larry r. squire- the history of neuroscience in autobiography: volume 1

8/3/2019 Larry R. Squire- The History of Neuroscience in Autobiography: Volume 1

1/34


2/34

EDITORIAL ADVISORY COM MITTEEAlbert J . AguayoBernice Grafstein

Theodore MelnechukDale Purves

Gordon M. Shep herdLa rry W. Swa nson (Chairperson)


3/34

T h e H is to r y o f N e u r o s c i en c ein A uto b io g ra phyVOLUME 1

E dited by L arry R. Squire

SOCIETY FOR NEUROSCIENCEWashington, D.C.

1996


4/34

Society for Neuroscience1121 14th Street, NW., Suite 1010

Washington, D.C. 20005

1996 by the Society for Neuroscience.

All rights reserved.

Printed in the United States of America.

Library of Congress Catalog Card Number 96-70950ISBN 0-916110-51-6


5/34

Contents

Denise Albe-Fessard 2

Julius Axelrod 50

Peter O. Bishop 80

Theodore H. Bullock 110

Irving T. Diamond 158

Robert Galambos 178

Viktor Hamburger 222

Sir Alan L. Hodgkin 252

David H. Hubel 294

Herbert H. Jasper 318

Sir Bernard Katz 348

Seymour S. Kety 382

Benjamin Libet 414

Louis Sokoloff 454

James M. Sprague 498

Curt von Euler 528

John Z. Young 554


6/34


7/34

J u l i u s A x e l r o dBORN: New York , New YorkMay 30 , 1912EDUCATION: Col lege of the Ci ty of Ne w York, B .S ., 1933

New York Univers i ty , M.A. , 1941George Wash ing ton Univer s i t y , Ph .D . , 1955APPOINTMENTS"

G o l d w a t e r M e m o r i a l H o s p i t a l, T h i r d N e w Y o rk U n i v e r s i t yResea rch Div i s ion (1946)N a t i o n a l H e a r t I n s t i t u t e ( 1 9 4 9 )N a t i o n a l I n s t i t u t e o f M e n t a l H e a l t h ; C h i e f, S e c ti o n onP h a r m a c o l o g y ( 19 5 5)N a t i o n a l I n s t i t u t e o f M e n t a l H e a l t h G u e s t R e s e a r c h e r(1984)S c i e n t i s t E m e r i t u s o f t h e N a t i o n a l I n s t i t u t e s o f H e a l t h(1996)

HONOR S AND AWARDS (SELECTED):Nobe l P r i ze fo r Phys io logy o r M edic ine (1970)A m e r i c a n A c a d e m y o f A r t s a n d S c ie n c es ( 19 7 1)N a t i o n a l A c a d e m y o f S c ie n c es U S A ( 1 9 71 )Fore ign M em ber o f t he Roya l Soc i e ty o f Lond on (1979)L e i b n iz M e d a l, A c a d e m y o f S c ie n ce s , E a s t G e r m a n y(1984)M a h o n e y A w a r d " D e ca d e o f t h e B r a i n " ( 1 9 91 )Ra lph W. Gera rd P r i ze , Soc i e ty fo r Neurosc i ence (1992)

Jul ius Axelrod has carr ied out extensive , fundamenta l research on a widerange o f topics, including b iochemical me cha nism s o f drug an d horm oneact ions and metabol i sm; enzymology; p inea l g la nd mem branes; an d

t ransduc t ion mechan i sms . He i s mos t we l l known fo r h i s Nobe lPr ize-w innin g e lucida t ion o f the s torage, re lease , and inact iva t ion o f

ca techo lamine neuro t ran smi t t ers and the ef fec t o f p sychoac t ive d rugs .


8/34

J u l i u s A x e l r o d

B e g i n n i n g s *

Su c c e s s f u l s c i e n t i s t s a r e g e n e r a l l y r e c o g n i z e d a t a y o u n g a g e . T h e yg o t o t h e b e s t s c h o o l s o n s c h o l a r s h i p s , r e c e i v e t h e i r p o s t d o c t o r a lt r a i n i n g f e l l o w s h i p s a t p r e s t i g i o u s l a b o r a t o r i e s , a n d p u b l i s h e a r l y .

N o n e o f t h i s h a p p e n e d t o m e .M y p a r e n t s e m i g r a t e d a t t h e b e g i n n i n g o f t h is c e n t u r y f ro m P o l i sh

G a l ic ia . T h e y m e t a n d m a r r i e d i n A m e r i ca , a n d e v e n t u a l l y s e t tl e d i n t h eL o w e r E a s t S i de o f N e w Y o rk , t h e n a J e w i s h g h e t to . M y f at h e r, I s a d o r e ,w a s a b a s k e t m a k e r w h o s ol d f lo w e r b a s k e t s t o m e r c h a n t s a n d g r o c e rs . Iw a s b o r n i n 1 9 12 in a t e n e m e n t o n E a s t H o u s t o n S t r e e t i n M a n h a t t a n .

I a t t e n d e d P S 2 2 , a s ch o o l b u i l t b e fo r e t h e C i v il W a r . A n o t h e r s t u d e n ta t t h a t s c ho o l b e f o re m y t i m e w a s I . I. R a b i , w h o l a t e r b e c a m e a w o r l d -r e n o w n e d p h y s i c is t . A f t e r P S 2 2 I a t t e n d e d S e w a r d P a r k H i g h S ch oo l. Ir e a l l y w a n t e d t o go t o S t u y v e s a n t , a h i g h s c ho o l f or b r i g h t s t u d e n t s , b u tm y g r a d e s w e r e n o t go od e n o u gh . S e w a r d P a r k H i g h S c ho ol h a d m a n yf a m o u s g r a d u a t e s , m o s t l y e n t e r t a i n e r s : Z e ro M o st el , W a l te r M a t t h a u , a n dT o n y C u r t i s . M y r e a l e d u c a t i o n w a s o b t a i n e d a t t h e H a m i l t o n F i s h P a r kL i b r a ry , a bl oc k f ro m m y h o m e . I w a s a v o ra c i o u s r e a d e r a n d r e a d t h r o u g hs e v e r a l b o o k s a w e e k , f r o m U p t o n S i n c la i r, H . L . M e n c k e n , a n d T o l s to y t op u l p n o v e l s s u c h a s t h e F r a n k M e r r i w e l l a n d N i c k C a r t e r s e r i e s .

A f t e r g r a d u a t i n g f r o m S e w a r d P a r k H i g h S c h o o l , I a t t e n d e d N e wY o r k U n i v e r s i t y i n t h e h o p e t h a t i t w o u l d g i v e m e a b e t t e r c h a n c e t o g e ti n t o m e d i c a l s c h oo l. A f t e r a y e a r m y m o n e y r a n o u t, a n d I t r a n s f e r r e dt o t h e t u i t i o n - f r e e C i t y C o l l e g e o f N e w Y o r k i n 1 9 3 0. C i t y C o l l eg e w a sa p r o l e t a r i a n H a r v a r d , w h i ch s u b s e q u e n t l y g r a d u a t e d s e ve n N o be lL a u r e a t e s . I m a j o r e d i n b io lo g y a n d c h e m i s t ry , b u t m y b e s t g r a d e sw e r e i n h i s to r y , p h il o so p h y , a n d l i t e r a t u r e . B e c a u s e I h a d t o w o r k a f t e rs ch o o l, I d i d m o s t o f m y s t u d y i n g d u r i n g t h e s u b w a y t r i p t o a n d f r o mu p t o w n C i t y C o ll eg e . S t u d y i n g i n a c r o w d e d , n o is y N e w Y o r k s u b w a yg a v e m e c o n s i d e r a b le p o w e r s o f c o n c e n t r a ti o n . W h e n I g r a d u a t e d f ro mC i t y C o l l e g e , I a p p l i e d t o s e v e r a l m e d i c a l s c h o o l s b u t w a s n o t a c c e p t e db y a n y .

*A m ajor por t ion of th is ar t ic le ha s been rep roduced, w ith p ermiss ion , f rom A n n R e vP h a r m a c o l T o x i c o l 1988;28:1-23 , by An nua l Reviews, Inc.


9/34

Jul ius Axelrod 53In 1933, the year I graduated from college, the country was in thedepths of a depression. More th an 20 percent of the w orkin g population

was unemployed, and ther e were few jobs available for City Collegegraduates. I had heard about a laboratory position that was availableat the Harriman Research Laboratory at New York University, andalthough the position paid $25 a month, I was happy to work in a lab-oratory. I assisted Dr. K.G. Falk, a biochemist, in his research onenzymes in malignant tumors. I also purified salts for the preparationof buffer solutions and determined their pH. The i nst rum en t used tomeasure pH at that time was a complex apparatus; the glass electrodeoccupied almost ha lf a room. In 1935 the labo ratory r an out of fundsand I was fortunate to get a position as a chemist in the Laboratory ofIndustrial Hygiene. This laboratory was a nonprofit organization andwas set up by New York City's De pa rt me nt of Hea lth to test vit ami nsupp lem ents added to foods. I worked in the La borator y of Ind ust ria lHygiene from 1935 to 1946.My duties there were to modify published methods for measuring vit-amins A, B, B2, C, and D so that they could be assayed in various foodproducts that city inspectors randomly collected. Vitamins had just beenintroduced at t ha t time, and the New York City Departmen t of Healthwanted to establish that accurate amounts of vitamins were added to milkand other food products. The methods used for measuring vitamins thenwere chemical, biological, and microbiological. It required some ingenuityto modify the methods described in the lit era ture to assays of food prod-ucts. This experience in modifying methods was slightly more than rou-tine, but it proved to be useful in my later research. The laboratory sub-scribed to the Jou rnal of Biological C hemistry, which I read with greatinterest. Reading this journal made it possible to keep up with advancesin enzymology, nutrition, and methodology. During the time I was in theLabora tory of Indust ria l Hygiene, I received an M.S. degree in chemis tryat New York Universi ty in 1942 by tak ing courses at night. My thesis wason the ester-hydrolyzing enzymes in tum or ti ssues. Because of the loss ofone eye in a laboratory accident, I was deferred from the draft duringWorld War II. In 1938 1 marr ied Sally Taub, a graduate of Hu nt er College,who later became an elementary school teacher. We had two sons, Pauland Alfred, born in 1946 and 1949.F i r s t E x p e r i e n c e i n R e s e a r c h : G o l d w a t e r M e m o r i a l H o s p i t a lI expected that I would remain in the Laboratory of Industrial Hygiene forthe rest of my working life. It was not a bad job, the work was moderatelyinteresting, and the salary was adequate. One day early in 1946 the Institutefor the Study of Analgesic and Sedative Drugs approached the president ofthe Laboratory of Industrial Hygiene with a problem. The president of the


