c l e contained the most ATP, followed by i n f e r i o r and superior c o l l i c u l u s . The caudate nucleus which contained the most vaso-d i l a t o r a c t i v i t y (see Table I) did not contain the amounts of ATP which one would expect consistent with i t s vasodilator - 33 -TABLE 2: ADENOSINE TRIPHOSPHATE CONTENT OP AREAS OP COW BRAIN EXPRESSED IN^g./gm. FRESH BRAIN Area of Brain Tested W a * e r D i a l y s a t e Acetone Powder X Cf 1 ( 0 IVth v e n t r i c l e f l o o r 25.3 76.0 0 0 Inf e r i o r c o l l i c u l u s 20.7 62.0 45.0 18.0 Superior c o l l i c u l u s 11.2 33.6 35.2 26.4 Caudate nucleus 4.1 12.2 26.7 20.0 Cerebral cortex 3.7 11.0 24.6 24.6 Thalamus 3.3 10.0 80.0 60.0 Subcortical white matter 2.5 7.4 48.0 48.0 Lenticular nucleus 2.0 6.0 39.5 29.6 Cerebellar cortex 1.7 5.0 16.0 24.6 Reticular formation 1.0 3.0 0 ' 0 Hypothalamus 0 0 0 ! 0 - 34 -a c t i v i t y i f ATP were the transmitter substance. The same can be s a i d for cerebral cortex,thalamus, and cerebellar cortex, the areas which also contained large amounts of vasodilator a c t i v i t y . The acetone powder of caudate nucleus also d i d not contain the amounts of ATP consistent with i t s vasodilator a c t i v i t y i f ATP were the transmitter substance concerned. The acetone powder of the thalamus contained the greatest amount of ATP but none of the other areas which had vasodilator a c t i v i t y contained large amounts of ATP. Caudate nucleus, cerebral cortex, and cerebellar cortex a l l contained relatisely small amounts of ATP by comparison with the other areas tested. It i s i n t e r e s t i n g to note that the amounts of ATP i n the acetone powders were a l l considerably larger than the amounts found in the water dialysates and a l s o that the order of decreas-ing amounts of ATP i s not the same i n both cases. This seems to indicate that the d i a l y s i s performed was not s u f f i c i e n t to ex-tract a l l the ATP present i n the brain homogenate inside the d i a l y s i s tubing. However, the d i a l y s i s was s u f f i c i e n t to ex-trac t the vasodilator a c t i v i t y present. This would tend to indicate that ATP and the vasodilator substance are two d i f f e r e n t compounds. 3t A comparison of vasodilator a c t i v i t y with adenosine triphosphate content of Ine central nervous system. It was thought that the d i s t r i b u t i o n of vasodilator a c t i v i t y could be best compared with the d i s t r i b u t i o n of ATP i n the central nervous system i n tabular form. The areas of the central nervous system are l i s t e d i n order of decreasing content - 35 -of the appropriate substances indicated. Table 3 shows that caudate nucleus, cerebral cortex, and cerebellar cortex were the areas where the greatest amount of vasodilator a c t i v i t y was consistently found, but they were never found to contain the most ATP. It i s apparent that there is l i t t l e c o r r e l a t i o n , d i r e c t or Inverse, between the ATP content of the water dialysates and acetone powders and the v a s o d i l a t o r substance content of areas i n the central nervous system. 4. A comparison of the chemical properties of the vaso-d i l a t o r a c t i v i t y and ATP. It was hoped that some ind i c a t i o n of the chemical nature of the vasodilator substance as compared to ATP would aid i n determining i f any chemical r e l a t i o n s h i p existed between the two compounds. The e f f e c t of several ion exchange resins was tested i n order to see i f the compounds had molecules which were charged and I f so, whether they were anions or cations. The s t a b i l i t y of the vasodilator a c t i v i t y t o acid, a l k a l i , and heat was also determined and compared to the s t a b i l i t y of ATP to these agents. Another simple experiment was done which gave a good in d i c a t i o n of the molecular size of the vasodilator agent. The homogenates of b r a i n were dialyzed against d i s t i l l e d water to prepare the extracts for t e s t i n g on the rabbit ear, so solu-tions of ATP were also dialyzed in the same manner to see i f enough ATP passed through the membrane and would then indicate i t s molecule was of a s i m i l a r s i z e . In these experiments only water dialysates of caudate nucleus and cerebral cortex were used as these were the two areas where vasodilator a c t i v i t y was consistently found. - 36 TABLE 3t COMPARISON OP THE AMOUNTS OF ATP AND VASODILATOR SUBSTANCE Water Dialysates of Areas of CNS Con-tain i n g ATP IVth v e n t r i c l e f l o o r i n f e r i o r c o l l