10/34

54 Julius Axelrodlaboratory at that time was George B. Wallace, a distinguished pharmacolo-gist who had just retired as chairman of the department of pharmacology atNew York University. Many analgesic preparations contained nonaspirinanalgesics, such as acetanilide or phenacetin. Some people who became habit-uated to these preparations developed methemoglobinemia. The Institu te forthe Study of Analgesic and Sedative Drugs offered a small grant to theLaboratory of Industria l Hygiene to find out why acetanilide and phenacetintaken in large amounts produced methemoglobinemia. Dr. Wallace asked meif I would like to work on this problem. I had little experience in this kind ofresearch, and he suggested that I consult Dr. Bernard "Steve" Brodie. Dr.Brodie was a former member of the department of pharmacology at New YorkUniversity and was doing research at Goldwater Memorial Hospital, a NewYork Universi ty division.

I met with Brodie in February 1946 to discuss the problem of anal-gesics. It was a fateful meeting for me. Brodie and I talked for severalhours about w hat kind of experiments could be done to find out howacetanilide might produce methemoglobinemia. Talking to Brodie aboutresearch was one of my most st imu lat ing experiences. He invited me tospend some time in his laboratory to work on this problem.

One of a number of possible products of acetanilide that would causethe toxic effects was aniline. It had previously been shown that anilinecould produce methemoglobinemia. Thus, one approach was to find outwhether acetanilide could be deacetylated to form aniline in the body.With the help and guidance of Steve Brodie, I developed a method for mea-suring aniline in nanogram amounts in urine and plasma. After theadministration of acetanilide to human subjects, aniline was found to bepresent in urine and plasma. A direct relationship between the level ofaniline in blood and the amo unt of methemoglobin present was soonobserved (Brodie and Axelrod, 1948). This was my first tas te of realresearch, and I loved it.Very little acetanilide was found in the urine, suggesting extensivemetabolism in the body. As acetanilide was almost completely trans-formed in the body, we looked for other metabolic products. Methods todetect possible metabolites, p-aminophenol and N-acetyl-p-aminophenol,were developed that were specific and sensitive enough to be used in theplasma and urine. Within a few weeks, we identified the major metaboliteas hydroxylated acetanilide N-acetyl-p-aminophenol and its conjugates.This metabolite was also found to be as potent as acetanilide in analgesicactivity. By taking serial plasma samples, acetanilide was shown to berapidly transformed to N-acetyl-p-aminophenol (Brodie and Axelrod,1948). After the administration of N-acetyl-p-aminophenol, negligibleamo unt s of methemoglobin were produced. As a resul t of these studies,Brodie and I stated in our paper (Brodie and Axelrod, 1948), "the resultsare compatible with the assumption that acetanilide exerts its action


11/34

Julius Axelrod 55mainly t hrough N-acetyl-p-aminophenol [now known as acetaminophen].The latter compound administered orally was not attended by the forma-tion of methemoglobinemia. It is possible therefore, th at it migh t have dis-tinct advantage over acetanilide as an analgesic." This was my first paper,and I was determined to continue doing research.

Soon after Brodie and I examined the physiological disposition andmetabolism of acetanilide, we turned our attention to a related analgesicdrug, phenacetin (acetophenetidin). I spent some time developing sensi-tive and specific methods for the identification of phenacet in and its pos-sible metabolite, p-phenetidine. Brodie and I soon found that in humans,the major metabolic product was also N-acetyl-p-aminophenol arisingfrom the deethyla tion of the parent compound (Brodie and Axelrod, 1949).A minor metabolite was p-phenetidine, which we found was responsiblefor the methemoglobinemia formed after the admin istr ation of largeamoun ts of phenaceti n to dogs. After the admin istr ation of phenacetin tohuman subjects, N-acetyl-p-aminophenol was rapidly formed. The speedand the amount with which N-acetyl-p-aminophenol was formed in thebody suggested that the analgesic activity resided in its deethylatedmetabolite.

The laboratories at Goldwater Memorial Hospital where I began myresearch career were set up during World War II to test newly synthesizedantimalarial drugs for their clinical effectiveness. Early in the war, theJapanese had cut off most of the world's supply of the antimalar ial qui-nine. James Shannon, then a renal physiologist at New York University,was put in charge of this program. Shannon had the remarkable capacityto pick the bright young people to carry out research on the antimalarialproject. Members of the team th at worked at Goldwater in addition toSteve Brodie were Sid Udenfriend, Robert Berliner, Bob Bowman, TomKennedy, and Gordon Zubrod. The atmosphere at Goldwater was highlystimulating, and an outpouring of importan t new findings resulted. It wasin this atmosphere tha t, in a period of a few years, I became a researcher.

After completion of the studies on acetanilide and phenacetin, Brodieinvited me to stay on at Goldwater to study the fate of other analgesicdrugs. We received a small grant from the Institute for the Study ofAnalgesic and Sedative Drugs, and the Laboratory of Indust ria l Hygienepaid my salary. Another drug we investigated was the analgesicantipyrine. A sensitive method for the detection of this drug was devel-oped, which has since been used by other investigators as a marker todetermine the activity of drug-metabolizing enzymes in vivo. We identi-fied 4-hydroxyantipyrine and its sulfate conjugate as metabolites ofantipyrine. We also observed tha t antipyrine di stributed in the same man-ner as body water. Because of this property, an tipy rine has been used forthe measu remen t of body water. Another analgesic we studied wasaminopyrine. Many of the drugs whose fate Brodie and I studied were


12/34

56 Julius Axelrodlater used by many investigators as substrates for the microsomal drug-metabolizing enzymes: aminopyrine for N-demethylation, phenacetin forO-dealkylation, and aniline for hydroxylation. Together with Jack Cooper,we developed a method for measuring the anticoagulant dicoumerol inplasma. In a study on the disposition of dicoumerol in hum ans , an exceed-ingly wide difference in the pla sma levels of this drug was found, sug-gesting genetic differences in drug metabolism.M o v e to t h e N a t i o n a l H e a r t I n s t i t u t eBecause I did not have a doctorate degree, I realized that I would have lit-tle chance for advancement in any hospital attached to an academic insti-tution. I had neither the inclination nor the money to spend several yearsgetting a Ph.D., so I decided to join the National Hea rt Ins tit ute as aresearch chemist. In 1949, Shannon was chosen as the director of thenewly organized National Heart Institute in Bethesda, and he offered mea position. Also coming to the National Ins titute s of Hea lth (NIH) at th attime were m any me mbers of the Goldwater staff--Brodie, SidneyUdenfriend, Robert Berliner, Thomas Kennedy, and Robert Bowman.

At the National Heart Institute from 1950 to 1952, I collaboratedwith Brodie and his s taff on the metabolism of analgesics and adrene r-gic blocking agents and the actions of ascorbic acid on drug metabolism.After a while, I became dissatisfied with working with a large team andwas allowed to work independently. The first problem I chose was anexam ina tion of the physiological disposition of caffeine in hu man s. Verylittle was known about the physiological disposition and metabolism ofthis widely used compound. A method for measuring caffeine in biologi-cal material was developed, and the plasma half-life and distributionwere determi ned (Axelrod and Reichenthal, 1953). Because of my workon analgesics and caffeine, I was delighted to be elected without a doc-tora te as a membe r of the American Society of Pharmacology andExperimental Therapeutics in 1953. K.K. Chen and Steve Brodie weremy sponsors.At that time, I became intrigued with the sympathomimetic amines.In 1910, George Barger and Henry Dale reported that numerous B-pheny leth anola mine derivatives si mulated the effects of sympathet icnerve stimul ation with varying degrees of intensi ty and precision. Theycoined the term sympathomimetic amines. Sympathomimetic aminessuch as amphetamine, mescaline, and ephedrine also produced unusualbehavioral effects. In 1952 very little informa tion concerning the metab-olism and physiological disposition of these amines was known. Becauseof my experience in drug metabolism, I decided to unde rt ake a study onthe fate of ephedrine and amphe tamin e. In retrospect, this was animportant decision.


13/34

Julius Axelrod 57The first amine that I studied was ephedrine. Ephedrine, the active

principle of Ma Huang, an herb used by ancient Chinese physicians, wasintroduced to modern medicine by Chen and Schmidt in 1930. I soonfound that ephedrine was transformed in animals by two pathways(demethylation and hydroxylation) to yield metabolic products that hadpressor activity. Various animal species showed considerable differencesin the relative importance of these two metabolic routes. The next sym-pathomimetic amines I examined were ampheta mine and methylamphet -amine. These compounds were shown to be metabolized by a variety ofmetabolic pathways including hydroxylation, demethylation, deamina-tion, and conjugation. Marked species variations in the transformation ofthese drugs were also observed.T h e D i s c o v e r y of t h e M i c r o s o m a l D r u g M e t a b o l i z i n g E n z y m e sWhen amphetamine was given to rabbits, it disappeared without a trace.This puzzled me, so I decided to look for enzymes that metabolized thisdrug. I had no experience in enzymology, but there were many outstand-ing enzymologists in Building 3 on the NIH campus where my laboratorywas located. Gordon Tomkins, who occupied the lab bench next to mine,offered me good advice. Gordon had the capacity of demystifying enzy-mology and told me that all I needed to start in vitro experiments was amethod of measuri ng amphetamine , an animal liver, and a razor blade. Idid my first in vitro experiment with rabbit liver in January 1953. Whenrabbit liver slices were incubated in Krebs-Ringer buffer solutions withamphetamine, the drug was almost completely metabolized. On homoge-nization of the rabbit liver, amphetamine was not metabolized unlesscofactors such as DPN (NAD), TPN (NADP), and ATP were added. I thendecided to examine which subcellular fraction was responsible for trans-forming amphetamine. Hogeboom and Schneider had just described areproducible method for separating the various subcellular fractions byhomogenizing tissue in isotonic sucrose and subjecting the homogenate todifferential centrifugation. After separation of nuclei, mitochondria,microsomes, and the cytosol, none of these f ractions were able to metabo-lize amphetam ine , even in the presence of added cofactors. However,when the microsomes and cytosol were combined, amphetamine rapidlydisappeared on the addition of DPN, TPN, and ATP. At tha t time Bert LaDu, a colleague at the NIH, observed tha t the demethyla tion of aminopy-rine in a dialyzed rat liver whole homogenate required TPN. In a subse-quent experiment I found that amphetamine was metabolized in a dia-lyzed preparation of microsomes and cytosol in the presence of TPN, butnot DPN or ATP. However, when microsomes and cytosol were separatelyincubated, little or no drug was metabolized, despite the addition of TPN.I realized then that I was dealing with a unique enzymatic reaction.


14/34

58 Julius AxelrodBefo re I wen t fu r the r , I dec ided t o i den t i fy t he me tabo l i c p roduc t s o fa m p h e t a m i n e p r o d u c e d w h e n t h e c o m b i n e d m i c r o s o m e s a n d c yt os ol ic f ra c -

t io n w e r e i n c u b a t e d w i t h T P N . O n e o f t h e p o s s ib l e m e t a bo l ic p a t h w a y sm i g h t b e d e a m i n a t i o n , l e a d i n g t o t h e f o r m a t i o n o f p h e n y l a c e to n e . A f t e ri n c u b a t i o n o f a m p h e t a m i n e w i t h th e a b o v e p r e p a r a t i o n s , p h e n y l a c e t o n ea n d a m m o n i a w e r e id e nt if ie d . T h es e r e s u l ts i n d i c a te d t h a t a m p h e t a m i n ew a s d e a m i n a t e d b y a n o x i d a t i v e e n z y m e r e q u i r i n g T P N e i t h e r i n t h em i c r o s o m e s o r c y to s o l t o f o r m p h e n y l a c e t o n e a n d a m m o n i a . B e c a u s e o f i t sp r o p e r t i e s a n d t h e s t r u c t u r e o f i ts s u b s t r a t e , i t w a s a p p a r e n t t h a t t h i se n z y m e d if fe re d fr o m a n o t h e r d e a m i n a t i n g e n z y m e , m o n o a m i n e o x id a se .

W h e r e w a s t h e e n z y m e l o c a t e d , i n t h e m i c r o s o m e s o r t h e s o l u b l es u p e r n a t a n t f ra c ti o n ? A n a p p r o a c h t h a t I u s e d t o l o ca te th e e n z y m e w a st o h e a t e a c h f r a ct io n fo r a f ew m i n u t e s a t 5 5 ~ a t e m p e r a t u r e t h a t w o u l dd e s t r o y h e a t - s e n s i t i v e e n z y m e s . W h e n t h e cy to s ol w a s h e a t e d to 55 ~ a n dt h e n a d d e d t o u n h e a t e d m i c r o s o m e s a n d T P N , a m p h e t a m i n e w a s d e a m i -n a t e d . W h e n t h e m i c r o s o m e s w e r e h e a t e d a n d a d d e d t o t h e c y to s ol ic fr ac -t i o n t o g e t h e r w i t h T P N , a m p h e t a m i n e w a s n o t m e t a b o l i z e d . T h i s w a s ac r u c i a l e x p e r i m e n t , w h i c h d e m o n s t r a t e d t h a t a h e a t - l a b i l e e n z y m e t h a td e a m i n a t e d a m p h e t a m i n e s w a s l o c a l i z e d i n t h e m i c r o s o m e s a n d t h a t t h ec y to s o l p r o v i d e d f a c t o r s i n v o l v i n g T P N n e c e s s a r y f o r t h i s r e a c t i o n .

B e r n a r d H o r e c k e r , t h e n w o r k i n g i n B u i l d i n g 3 , p r e p a r e d s e v e r a l s u b -s t r a t e s f o r t h e T P N - r e q u i r i n g d e h y d r o g e n a s e f o r h i s c l a s s i c w o r k o n t h ep e n t o s e p h o s p h a t e p a t h w a y . H e g e n e r o u s l y s u p p l i e d m e w i t h t h e s e s u b -s t r a t e s , w h i c h I c o ul d t e s t o n m y p r e p a r a t i o n . I f o u n d t h a t t h e a d d i t i o n o fg l u c o s e - 6 - p h o s p h a t e , i s o c it ri c a c id , o r p h o s p h o g l u c o n i c a c i d , t o g e t h e r w i t hT P N , t o u n w a s h e d m i c r o so m e s tr a n s f o r m e d a m p h e t a m i n e . A r e a c ti o nc o m m o n to t h e s e s u b s t r a t e s is t h e g e n e r a t i o n o f T P N H , s u g g e s t i n g t h a tt h e e n z y m e s i n t h e c y to s ol f r a c ti o n w e r e r e d u c i n g T P N . I n c u b a t i n g m i c ro -s o m e s w i t h a T P N H - g e n e r a t i n g s y s t e m u s i n g g l u c o s e - 6 - p h o s p h a t e a n dg l u c o se - 6 -p h o s p h a te d e h y d r o g e n a s e r e s u l te d i n t h e d e a m i n a t i o n o f 'a m p h e t a m i n e . O n i n c u b a t i o n o f c h e m i c al ly s y n t h e s iz e d T P N H , m i cr o-s o m es , a n d o x yg en , a m p h e t a m i n e w a s d e a m i n a t e d . A t a b o u t t h e s a m et i m e , I a ls o f o u n d t h a t e p h e d r i n e w a s d e m e t h y l a t e d to n o r e p h e d r i n e a n df o r m a l d e h y d e b y e n z y m e s p r e s e n t i n r a b b i t m i c r o s o m e s t h a t r e q u i r e dT P N H a n d o x y g e n . B y t h e e n d o f J u n e 1 9 53 , I f e lt c o n f i d e n t t h a t I h a dd e s c r i b e d a n e w e n z y m e t h a t w a s l o c a l i z e d i n t h e m i c r o s o m e s , r e q u i r e dT P N H a n d o x y g en , a n d c o u ld d e a m i n a t e a n d d e m e t h y l a t e d r u g s. I re p o r t-e d t h e s e f i n d i n g s a t t h e 1 9 5 3 f a ll m e e t i n g o f t h e A m e r i c a n S o c i e t y o fP h a r m a c o l o g y a n d E x p e r i m e n t a l T h e r a p e u t i c s ( A x e l r o d , 1 9 5 4 ) .A f t e r t h e d e s c ri p ti o n o f t h e T P N H - r e q u i r i n g m i c ro s o m a l e n z y m e s t h a td e a m i n a t e d a m p h e t a m i n e a n d d e m e t h y l a t e d e p h e d r i n e , s e v e r a l m e m b e r so f t h e L a b o r a t o r y o f C h e m i c a l P h a r m a c o l o g y a t t h e N I H d e s c ri b e d s i m i l a re n z y m e s y s t e m s t h a t c o ul d m e t a b o l i z e o t h e r d r u g s b y a v a r i e t y o f p a t h -w a y s : N - d e m e t h y l a t i o n o f a m i n o p y r i n e ( L a D u, G a u d e t t e , T ro u so f, a n d


15/34

Julius Axelrod 59B r o d ie ), o x i d a t i o n o f b a r b i t u r a t e s ( C o o p e r a n d B r o d ie ), a n d t h e h y d r o x y l a -t io n o f a n i l in e ( M i t o m a a n d U d e n f r ie n d ) a s r e v ie w e d i n t h e Annual Reviewof Biochemistry ( B ro d i e e t al ., 1 9 5 8). I n a s t u d y o f t h e N - d e m e t h y l a t i o n o fn a r c o ti c d r u g s t h a t I m a d e s oo n af te r, it b e c a m e a p p a r e n t t h a t t h e r e w e r em u l t ip l e m i c r o s o m a l e n z y m e s t h a t r e q u i r e d T P N H a n d 0 2 (A x e lr od ,1 9 5 6 a) . R e s e a r c h o n t h e m i c r o s o m a l e n z y m e s ( n o w c a ll e d c y to c h r o m e - P 4 5 0m o n o - o x y g e n a s e s) h a s e x p a n d e d e n o r m o u s l y a n d h a s h a d a p r o f o u n d in f lu -e n c e o n b i o m e d i c a l s c i en c e, r a n g i n g f r o m s t u d i e s o f m e t a b o l i s m o f n o r m a l -l y o c c u r r i n g c o m p o u n d s t o c a r c i n o g e n e s i s . I n r e t r o s p e c t , t h e d i s c o v e r y o ft h e m i c ro s o m a l e n z y m e s i s a m o n g t h e b e s t w o r k I d id .