i c u l u s superior c o l l i c u l u s caudate nucleus cerebral cortex thalamus subcortical white matter l e n t i c u l a r nucleus cerebellar cortex r e t i c u l a r formation hypothalamus Areas of CNS Con-tain i n g Vasodilator Substance caudate nucleus cerebral cortex cerebellar cortex thalamus l e n t i c u l a r nucleus r e t i c u l a r formation subcortical white matter i n f e r i o r c o l l i c u l u s superior c o l l i c u l u s IVth v e n t r i c l e f l o o r hypothalamus Acetone Powders of Areas of CNS Con-ta i n i n g ATP thalamus subcortical white matter i n f e r i o r c o l l i c u l u s l e n t i c u l a r nucleus superior c o l l i c u l u s caudate nucleus cerebral cortex cerebellar cortex IVth v e n t r i c l e f l o o r r e t i c u l a r formation hypothalamus - 37 -(a) Table 4 shows the effect of strong and weak cation and anion exchange resins on dialysates containing vasodilator a c t i v i t y and on solutions of ATP. The vasodilator a c t i v i t y was tested on the rabbit ear preparation as described above, the amount of d i l a t a t i o n i s shown by +'s, being the strongest d i l a t a t i o n effect and the weakest as recorded on a f i l m s t r i p moving past an oscilloscope screen. The amount of ATP present was measured by the amount of l i g h t passing through a known solution path at a wavelength of 260 m^u . The concentration of the ATP can be calculated from the o p t i c a l density of the solution, the molecular extinction c o e f f i c i e n t at pH 7.0, and the measured path length. Thus, a solution having an op t i c a l density of 0.090, from the equation A = k c l ; where A a o p t i c a l density, k = molecular extinction c o e f f i c i e n t , c = concentration in moles/litre, and 1 = length of l i g h t path i n cms., w i l l have a concentration of 0.090. Where „ k l k f o r ATP = 15.4 x 10° at pH 7.0 and 1 = 0 . 5 cm., the concentra-t i o n of ATP w i l l therefore be °^° 9° x ^ Q < 5 ^ or 0.011 x 10" 3 m/l or expressed i n g./ml. 6.85. It can be seen from Table 4 that ATP was completely taken out of solution by the two anion exchange resins Dowex-1 and Amberlite IR-45. The strong cation exchange r e s i n Dowex-50, on the other hand, removed less than half the ATP present. The weak cation exchange r e s i a , Amberlite IRC-50 removed less than one-tenth of the t o t a l ATP present. These data indicate that the ATP molecule under neutral conditions Is an anion, and there-fore under these conditions i s sim i l a r to the vasodilator substance. - 38 -TABLE 4: THE EFFECT OF VARIOUS ION EXCHANGE RESINS ON ACTIVE DIALYSATES AND SOLUTIONS OF ATP Sample (Water Dialysates) Vasodilatation i n Rabbit Ear caudate nucleus treated (anion exchanger) wi th Dowex-1 0 caudate nucleus treated (cation exchanger) with Dowex-50 cerebral cortex treated (anion exchanger) with Dowex-1 0 cerebral cortex treated (cation exchanger) with Dowex-50 +- +•+• caudate nucleus treated anion exchanger) with IR-45 (weak 0 caudate nucleus treated cation exchanger) with IRC-50 (weak -t- + cerebral cortex treated anion exchanger) with IR-45 (weak 0 cerebral cortex treated cation exchanger) with IRC-50 (weak -t-Amount of ATP 10T/ml.) ATP solution treated with Dowex-1 (anion exchanger) 0 " ATP solution treated with Dowex-50 (cation exchanger) 6.85 " ATP solution treated with IR-45 (weak anion exchanger) 0 " ATP'solution treated with IRC-50 (weak cation exchanger) 9.34 - 39 -(b) A comparison of the s t a b i l i t i e s of the vasodila-tor material and ATP t o conditions of alkaline and acidic hydro-l y s i s as well as heat was also thought to give an Insight into a possible r e l a t i o n s h i p between the two compounds. In these experiments, as mentioned above, successive portions of water dialysates from caudate nucleus and cerebral cortex were heated to b o i l i n g wibh 2N.HC1 and IN.NaOH as well as being heated to b o i l i n g with no hydrolyzing agent present. The same thing was also done with solutions of ATP. The method of determining the remaining vasodilator a c t i v i t y was to in j e c t the neutralized solutions i n t o the rabbit ear preparation, measuring any vaso-d i l a t a t i o n as mentioned before. The water dialysates which had been subjected to con-d i t i o n s of alkaline hydrolysis and to heat alone showed no loss of vasodilator a c t i v i t y . The dialysates which had been subjected to conditions of a c i d i c hydrolysis, however, showed a complete absence of any vasodilator a c t i v i t y . S i m i l a r l y , a solution of ATP when subjected to alkaline hydrolysis or heat alone showed l i t t l e decrease i n vasodilator a c t i v i t y , while a solution of ATP subjected to a c i d i c hydrolysis showed a marked decrease of vasodilator a c t i v i t y . (c) To prepare the water dialysates homogenates of tissue from the areas of the brain indicated were dialyzed i n the cold against d i s t i l l e d water for 48 hours. The r e s u l t i n g dialysates were concentrated down to a f i n a l volume which was one-third the volume of the f r e s h t i s s u e . A solution of ATP was also treated in the same manner - 40 -to determine whether the ATP molecule would behave in the same way against a concentration gradient as d i d the vasodilator substance. The amount of ATP which passed through the d i a l y s i s membrane was measured by the f i r e f l y luminescence enzyme tech-nique (see materials & methods). The amounts of ATP which were found to pass through the membrane were so small as to render i t most u n l i k e l y that ATP was the vasodilator substance dialyzed out of the b r a i n tissue homogenates. It would indicate also that the substance responsible for the vasodilatation had a smaller molecular diam-eter than ATP as i t passed through the d i a l y s i s membrane almost completely after 48 hours. 5. Chromatography of active extracts for vasodilator material and ATP An attempt was made to i s o l a t e the vasodilator sub-stance as well as ATP chromatographically from the active water dialysates. It was thought that i f the active vasodilator mater-i a l and ATP could be i s o l a t e d then these substances could be eluted from the chromatograms and injected into the rabbit ear preparation to determine whether any • s i m i l a r i t y could be found between the eluted material and the vasodilator effects of the water dialysates. Chromatograms of water dialysates were developed i n n-propanol, ammonia, water (60^30:10) for 18 hours and upon being dried were scanned under u l t r a v i o l e t for l i g h t absorbing areas (which Is c h a r a c t e r i s t i c of ATP). ATP was also run along one border as a marker compound. Several l i g h t absorbing areas - 41 -were found although none corresponded to ATP. Neither did any areas upon elut i o n produce vasodilatation i n the rabbit ear preparation. S i m i l a r l y , water dialysates were chromatographed and developed in isopropanol, (NR^^SC^ saturated solution, water (2:79:19) for 4-| hours. U l t r a v i o l e t l i g h t absorbing areas were again tested unsuccessfully for vasodilator a c t i v i t y . S trips of chromatogram paper were spotted with active water dialysates and developed i n the two solvent systems men-tioned above. On drying the s t r i p s were cut into ten sections between the o r i g i n and the solvent front and each section was eluted with isotonic s a l i n e . "Each elut i o n i n turn was tested on the rabbit ear preparation for vasodilator a c t i v i t y without success. It was hoped that a c o r r e l a t i o n could be established between an area whose eluate caused vasodilatation and a reac-t i o n of the same area to s p e c i f i c colourizing agents. Similar s t r i p s of paper were prepared at the same time and treated with various colourizing agents s p e c i f i c for certain species of com-pounds. Ninhydrin solution which y i e l d s coloured compounds with molecules possessing free-NHg groups was used. Most dia-lysates yielded 4-6 ninhydrin positive areas, probably indicating the presence of amino acids which most c e r t a i n l y would be there. Molybdic acid spray was used to indicate the presence of organic phosphorous compounds, of which most dialysates gave four d i s t i n c t areas. A Hg, containing reagent was used, which Indicated the presence of t h i o u r a c i l , u r a c i l , uridine-5-P0 4, 3,4 dihydroxy-- 42 -phenylalanine, and L-adrenaline. Most water dialysates yielded four areas of positive reaction to t h i s reagent, Ehrlich's reagent, containing p-dimethylaminobenzalde-hyde, whose positive reaction gives a yellow colour to dihydro-u r a c i l , thymine, phenylalanine, tyrosine, quanosine, and quanine ribotide was also used. The chromatograms of active water dia-lysates a l l yielded only one area of positive reaction t o t h i s reagent. Sakaguchi reagent whose posit ive,. react ion yields an orange colour with quanidine type compounds was also t r i e d . The water dialysates a l l had only one area which reacted with the reagent. As no vasodilator a c t i v i t y could be found i n any of the eluates, no c o r r e l a t i o n could be made with the areas of the chromatograms which had reacted with the various species' spec-i f i c colouring reagent. 