B r o d ie a n d I w e r e s t r u c k b y t h e f i n d i n g s o f i n v e s t i g a t o r s a t S m i t hK l i n e & F r e n c h t h a t S K F 5 2 5 A , a c o m p o u n d w i t h l i t t l e p h a r m a c o l o g i c a la c t i o n o f i t s o w n , p r o l o n g e d t h e d u r a t i o n o f a c t i o n o f a w i d e v a r i e t y o fd r u g s . W e c o n j e c t u r e d t h a t t h e c o m p o u n d m i g h t e x e r t i t s e f f e c t s b yi n h i b i t i n g t h e m e t a b o l i s m o f d r u g s . T h e e ff ec ts o f S K F 5 2 5 A o n th e m e t a b -o l is m o f e p h e d r i n e i n d og s a n d o n t h e m e t a b o l i s m a n d d u r a t i o n o f a c t io no f h e x a b a r b i t a l i n th e p l a s m a a n d t h e s l e e p i n g t i m e i n r a t s a n d d o g s w a se x a m i n e d . W e f o u n d t h a t S K F 5 2 5 A s lo w e d th e m e t a b o l i s m o f e p h e d r i n e i nd o gs . I t p ro l o n g e d t h e p r e s e n c e o f h e x a b a r b i t a l i n t h e p l a s m a a n d s le e p-i n g t i m e i n r a t s a n d d o gs . T h u s , t h e a b i li ty o f S K F 5 2 5 A to p r o l o n g th ea c t i o n o f d r u g s c o u l d b e e x p l a i n e d b y it s a b i l i ty t o sl o w t h e i r m e t a b o l i s m .A s s o o n a s t h e m i c r o s o m a l e n z y m e s w e r e d e s c r i b e d , i t w a s o b s e r v e d t h a tS K F 5 2 5 A in h i b i t e d t h i s c l as s o f e n z y m e s . S u b s e q u e n t l y , S K F 5 2 5 A w a sw i d e l y u s e d a s a n i n h i b i t o r o f t h e m i c r o s o m a l e n z y m e s .

T h e e ff ec t o f t h e m i c r o s o m a l e n z y m e s o n t h e d u r a t i o n o f d r u g a c t i o n sw a s e x a m i n e d w i t h th e c o l la b o ra ti o n o f G e r t r u d e Q u i n n , a g r a d u a t e s t u d e n ta t G e o r g e W a s h i n g t o n U n i v e r si ty , a n d S t e v e B ro d ie . B e c a u s e s l e ep i n g ti m eo f h e x a b a r b i t a l w a s e a s y t o m e a s u r e , w e c h o se t h a t d r u g t o m a k e t h i s s tu d y.J a c k C o o p e r a n d B r o d i e h a d f o u n d t h a t h e x a b a r b i t a l w a s m e t a b o l i z e d b ym i c r os om a l e nz ym e s i n t he l ive r. The s l e e p i ng t i m e o f a g i ve n dos e o f he xa -b a r b i t a l w a s c o m p a r e d w i t h i ts p l a s m a h a l f- li fe a n d w i t h t h e a c t iv i t y o f al iv e r e n z y m e p r e p a r a t i o n u s i n g th e b a r b i t u r a t e a s a s u b s t r a t e i n a n u m b e ro f m a m m a l i a n s p ec ie s. T h e r e w e r e c o n s i d e ra b l e d i f fe r e n ce s in t h e p l a s m ah a l f - l i f e , s l e e p i n g t i m e , a n d e n z y m e a c t i v i t y a m o n g t h e v a r i o u s s p e c i e s( Q u i n n e t a l. , 1 95 8 ). A h i g h c o r r e la t io n w a s o b s e r v e d b e t w e e n t h e p l a s m ah a lf -l if e a n d s l e e p i n g t i m e o f t h e b a r b i t u r a t e . T h e r e w a s a l so a n i n v e r s er e l a t i o n s h i p b e t w e e n t h e d u r a t i o n o f a c t io n o f h e x a b a r b i t a l a n d i ts a b i l it y t ob e m e t a b o l i z e d b y t h e m i c r o s o m a l e n z y m e s .I n 1956 , I r e por t e d t ha t na r c o t i c d r ugs s uc h a s mor ph i ne , me pe r i d i ne ,a n d m e t h a d o n e w e r e N - d e m e t h y l a t e d b y t h e liv er , r e q u i r i n g T P N H a n d 0 2( Axel rod , 1956a ). D i f f er e nc es i n t he r a t e o f N - de m e t hy l a t i on o f va r i ous na r -c o t i c d r u g s i n s e v e r a l s p e c i e s m a d e i t a p p a r e n t t h a t m o r e t h a n o n e e n z y m ewa s i nvo l ve d i n t he i r de me t hy l a t i on . The r e wa s a l s o a ma r ke d s e x d i f f e r e nc ei n N- de m e t hy l a t i on o f na r c o t ic d r ug s by r a t l i ve r mi c r os om a l e nz yme s .


16/34

60 J u l i u s A x e l r o dMicrosomes obtained from male rats were found to N-demethylate narcoticdrugs much faster than those from female rats. When testosterone wasadministered to oophorectomized female rats, the activity of the demethylat-ing enzyme was markedly increased. Estradiol given to male rats decreasedthe enzyme activity. Subsequent work by many investigators found similarsex differences in microsomal enzyme activity for many metabolic pathways.

While working on the metabolism of narcotic drugs, I observed tha trepeated admin istration of narcotic drugs not only produced tolerance tothese drugs, but also markedly reduced the ability to N-demethylate themenzymatically (Axelrod, 1956b). There was also a correlation between therate of demethylation of opiate subst rates and their cross-tolerance to mor-phine. Opiate antagonists not only blocked the development of tolerance,but also prevented the reduction of enzyme activity. On the basis of theseobservations, a mechanism for tolerance to narcotic drugs was proposed. Ina paper reporting these experiments (Axelrod, 1956b) the following state-ment was made: "The changes in enzyme activity in morphine-treated ratssuggests a mechanism for the development of tolerance to narcotic drugs. Ifone assumes that enzymes which N-demethylate narcotic drugs and thereceptors for these drugs are closely related, then the continuous interac-tion of narcotic drugs with the demethylating enzymes inactivates theenzymes. Likewise, the continuous interaction of narcotic drugs with theirreceptors may inactivate the receptors. Thus, a decreased response to nar-cotic drugs may develop as a result of unavailabili ty of receptor sites." Thishypothesis stimulated considerable critical reaction, mostly negative.

Although I had just described the physiological disposition of caffeine,demonstrated the variety of metabolic pathways of amphet amine andephedrine, and independently described the microsomal enzymes andthe ir role in drug metabolism, it was difficult for me to obtain a promotionto a higher rank at the National Heart Institute because I had no doctor-ate. I decided to get a Ph.D. degree at George Washington University,because few courses were required if a candidate already had an M.S.degree. However, it would be necessary to take demanding comprehensiveexaminat ions in several subjects. Paul K. Smith, then chai rman of phar-macology, accepted me as a graduate student in his department. Heallowed me to submit my work on the metabolism of sympathomimeticamines and the microsomal enzymes for my dissertation. I took a year offto attend courses at George Washington University, and I found goingback to school pleasant and challenging. A few of the medical s tudent s didbetter than I did on the pharmacology examinations. On one occasion aquestion was asked on a multiple-choice examination on antipyrine, acompound on which I published several papers, and I gave the wronganswer. After a year's study, I passed a tough comprehensive examinat ion,and my thesis, "The Fate of Phenylisopropylamines," was accepted. In1955, at the age of 42 years, I received my Ph.D.


17/34

Julius Axelrod 61S e t t i n g U p A L a b or a to r y a t th e N a t i o n a l I n s t i t u t e o f M e n t a lH e a l t hW h i l e s t u d y i n g f o r m y P h . D ., I w a s i n v i te d b y E d w a r d E v a r t s t o s e t u p aS e c t i o n o f P h a r m a c o l o g y i n h i s L a b o r a t o r y o f C l i n ic a l S c i e n c e s a t t h eN a t i o n a l I n s t i t u t e o f M e n t a l H e a l t h ( N I M H ) . To g e t s t a r t e d i n m y n e wp o s i t io n a t t h e N I M H I t o o k a f e w a f t e r n o o n s o ff m y c l a s s e s a t G e o r g eW a s h i n g t o n U n i v e r s i t y to do l a b o r a t o r y w o r k . I t h o u g h t t h a t a s t u d y o ft h e m e t a b o l i s m a n d d i s t r ib u t i o n o f L S D w o u l d b e a n a p p r o p r i a t e p r o b l e mf or m y n e w l a b o r a t o r y a t t h e N I M H . L S D w a s t h e n u s e d a s a n e x p e ri -m e n t a l d r u g by p s y c h i a t r i s t s t o s t u d y a b n o r m a l b e h av i o r. B o b B o w m a n a tt h e N I H w a s i n t h e p r o c e s s o f b u i l d i n g a s p e c t r o f l u o r o m e t e r . H e w a s k i n de n o u g h t o l e t m e u s e h i s e x p e r i m e n t a l m o d e l , w h i c h a l l o w e d m e t o d e v e l -o p a v e r y s e n s i t i v e f l u o r o m e t r i c a s s a y f o r L S D . T h i s m a d e i t p o s s i b l e t om e a s u r e t h e n a n o g r a m a m o u n t s f o u n d i n b r a i n a n d o t h e r t i ss u e s. T h i si n s t r u m e n t l a t e r b e c a m e t h e w e l l - k n o w n A m i n c o B o w m a n s p e c tr o fl u o-r o m e t e r . T h e a v a i l a b i li t y o f t h i s i n s t r u m e n t m a d e i t p o s si b le f o r m a n y l a b -o r a t o ri e s to d e v is e s e n s it i v e m e t h o d s f or t h e m e a s u r e m e n t o f e n d o g e n o u se p i n e p h r i n e , n o r e p i n e p h r i n e , d o p a m i n e , a n d s e r o t o n i n i n b r a i n a n d o t h e rt i s s u e s . T h e s e n e w l y d e v e l o p e d m e t h o d s f or b i o g en i c a m i n e s w e r e c r u c i a li n t h e s u b s e q u e n t r a p i d e x p a n s i on i n n e u r o t r a n s m i t t e r r e s ea r c h .

J u s t b e f o r e I l e f t t h e H e a r t I n s t i t u t e , I r e a d a r e p o r t i n t h e l i t e r a t u r et h a t u r i d i n e d i p h o s p h a t e g l u c u ro n i c a c id ( U D P G A ) w a s a n e c e s s a r y c ofa c-t o r f or t h e f o r m a t i o n o f p h e n o l i c g l u c u r o n i d e i n a c el l- fr e e p r e p a r a t i o n o fl i v e r s . J a c k S t r o m i n g e r , a b i o c h e m i s t t h e n a t t h e N I H , a n d I d i s c u s s e d t h ep o s si b le m e c h a n i s m f or t h e e n z y m a t i c s y n t h e s i s o f U D P G A . W e s u s p e c t-e d t h a t i t w o u l d a r i s e f r o m t h e o x i d a t i o n o f u r i d i n e d i p h o s p h a t e g l u co s e( U D P G ) b y e i t h e r T P N o r D P N . W e o b t a i n e d a s a m p l e o f U D P G f ro mH e r m a n K a l c k a r a n d d i d a p r e l i m i n a r y e x p e r i m e n t in w h i c h I m e a s u r e dt h e d i s a p p e a r a n c e o f m o r p h i n e i n g u i n e a p i g liv er . W h e n m o r p h i n e w a si n c u b a t e d w i t h g u i n e a p ig li v er m i c r o s o m e s a n d t h e s o lu b le f r a c t io n w i t hD P N a n d U D P G , m o r p h i n e w a s m e t a b o l i z e d ; T P N h a d n o e f f e c t . W h e ne i t h e r D P N o r U D P G , s o l u b l e f r a c t i o n , o r l i v e r w a s o m i t t e d , t h e d i s a p -p e a r a n c e o f m o r p h i n e w a s n e g l i gi b le . A f t e r a p e r io d o f i n c u b a t i o n d u r i n gw h i c h t h e m i x t u r e w a s h e a t e d i n 1 N H C1, t h e m o r p h i n e t h a t d i s a p p e a r e dw a s r e c o v e r e d . T h e s e e x p e r i m e n t s s u g g e s t e d t h a t m o r p h i n e w a s e n z y -m a t i c a l l y c o n j u g a te d i n t h e p r e s e n c e o f U D P G a n d D P N , p r e s u m a b l y b yt h e f o r m a t i o n o f U D P G A f o ll ow e d b y m o r p h i n e g l u c u r o n i d e f o r m a t i o n . Ih a d l i t t l e t i m e t o c o n t i n u e t h i s p r o b l e m b e c a u s e I w a s i n t h e p r o c e s s o fg e t t i n g m y P h . D . S t r o m i n g e r a n d c o - w o r k e r s t h e n w e n t o n t o p u r i f y a ne n z ym e U D P G d e h y d r o ge n a s e t h a t f o rm e d U D P G A fr om U D P G a n d D P N .

A f t e r c o m p l e t i o n o f m y P h .D . , I r e t u r n e d t o t h e g l u c u r o n i d e p r o b l e mi n m y n e w l a b o r a t o r y a t t h e N I M H . A s e x p e c t e d f r o m m y p r e l i m i n a r ye x p e r i m e n t w i t h m o r p h i n e , I f o u n d t h a t m o r p h i n e a n d o t h e r n a rc o t ic


18/34

62 Julius Axelrodd r u g s f o r m e d g l u c u r o n id e c o n j u g a t e s b y a n e n z y m e p r e s e n t i n li v e r m i c ro -s o m e s t h a t r e q u i r e d U D P G A . W o r k i n g t o g e th e r , Jo e I n sc o e, a g r a d u a t es t u d e n t a t G e o r g e W a s h i n g t o n U n i v e r s i t y , a n d I s h o w e d t h a t g l u c u r o n i d ef o r m a t i o n c o u l d b e i n d u c e d b y b e n z p y r e n e a n d 3 - m e t h y l c h o l a n t h r e n e .

T h e w o r k o n g l u c u r o n i d e c o n j u g a t i o n l e d to a s t u d y o n t h e r o le o f g l u -c u r o n i c a c i d c o n j u g a t i o n o n b i l i r u b i n m e t a b o l i s m . R u d i S c h m i d , t h e n a tt h e N I H , m a d e t h e i n t e r e s t i n g o b s er v a ti o n t h a t b i l ir u b in w a s t r a n s f o r m e dt o a g lu c u r o n i d e . S c h m i d a n d I t h e n w e n t o n to d e s c ri b e t h e e n z y m a t i c fo r-m a t i o n o f b i l i r u b i n g l u c u r o n i d e b y e n z y m e s i n t h e l i v er r e q u i r i n gU D P G A . T h i s c o n j u g a t i n g e n z y m e s e r v e d a s a m e c h a n i s m f o r i n a c t i v a t -i n g b i l i r u b i n . T h i s f i n d i n g l e d t o a n i n t e r e s t i n g c l i n i c a l o b s e r v a t i o n c o n -c e r n i n g a d e f ec t in g l u c u r o n i d e f o r m a t i o n . I n c o n g e n i t a l j a u n d i c e t h e r e i sa m a r k e d e l e v a t i o n o f f r ee b i l i r u b i n i n t h e b lo o d. T h i s f a c t s u g g e s t e d t o u st h a t s o m e t h i n g m i g h t b e w r o n g w i t h g l u c u r o n i d e f o r m a t i o n i n t h i s d i s -e a s e. T h e a v a i l a b i l i ty o f a m u t a n t s t r a i n o f r a t s ( G u n n r a t s ) t h a t e x h i b i t -e d c o n g e n i t a l j a u n d i c e m a d e i t p o s si b le to e x a m i n e w h e t h e r t h e g l u -c u r o n i d e - f o r m i n g e n z y m e w a s d e f e c t i v e . W e t h e n w e n t o n t o d e m o n s t r a t et h a t t h e s e r a t s s h o w e d a m a r k e d d e fe c t i n t h e a b i l it y t o s y n t h e s i z e g lu -c u r o n i d e s f r o m U D P G A ( A x e l r o d e t a l . , 1 9 5 7 ) . G l u c u r o n i d e f o r m a t i o n w a sa ls o e x a m i n e d i n h u m a n s w i t h c o n g e n i ta l j a u n d i c e b y m e a s u r i n g t h e r a t ea n d m a g n i t u d e o f p l a s m a a c e t a m i n o p h e n g l u cu r o n id e a f t er th e a d m i n i s -t r a t i o n o f t h e a c e t a m i n o p h e n . A d ef ec t i n g l u c u r o n i d e f o r m a t i o n i n t h i sd i s e a s e w a s d e m o n s t r a t e d .C a t e c h o l a m i n e R e s e a r c hW h e n I j o in e d t h e N I M H , I k n e w v e r y li t t le a b o u t n e u r o sc i e n ce . M yi m p r e s s i o n o f n e u r o s c ie n c e t h e n w a s t h a t i t w a s c o n c e r n e d m a i n l y w i t he l e ct r op h y s i o lo g y , b r a i n a n a t o m y , a n d b e h a v i o r . To m e t h e s e s u b j e c t sw e r e s o m e w h a t s t r a n g e a n d e s o t e r i c a n d c o n c e r n e d w i t h c o m p l i c a t e de l e c t r o n i c e q u i p m e n t . I b e l i e v e d t h a t a n i n v e s t i g a t o r h a d t o b e a g i f t e de x p e r i m e n t a l i s t a n d t h e o r i s t t o d o r e s e a r c h i n t h e n e u r o s c i e n c e s . E dE v a r t s , m y la b c hie f, a s s u r e d m e t h a t I c o ul d w o r k o n w h a t e v e r p r o b l e mI t h o u g h t w o u l d b e l i k e ly t o y i e ld n e w i n f o r m a t i o n . T h e p h i l o s o p h y o fS e y m o u r K e ty , t h e n h e a d o f t h e I n t r a m u r a l P r o g r a m s o f t h e N I M H , w a sto a l lo w i n v e s t i g a t o r s w o r k i n g i n t h e l a b o r a t o r i e s o f t h e N I M H t o dot h e i r r e s e a r c h o n w h a t e v e r w a s p o t e n t i a l l y p r o d u c t i v e a n d i m p o r t a n t .K e t y b e l i e v e d t h a t w i t h o u t s u f f i c i e n t b a s i c k n o w l e d g e a b o u t t h e l i f ep r o c es s e s, d o i ng t a r g e t e d r e s e a r c h o n m e n t a l i l ln e s s w o u l d be a w a s t e o ft i m e a n d m o n e y .