6 . Recordings of vasodilatation produced i n the rabbit ear preparation. (a) This t r a c i n g is an example of the vasodilatation produced by stimulating the great a u r i c u l a r nerve antidromic-a l l y . There is a lapse of 5 seconds between the time of nerve stimulation and the onset of v a s o d i l a t a t i o n . At the end of 55 seconds the v a s o d i l a t a t i o n is s t i l l very marked. (b) This curve i s a recording of the vasodilatation caused by Injection of a solution of ATP ( l mg./ml.). At f i r s t there i s a small drop i n the curve caused by the entry of the clear solution into the blood vessels, this appears as a - 42(a) -• i 10 SEC. Figure 6A. This tracing i s an example of the vasodilatation produced i n the rabbit ear preparation by stimu-l a t i o n of the auricular nerve; the arrow indicates the point of stimulation. - 42(b) -10 S E C . Figure 6B. This tracing i s an example of vasodilatation produced i n the rabbit ear preparation by-in j e c t i o n into the f a c i a l artery of ATP solution ( l mg./ml.). The arrow indicates the point of i n j e c t i o n . - 42(c) -Figure 6C. This tracing i s an example of the vasodilatation produced i n the rabbit ear preparation by i n j e c -t i o n of extract of the caudate nucleus. The arrow indicates the point of i n j e c t i o n . - 42(d) -Figure 6D. This tracing i s an example of the vasodilatation produced i n the rabbit ear preparation by i n j e c -t i o n of extract of posterior roots of the spinal cord. The arrow indicates the point of i n j e c t i o n . - 43 -co n s t r i c t i o n , the d i l a t a t i o n is delayed by 5 seconds. Unlike •the d i l a t a t i o n produced by nerve stimulation, the d i l a t a t i o n produced by ATP starts to subside after 13 seconds. (c) Shows the d i l a t a t i o n produced by i n j e c t i o n of the extract of the caudate nucleus. Here there Is a short constric-t i o n again caused by the entry of the clear solution, again the onset of the d i l a t a t i o n is delayed by 3-4 seconds. The peak vasodilatation is over after 13 seconds. (d) This t r a c i n g i l l u s t r a t e s the v a s o d i l a t a t i o n pro-duced b?/ i n j e c t i o n of an extract of posterior roots. The onset of the d i l a t a t i o n is delayed by 5 seconds and the d i l a t a t i o n i t s e l f subsides aft e r 8 seconds. - 44 -IV. DISCUSSION: Dale (48) and Feldberg (65) have both put forward the same concept, that a neurone could be expected to release the same transmitter agent at a l l synapses formed by the d i f f e r -ent branches of the axon, and further, that a b i p o l a r neurone would possess the same transmitter material at the central end as well as at the peripheral end of i t s processes. It would therefore be expected that any information obtained about the transmitter released at the peripheral terminals of a primary sensory neurone could also be applied to the transmitter at the central synapse. Evidence has come to l i g h t recently which substan-t i a t e s Dale's hypothesis. Among spinal neurones acetylcholine i s known to be the transmitter substance elaborated for the propagation of a t r a i n of impulses from the motor neurones and libe r a t e d from t h e i r axonal endings to cause transmission across neuromuscular junctions leading t o the ac t i v a t i o n of the muscle. Inhibitory Interneurones (Renshaw c e l l s ) , whose action i n h i b i t s the a c t i v i t y of the motor neurones, are present and are served by c o l l a t e r a l f i b r e s from the same motor neurones. Acetylcholine has been found to activate the muscle at the neuromuscular junc-t i o n and i t w i l l also activate the Renshaw c e l l s (75); t h i s indicates that acetylcholine is acting as the transmitter sub-stance at more than one branch of the axon of the motor neurone. It i s but a short step to postulate that the bipolar sensory neurone with i t s branches going to the periphery and also synaps-ing c e n t r a l l y i n the spinal cord, could probably also employ the - 45 -same transmitter substance at either end of what is e s s e n t i a l l y one continuous process. Sensory information reaches the higher centres of the central nervous system by a three neurone pathway, the f i r s t synapse can either be within the segment at which the posterior root f i b r e enters the s p i n a l cord, or higher up i n the n u c l e i cuneatus et g r a c i l i s . The second synapse i n the pathway is in the thalamus and the f i n a l synapse i n the appropriate area of the cerebral cortex. Evidence in cholinergic transmission i n the central nervous system has shown that not a l l the neurones of the path-way are capable of synthesizing acetylcholine, the synthesis of which i s taken t o indicate a transmitter role for acetylcholine (63). A tendency appears to e x i s t , In some cases at any rate, for neurones of high choline acetylase a c t i v i t y to alternate i n a chain with neurones.which' contain no synthesizing enzyme (71,72). Of the three neurones involved in the primary sensory pathway, the f i r s t and t h i r d are d e f i c i e n t i n choline acetylase while the second neurone has a high a c t i v i t y of t h i s enzyme. An alterna-t i o n here seems to ex i s t , of neurones having di f f e r e n t enzyme a c t i v i t i e s which i s reasonable to believe corresponds with an alternation of cholinergic and non-cholinergic elements of trans-mission. The d i s t r i b u t i o n of vasodilator substance i n beef brain was determined in the present experiments (see Table 1, r e s u l t s ) . The results agreed roughly with those reported by Holton (57), Table 5, In that the area found to contain the most a c t i v i t y was - 46 -the caudate nucleus, while the thalamus also contained a degree of a c t i v i t y , as did the dorsal and ventral roots. However, here the agreement ends. Holt on found the nucleus cuneatus to have much vasodilator material while i n these experiments the nu c l e i cuneatus et g r a c i l i s was found to possess almost no vasodilator material. Holton also reports l i t t l e a c t i v i t y i n the cerebral cortex whereas i n the above experiments the cerebral cortex contained almost as much vasodilator substance as the caudate nucleus. At this point i t i s of interest to compare the d i s t r i -bution of vasodilator material in areas of the central nervous system with the content of other neurologically active substan-ces. Table 6 shows the d i s t r i b u t i o n of some compounds l i k e l y to be of importance at central synapses. There does not appear to be any co r r e l a t i o n between the d i s t r i b u t i o n of the various substances given i n Table 6 and the d i s t r i b u t i o n of vasodilator •material from the above experiments. This i s not surprising i n view of the fact that the substances mentioned have a l l been ruled out as possible vasodilator agents either because of t h e i r chemical or pharmacological properties. Hence, histamine could not be the vasodilator material because vasodilatation w i l l s t i l l occur in the presence of antihistaminics compounds; or substance P cannot be the substance as i t w i l l not dialyze through a semi-permeable membrane while the active material w i l l dialyze through. It i s tempting to postulate that the transmitter sub-stance responsible f o r antidromic vasodilatation may also be res-ponsible for synaptic transmission at the f i r s t and t h i r d neurones - 47 -TABLE 5: VASODILATOR ACTIVITY, NOT DUE TO ACETYLCHOLINE OR HISTAMINE, OF EXTRACTS FROM DIFFERENT REGIONS OF THE HORSE BRAIN"' caudate nucleus nucleus cuneatus nucleus g r a c i l i s fasiculus g r a c i l i s pyramidal t r a c t s dorsal root optic tract trapezoid fimbria thalamus cerebral cortex i n t e r n a l capsule, post, limb anterior pituitary-hyp ot ha lamus medial eminence of tuber cinereum standard acetone-dried powder of dorsal roots Exp. #1 100 300 5 1 2 3 5 1 2 #2 100 10 <2 20 #3 100 #4 100 40 100 constric-tor 80 30 4 4 8 4 4 20 20* 20 #5 100 400 100 " after treatment which inactivated the posterior p i t u i t a r y p r i n c i p l e - extract of caudate nucleus taken as the standard for each brain, and the a c t i v i t y of the other samples was found by matching each with a d i l u t i o n of the standard. The a c t i v i t y i s therefore expressed as a percentage of that i n the caudate nucleus. In addition, the caudate nucleus extract was assayed against a saturated acetone-dried preparation of dorsal roots 1 P. H o l l i n , G.W. Harris, Journal of Physiology, Vol; 120, page 254, (1953). TABLE 6: DISTRIBUTION OF NEUROLOGICAL ACTIVE COMPOUNDS IN AREAS OF CENTRAL NERVOUS SYSTEM2 Choline acetylase ^g./gm. powder Nor-adrenaline •^g./gm. 5 HT ^g./gm. Histamine , ^«rg./gm. Subst. P unit/gm. Holt on's v a s o d i l . subst. % CEF % deep roots - ^20 0.01 0 4-11 40 10 100 optic nerves 16 0 0.02 0 9 6 1 100 dorsal columns 33 - mm 0 0.3 27 30 0 Internal capsule (posterior) 70 - - - - - - 100 cerebellum 26 90 0.07 0.01 ^0.1 1.6 1 100 pyramids 42 - 0.06 0 - - 3 15 ven t r a l roots 573 11000 0.06 0 6-9 6 0 100 sympathetic ganglia - - 6 0 61 - - 0 l a t e r a l geniculate body 325 2600 0.07 - - - - 100 midbrain 170 - 0.37 0.20 - - - 0 hypothalamus - 2000 1.03 0.28 12 70 20 -thalamus area postrema 823 3000 fO.24 med. 1-0.28 l a t . 