I n s t e a d o f w o r k i n g o n a n e u r o b io l o g i c a l p r o b l e m , I t h o u g h t i t w o u l d b eb e s t t o w o r k o n o n e t h a t I k n e w s o m e t h i n g a b o u t , a n d t h a t m i g h t b ea p p r o p r i a t e t o t h e m i s s io n o f t h e N I M H . I b e g a n t o e x p e r i m e n t o n t h em e t a b o l i s m a n d p h y s io l o g ic a l d i s p o s it i o n of L S D a n d t h e e n z y m e s


19/34

J u l i u s A x e l r od 63i n v o l v e d in t h e m e t a b o l i s m o f n a r c o t i c d r u g s . I a l so w o r k e d o n t h e e n z y -m a t i c s y n t h e s i s o f g l u c u r o n i d e s d e s c r i b e d a b o v e.

A l t h o u g h t h e N I M H a d m i n i s t r a t o r s w e r e s u p p o r t iv e o f t h e t y p e o fr e s e a r c h I w a s d o i n g , I s t il l f e lt g u i l t y t h a t I w a s n o t w o r k i n g o n s o m ea s p e c t o f t h e n e r v o u s s y s t e m o r m e n t a l i l ln e s s . D r. K et y , i n a s e m i n a r too u r l a b o r a t o r y , g a v e a f a s c i n a t i n g a c c o u n t o f t h e f i n d i n g s o f t w o C a n a d i a np s y c h i a t r i s ts . T h e y r e p o r t e d t h a t a d r e n o c h r o m e p r o d u c e d s c h iz o p h r e n ic -l ik e h a l l u c i n a t i o n s w h e n i t w a s i n g e s te d . B e c a u s e o f t h e s e b e h a v i o r a le ff ec ts , th e y p r o p o s e d t h a t s c h i z o p h r e n i a c o u ld b e c a u s e d b y a n a b n o r m a lm e t a b o l i s m o f e p i n e p h r i n e t o a d r e n o c h r o m e . I w a s i n t r i g u e d b y th i s p r o -p o s a l . I n s e a r c h i n g t h e l i t e r a t u r e , I w a s s u r p r i s e d t o f i n d t h a t l i t t l e w a sk n o w n a b o u t t h e m e t a b o l i s m o f e p i n e p h r i n e a t t h a t t i m e , i n 1 95 7. I n v i ewo f t h e p r o v o c a ti v e h y p o t h e s i s a b o u t t h e a b n o r m a l m e t a b o l i s m o f e p i n e p h -r i n e i n s c h i z o p h r e n i a , I d e c id e d t o w o r k o n t h e m e t a b o l i s m o f e p i n e p h r i n e .E p i n e p h r i n e w a s t h e n b e l i e v e d t o b e m e t a b o l i z e d a n d i n a c t i v a t e d b yd e a m i n a t i o n b y m o n o a m i n e o x i d a s e . H o w e v e r , w i t h t h e i n t r o d u c t i o n o fm o n o a m i n e o x i d a se i n h i b i t o r s b y A l b e r t Z e l l er a n d c o -w o r k e rs , i t w a so b s e r v ed t h a t , a f t e r t h e i n h i b i t io n o f m o n o a m i n e o x i d a s e in v ivo, t h e p h y s -i o lo g ic a l a c t i o n s o f a d m i n i s t e r e d e p i n e p h r i n e w e r e s t i ll r a p i d l y e n d e d .T h i s f i n d i n g i n d i c a t e d t h a t e n z y m e s o t h e r t h a n m o n o a m i n e o x i d a s em e t a b o l i z e d e p i n e p h r i n e . A p o s si b le r o u t e o f m e t a b o l i s m o f e p i n e p h r i n em i g h t b e v i a o x i d a t i o n . I s p e n t s e v e r a l m o n t h s l o o k i n g a t o x i d a t i v ee n z y m e s f o r e p i n e p h r i n e w i t h o u t a n y s u c c e s s .A n a b s t r a c t i n t h e M a r c h 1 9 5 7 Federat ion Proceedings g a v e m e a ni m p o r t a n t c lu e r e g a r d i n g a p o ss ib l e p a t h w a y fo r t h e m e t a b o l i s m o f e p i-n e p h r i n e . I n t h i s a b s t r a c t , A r m s t r o n g a n d M c M i l l a n ( 1 9 5 7 ) r e p o r t e d t h a tp a t i e n t s w i t h n o r e p i n e p h r i n e - f o r m i n g t u m o r s ( p h e o c h ro m o c y t o m a s )e x c r e te d l a r g e a m o u n t s o f a n O - m e t h y l a t e d p r o d u c t, 3 - m e t h o x y -4 - h y d ro x -y m a n d e l i c a c id (V M A ). T h i s f i n d i n g s u g g e s t e d t h a t t h i s m e t a b o l i t e c ou l d b ef o rm e d b y th e O - m e t h y l a t i o n a n d d e a m i n a t i o n o f e p i n e p h r i n e o r n o r e p i-n e p h r i n e . T h e O - m e t h y l a t i o n of c a t e c h o l a m i n e s w a s a n i n t r i g u i n g p o ss i-b i l i t y t h a t c o u l d b e e x p e r i m e n t a l l y t e s t e d . A p o t e n t i a l m e t h y l d o n o r c o u l db e S - a d e n o s y l m e t h i o n i n e . T h a t a f t e r n o o n I i n c u b a t e d e p i n e p h r i n e w i t h ah o m o g e n a t e o f r a t l iv e r, A T P, a n d m e t h i o n i n e . I d id n o t h a v e S - a d e n o s y l -m e t h i o n i n e a v a il a b le , b u t C a n t o n i ( 19 53 ) h a d s h o w n t h a t a n e n z y m e in t h el iv e r co u ld c o n v e r t A T P a n d m e t h i o n i n e t o a d e n o s y l m e t h i o n i n e . I fo u n dt h a t e p i n e p h r i n e w a s r a p i d l y m e t a b o l i z e d in t h e p r e s e n c e o f A TP , m e t h i o -n i n e a n d l iv e r h o m o g e n a t e . W h e n e i t h e r A T P o r m e t h i o n i n e w a s o m i t te d o rt h e h o m o g e n a t e w a s h e a t e d , t h e r e w a s a n e gl ig i b le d i s a p p e a r a n c e o f e p i-n e p h r i n e . T h i s e x p e r i m e n t s u g g e s t e d t h a t e p i n e p h r i n e w a s O - m e t h y l a t e di n th e p r e s e n c e o f a m e t h y l d o n or , p r e s u m a b l y S - a d e n o s y l m e t h i o n i n e . I n as u b s e q u e n t e x p e r i m e n t , I o b t a i n e d S - a d e n o s y l m e t h i o n i n e a n d o b s e r v e dt h a t i n c u b a t i n g l i v e r h o m o g e n a t e w i t h t h e m e t h y l d o n o r r e s u l t e d i n t h em e t a b o l i s m o f e p i n e p h r i n e . T h e m o s t l i ke l y s it e o f m e t h y l a t i o n w o u l d b e o n


20/34

64 J u l i u s A x e lr o dt h e m e t a h y d r o x y l g r o u p o f e p i n e p h r i n e t o f o r m 3 - O - m e t h y l e p i n e p h r i n e . Ip r e v a i l e d o n m y c o l l e a g u e B e r n h a r d W i t k o p , a n o r g a n i c c h e m i s t , t o s y n -t h e s i z e t h e O - m e t h y l m e t a b o l i t e o f e p i n e p h r i n e . A f ew d a y s l a t e r S h e r o hS e n o h , a v i s i t i n g s c i e n t i s t i n W i t k o p ' s l a b o r a t o r y , s y n t h e s i z e d m e t a - O -m e t h y l e p i n e p h r i n e . A f t e r i n c u b a t i n g l i v e r a n d S - a d e n o s y l m e t h i o n i n e , t h em e t a b o l i t e f o r m e d f r o m e p i n e p h r i n e w a s i d e n t i f i e d a s m e t a - O - m e t h y l e p i -n e p h r i n e , w h i c h w e n a m e d metanephr ine , i n d i c a t i n g t h e e x i s t e n c e o f a n O -m e t h y l a t i n g e n z y m e . T h e O - m e t h y l a t i n g e n z y m e w a s p u r if ie d a n d f o u n d t oO - m e t h y l a t e c a te c h o ls , i n c l u d i n g n o r e p i n e p h r i n e , d o p a m i n e , L -D O P A , a n dsyn the t i c ca t echo l s , bu t n o t m ono phe no l s (Axe lrod , 1971 ). I n v iew o f t hes u b s t r a t e s pe cif ic ity , t h e e n z y m e w a s n a m e d catechol-O-methyl trans ferase(COMT) . T h e e n z y m e w a s f o u n d t o b e w i d e l y d i s t r i b u t e d i n ti s s u e s , i n c l u d -i n g t h e b r a i n .

I n j e c t in g c a t e c h o l a m i n e s i n t o a n i m a l s r e s u l t e d i n t h e e x c r e t i o n o f t h er e s p e c t i v e O - m e t h y l a t e d m e t a b o l i t e s . W e s o o n i d e n t i f i e d n o r m a l l y o c c u r -r i n g O - m e t h y l a t e d m e t a b o li t es s u ch a s n o r m e t a n e p h r i n e , m e t a n e p h r i n e ,3 - m e t h o x y t y r a m i n e , a n d 3 - m e t h o x y - 4 - h y d r o x y p h e n y l g ly c o l ( M H P G ) i nl iv e r a n d b r a i n . A s a r e s u l t o f t h e d i sc o v e ry o f t h e O - m e t h y l a t e d m e t a b o -l it es , t h e p a t h w a y s o f c a t e c h o l a m i n e m e t a b o l i s m w e r e c la r if ie d ( A x elr od ,1 9 7 1 ) . C a t e c h o l a m i n e s w e r e m e t a b o l i z e d b y O - m e t h y l a t i o n , d e a m i n a t i o n ,g ly c o l f o r m a t i o n , o x i d a t io n , a n d c o n j u g a t i o n . A s a r e s u l t o f t h e s e f i n d in g s ,I t h e n c o n s i d e re d m y s e l f a n e u r o c h e m i s t . T h i s w o r k a ls o g a v e m e a l o n g-l a s t i n g i n t e r e s t i n m e t h y l a t i o n r e a c t i o n s t h a t I d e s c ri b e la t e r. T h e m e t a b o -l it e s o f c a t e c h o l a m i n e s , p a r t i c u l a r l y M H P G , h a v e b e e n u s e d a s a m a r k e ri n m a n y s t u d i e s i n b i o l o g i c a l p s y c h i a t r y .

A m a j o r p r o b l e m i n n e u r o b i o l o g y r e s e a r c h i s t h e m e c h a n i s m b y w h i c hn e u r o t r a n s m i t t e r s a r e i n a c t iv a t e d . A t t h e t i m e I d e sc r ib e d t h e m e t a b o l i cpa th w ay fo r ca t ech o lam ines i n 1957 , i t was be l i eved t h a t t he ac t i ons o f neu -r o t r a n s m i t t e r s w e r e t e r m i n a t e d b y e n z y m a t i c t r a n s fo r m a t i o n . A c e ty lc h ol in ew a s a l r e a d y k n o w n t o b e r a p i d l y i n a c t i v a t e d b y a c e t y l c h o l i n e s t e r a s e .H o w e v e r, w h e n t h e p r i n c ip a l e n z y m e s f or th e m e t a b o l i s m o f c a t e c h o l a m i n e s ,c a t e c h o l - O - m e t h y l t r a n s f e r a s e a n d m o n o a m i n e o x i d a s e , w e r e a l m o s t c o m -p le t e ly i nh ib i t ed in vivo, t he phys io log i ca l a c t i ons o f i n j ec ted ep in eph r inew e r e r a p i d l y e n d e d . T h e s e e x p e r i m e n t s i n d i c a t e d t h a t t h e r e w e r e o t h e rm e c h a n i s m s f o r t h e r a p i d i n a c t i v a t i o n o f c a t e c h o la m i n e s .

T h e a n s w e r t o t h e q u e s t i o n o f t h e i n a c t i v a t i o n o f c a t e c h o l a m i n e s c a m ei n a n u n e x p e c t e d w a y. W h e n t h e m e t a b o l i s m o f c a t e c h o la m i n e s w a sd e s c r i b e d , S e y m o u r K e t y a n d c o - w o r k e r s s e t o u t t o e x a m i n e w h e t h e r o rn o t t h e r e w a s a n a b n o r m a l m e t a b o l i s m o f e p i n e p h r i n e i n s ch i z o p h r e n icp a t i e n t s . T o c a r r y o u t t h i s s t u d y , K e t y a s k e d t h e N e w E n g l a n d N u c l e a rC o r p o r a t io n t o p r e p a r e t r i t i u m - l a b e l e d e p i n e p h r i n e a n d n o r e p i n e p h r i n e o fh i g h s p ec if ic a c ti v it y . T h e f i r s t b a t c h o f 3 H - e p i n e p h r i n e t h a t a r r i v e d i n l a t e1 9 5 7 w a s l a b e l e d o n t h e 7 p o s i ti o n , w h i c h w e f o u n d t o b e s ta b l e . K e t y w a sk i n d e n o u g h t o gi ve m e s o m e o f t h e 3 H - e p i n e p h r i n e f o r m y s t u d i e s . I


21/34

Julius Axelrod 65t h o u g h t i t w o u l d b e a g o o d i d e a t o e x a m i n e t h e t i s s u e d i s t r i b u t i o n a n dh a l f- li fe o f 3 H - e p i n e p h r i n e i n a n i m a l s .

A b o u t t h a t t im e , H a n s W e i l -M a l h e rb e s p e n t t h r e e m o n t h s i n m y la b -o r a t o r y a s a v i s it i n g s c ie n t is t , a n d t o g e t h e r w e d e v e l o p e d m e t h o d s o f m e a -s u r i n g 3 H - e p i n e p h r i n e a n d i ts m e t a b o l i t e s i n t i s s u e s a n d p l a s m a . To o u rs u r p r i s e , w h e n 3 H - e p i n e p h r i n e w a s i n j e c t e d i n t o c a t s , i t p e r s i s t e du n c h a n g e d i n t h e h e a r t , s p l e e n , a n d t h e s a l i v a r y a n d a d r e n a l g l a n d s l o n ga f t e r i t s p h y s i o l o g i c a l e f f e c t s w e r e e n d e d . T h i s p h e n o m e n o n p u z z l e d u s .W e a l s o f o u n d t h a t 3 H - e p i n e p h r i n e d i d n o t c r o s s t h e b l o o d - b r a i n b a r r i e r .J u s t a b o u t t h is t i m e G o r do n W h it by , a g r a d u a t e s t u d e n t f r om C a m b r i d g eU n i v e r s i t y , c a m e t o o u r l a b o r a t o r y t o d o h i s P h . D . t h e s i s . I s u g g e s t e d t h a th e u s e m e t h o d s f o r a s s a y i n g 3 H - n o r e p i n e p h r i n e s i m i l a r t o t h o s e w e u s e df or 3 H - e p i n e p h r i n e t o s t u d y i t s ti s s u e d i s t r i b u t i o n . A s i n t h e c a s e o f 3 H -e p i n e p h r i n e , 3 H - n o r e p i n e p h r i n e p e r s i s t e d i n o r g a n s r i c h i n s y m p a t h e t i cn e r v e s ( h e a r t , s p l e e n , s a l i v a r y g l a n d ). T h e s e s t u d i e s g a v e u s a c l u e r e g a r d -i n g t h e i n a c t i v a ti o n o f c a t e c h o la m i n e n e u r o t r a n s m i t t e r s : u p t a k e a n dr e t e n t i o n i n s y m p a t h e t i c n e r v e s .T h e c r u c i a l e x p e r i m e n t t h a t e s t a b l i s h e d t h a t c a t e c h o l a m i n e s w e r es e le c t iv e l y t a k e n u p i n s y m p a t h e t i c n e u r o n s w a s s u g g e s t e d b y G e o rg eH e r t t i n g f r om t h e U n i v e r s i t y o f V i e n n a , w h o j o i n e d m y l a b o r a t o r y a s a v is -i t i n g s c i e n t i s t . I n t h e n e x t e x p e r i m e n t , t h e s u p e r i o r c e r v i c a l g a n g l i a o fc a t s w e r e t a k e n o u t o f o n e si de , r e s u l t i n g i n a u n i l a t e r a l d e g e n e r a t i o n o fs y m p a t h e t i c n e r v e s i n t h e s a l i v a r y g l a n d a n d e y e m u s c l e s . O n t h e i n j e c -t io n o f 3 H - n o r e p i n e p h r i n e , r a d i o a c t i v e c a t e c h o l a m i n e a c c u m u l a t e d o n t h ei n n e r v a t e d s i d e , b u t v e r y l i t t l e a p p e a r e d o n t h e d e n e r v a t e d s i d e ( H e r t t i n ge t a l . , 1 9 6 1 ) . T h i s s i m p l e e x p e r i m e n t c l e a r l y s h o w e d t h a t s y m p a t h e t i cn e r v e s t a k e u p a n d s to r e n o r e p i n e p h r i n e . I n a n o t h e r s e r i es o f e x p e r i m e n t s ,H e r t t i n g a n d I f o u n d t h a t i n j e c t e d 3 H - n o r e p i n e p h r i n e t a k e n u p b y s y m p a -t h e t i c n e r v e s w a s r e l e a s e d w h e n t h e s e n e r v e s w e r e s t i m u l a t e d ( H e r t t i n ga n d A x e l ro d , 1 96 1) . A s a r e s u l t o f t h e s e e x p e r i m e n t s , w e p r o p o s e d t h a t n o r -e p i n e p h r i n e i s r a p i d l y i n a c t i v a t e d b y r e u p t a k e i n t o s y m p a t h e t i c n e r v e s .O t h e r s lo w e r m e c h a n i s m s fo r t h e i n a c t i v a ti o n o f c a t e c h o l a m i n e s p r o p o s e dw e r e r e m o v a l b y t h e b l o o d s t r e a m , m e t a b o l i s m b y O - m e t h y l a t i o n , a n d / o rd e a m i n a t i o n b y li v e r a n d k i d ne y .