1.04 f0.07 l o 0.24 med, <0.4 l a t . 12.5 460 6 100 caudate nucleus 437 13300 0.10 0 ^0.2 46 10.0 -2 from Crossland, Journal of Pharmacy & Pharmacology, Vol. 12, page 10, (1960/. - 49 -of the sensory pathway. Holt on has Indeed proposed such a role for ATP l a r g e l y on the grounds that i t gives r i s e to a c a p i l l a r y d i l a t a t i o n and no pharmacological agents are known which block i t s action. She has also produced some evidence, which has been discussed above (see page 15, Introduction), that ATP i s released upon stimulation of sensory f i b r e s . Measurements of the amounts of ATP were carried out on various areas of the brain with the idea i n mind that i f ATP were the transmitter responsible for antidromic vasodilatation, or related to i t , the areas containing the most vasodilator a c t i v i t y should also contain the most ATP. As can be seen from Table 3 i n the r e s u l t s , no r e l a t i o n s h i p i s apparent between the location and concentration of the two substances. In postulating the presence of the vasodilator "material i n the sensory pathways i t Is necessary to comment on the possible significance of d i s t r i b u t i o n i n the areas.tested. I f one accepts Dale's hypothesis that a neurone w i l l u t i l i z e the same trans-mitter substance at a l l branches of the axon, and there i s some evidence to support t h i s , then one might well expect to f i n d the active substance at the peripheral ends of the sensory neurones and at the central ends, i n which case the vasodilator substance would be present i n the posterior roots. In considering the three neurone sensory pathways the f i r s t neurone synapses with the second neurone i n the thalamus, hence i t would seem reason-able the vasodilator material be i n the thalamus at the central synapse of the f i r s t sensory neurone. This has been shown (see Table l) i n the r e s u l t s . I f the d i s t r i b u t i o n of the vasodilator - 50 -substance alternated with the d i s t r i b u t i o n of acetylcholine, then one would expect to f i n d vasodilator material i n the f i r s t and t h i r d neurone of the three neurone pathway; in which case the cerebral cortex should a l s o contain vasodilator material. This has been demonstrated, i n fact the cerebral cortex possesses a large amount of vasodilator material. However, one enigma s t i l l remains. Of a l l the areas tested f o r vasodilator material the caudate nucleus was consistently found to contain the great-est amount. A 3 can be seen from Table 6. the caudate nucleus contains appreciable amounts of most of the active substances. The presence of the vasodilator material in the caudate nucleus i s d i f f i c u l t to reconcile with the fact that It has been shown to date to have motor functions only. However, there i s no reason why the material, i f i t is an excitatory transmitter, should only be i n sensory pathways. In view of the above evidence and from the fact that ATP has such an important role as a source of energy for the body i n general, i t seems u n l i k e l y that i t can be the substance res-ponsible for sensory nerve transmission. As ATP i s present i n a l l c e l l s , including neurones, i t i s obvious that i t would meet some of the c r i t e r i a for a transmitter agent. Some of the c r i t e r i a are; there must be some system for synthesizing the substance, and there must also be some mechanism f o r destroying the substance. Since neurones must be able to control t h e i r source of energy accurately they possess a system for synthesiz-ing ATP and also a mechanism for destroying ATP thereby, iri e f f e c t , f u l f i l l i n g some of the c r i t e r i a f o r a transmitter agent. - 51 -The two most important questions for which answers were sought i n t h i s research were: 1. i s ATP the substance responsible for antidromic vasodilatation? and 2. i f i t i s , i s i t also a sensory synaptic transmitter substance? It appears from the above research that ATP i s u n l i k e l y to be the substance responsible f o r the vasodilatation, which would seem to rule i t out as the sensory synaptic transmitter substance. 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