In 1961, t he f i r s t pos tdoc to ra l f ellow , L inco ln Po t t e r , j o ined m y l abo ra -t o ry v ia t h e N I H R e s e a r c h A s so c ia te s P r o g r a m . T h e N I H R e s e a r c hA s s o ci at es P r o g r a m a n d t h e P h a r m a c o l o g y R e s e a r c h A s s o ci a te s P r o g r a mp r o v i d e d a n o p p o r t u n i t y f o r r e c e n t P h . D . a n d M . D . g r a d u a t e s t o s p e n d t w oo r t h r e e y e a r s i n B e t h e s d a d o i n g f u l l- t im e r e s e a r c h . B e c a u s e o f t h e n u m b e ro f a p p l i c a n ts f o r t h i s p r o g r a m , t h e i n v e s t ig a t o r s i n th e I n t r a m u r a l P r o g r a ma t t h e N I H w o u l d g e t t h e b e s t a n d b r i g h t e s t p o s t d o c t o r a l f e l l o w s . D u r i n gt h e p a s t 2 5 y e a r s m o r e t h a n 6 0 p o s td o c t o r a l fe ll ow s j o i n e d m y l a b o r a t o r y t odo fu l l - t ime r e sea rch . W i th one o r two excep t ions , m os t o f t he pos tdocs whow o r k e d i n m y l a b o r a t o r y w e n t o n t o p r o d u c t i v e c a r e e r s i n r e s e a r c h .


22/34

66 Julius AxelrodW h e n L i n c P o t t e r j o i n e d m y l a b o ra t o ry , w e d i r e c t e d o u r a t t e n t i o n t ot h e s i t e s o f t h e i n t r a n e u r a l s t o r a g e o f n o r e p i n e p h r i n e . W e s u s p e c t e d t h a t

3 H - n o r e p i n e p h r i n e , a l r e a d y s h o w n t o b e t a k e n u p b y s y m p a t h e t i c n e u -r o n s , w o u l d l a b e l i n t r a c e l l u l a r s t o r a g e s i t e s . 3 H - n o r e p i n e p h r i n e w a si n j e c t e d i n t o r a t s , a n d t h e i r h e a r t s w e r e h o m o g e n i z e d i n i s o t o n i c s u c r o s e .T h e v a r i o u s c e l lu l a r fr a c ti o n s w e r e t h e n s e p a r a t e d i n a c o n t i n u o u s s u c r o s eg r a d i e n t . T h e r e w a s a s h a r p p e a k o f r a d i o a c t iv e n o r e p i n e p h r i n e i n a fr ac -t i o n t h a t c o in c id e d w i t h e n d o g e n o u s c a t e c h o l a m i n e s a n d d o p a m i n e - B -h y d r o x y l a s e , t h e e n z y m e t h a t c o n v e r ts d o p a m i n e to n o r e p i n e p h r i n e . T h en o r e p i n e p h r i n e - c o n t a i n i n g p a r t i c le s e x e r t e d a p r e s s o r r e s p o n s e o n l y w h e nt h e y w e r e l y s ed . I n a n o t h e r e x p e r i m e n t , 3 H - n o r e p i n e p h r i n e w a s i n je c te d ,a n d t h e p i n e a l g l a n d , a n o r g a n r i c h i n s y m p a t h e t i c n e r v e t e r m i n a l s , w a ss u b j e c t e d t o r a d i o a u t o g r a p h y a n d e l e c t r o n m i c r o s c o p y . P h o t o g r a p h i cg r a i n s o f 3 H - n o r e p i n e p h r i n e w e r e h i g h l y l o c al iz e d o v e r d e n s e c o r e - g r a n u -l a t e d v e s i cl e s o f a b o u t 5 0 0 a n g s t r o m s ( A x e lr o d, 1 9 71 ). A l l t h e s e e x p e r i -m e n t s i n d i c a t e d t h a t n o r e p i n e p h r i n e i n s y m p a t h e t i c n e r v e s w a s s t o re d i nsma l l , dense co re ve s i c l e s .

S u b s e q u e n t s t u d i e s w i t h a n o t h e r p o st do c , D i c k W e i n s h i l b o u m ,s h o w e d t h a t o n s t i m u l a t i o n o f t h e h y p o g a s t r i c n e r v e o f t h e v a s d e f e re n s ,b o t h n o r e p i n e p h r i n e a n d d o p a m i n e - B - h y d r o x y l as e w e r e d i s c h a r g e d f r o mt h e n e r v e t e r m i n a l s . T h i s f i n d i n g s u g g e s t e d t h a t n o r e p i n e p h r i n e a n dd o p a m i n e - B - h y d r o x y l a s e w e r e c o lo c al iz e d in t h e c a t e c h o l a m i n e s t o r a g ev e s ic l es o f s y m p a t h e t i c n e r v e s a n d w e r e t h e n d i s c h a r g e d t o g e t h e r b y ex o-c y t o s i s ( W e i n s h i l b o u m e t a l . , 1 9 7 1 ) . T h e s e f i n d i n g s l e d u s t o t h e p o s t u l a -t i o n t h a t t h e r e l e a s e d d o p a m i n e - B - h y d r o x y l a s e w o u l d a p p e a r i n t h e b lo o d,w h i c h w a s s oo n c o nf ir m e d . L a t e r , o u r l a b o r a t o r y a n d o t h e r s f o u n d a b n o r -m a l l y l ow l e v e ls o f p l a s m a d o p a m i n e - B - h y d r o x y l a s e in f a m i l ia l d y s a u -t o n o m i a a n d D o w n ' s s y n d r o m e , a n d h i g h l e v e l s i n p a t i e n t s w i t h t o r s i o nd y s t o n i a , n e u r o b l a s t o m a , a n d c e r t a i n f or m s o f h y p e r t e n s i o n .A s s oo n a s it w a s f o u n d t h a t c a t e c h o l a m i n e s c o ul d b e t a k e n u p a n di n a c t i v a te d b y r e u p t a k e i n to s y m p a t h e t i c n e r v e t e r m i n a l s , m y c o -w o r k er sa n d I t u r n e d o u r a t t e n t i o n t o t h e e ff ec t o f a d r e n e r g i c d r u g s o n t h i s p r o c e s s .W e d e s i g n e d r e l a t i v e l y s i m p l e e x p e r i m e n t s f o r t h i s s t u d y , i n j e c t i n g t h ed r u g i n t o r a t s a n d t h e n m e a s u r i n g t h e u p t a k e o f i n je c te d 3 H - n o r ep i n ep h -r i n e i n t is s u e s . C o c a in e w a s t h e f i r s t d r u g w e e x a m i n e d . I t h a d b e e n p os -t u l a t e d t h a t c o c ai ne c a u se s s u p e r s e n s i t i v i t y to n o r e p i n e p h r i n e b y in t e r -f e r in g w i t h i t s i n a c t i v a t io n . A f t e r p r e t r e a t m e n t o f c a t s w i t h c o ca in e , t h e r ew a s a m a r k e d r e d u c t i o n o f 3 H - n o r e p i n e p h r i n e i n t i s s u e s t h a t w e r e i n n e r -v a t e d b y s y m p a t h e t i c n e r v e s a f t e r t h e i n j e c ti o n o f t h e r a d i o a c t iv e c a t e -c h o l a m i n e ( W h i t b y e t a l . , 1 9 6 0 ) . T h i s e x p e r i m e n t i n d i c a t e d t h a t c o c a i n eb l o c ke d t h e r e u p t a k e o f n o r e p i n e p h r i n e i n n e r v e s a n d t h u s a l lo w e d la r g ea m o u n t s o f c a t e c h o l a m i n e to r e m a i n a t t h e s y n a p t i c c le ft a n d a c t o n t h ep o s t s y n a p t i c r e c e p t o r s f o r l o n g e r p e r i o d s o f t i m e . U s i n g a s i m i l a ra p p r o ac h , w e o bs e rv e d t h a t a n t i d e p r e s s a n t d r u g s a m p h e t a m i n e a n d o t h e r


23/34

Julius Axelrod 67sympathomimeti c amines also blocked the upt ake of norepinephrine(Axelrod, 1971). In another type of experiment, using an isolated perfusedbeating rat heart whose nerves had previously been labeled with 3H-nor-epinephrine, we found that the physiological action of sympathomimeticamines, such as tyramine, was mediated by releasing the norepinephrinefrom sympathetic nerves (Axelrod et al., 1962). After repeated treatmentof the isolated hear t wi th tyramine, the hea rt rate and amplitude of con-traction were gradual ly reduced, presumably by the depletion of thereleasable stores of the neuro transmit ters . After replenishing the isolatedheart with exogenous norepinephrine, the heart rate and amplitude ofcontraction of the isolated hea rt were restored. Amph etamine alsoreleased norepinephrine, and it was later shown by others that the phys-iological effects of the amine were due to the release of dopamine.

Most of my early work in catecholamines was done in the periphera lsympathetic nervous system. Hans Weil-Malherbe and I had found thatcatecholamines did not cross the blood-brain barrier. This finding made itimpossible to study the metabolism, storage, and release of norepineph-rine in the brain by peripheral administ ration of 3H-norepinephrine. Itwas Jacques Glowinski, a visiting scientist from France, who circumvent-ed this problem. He devised a technique to introduce 3H-norepinephrinedirectly into the brain by injection into the lateral ventricle. Subsequentexperiments showed that 3H-norepinephrine was mixed with the endoge-nous catecholamines in the brain. As in the peripheral nervous system,the 3H-norepinephrine was found to be metabolized by O-methylation anddeaminat ion. In a series of exper iments we estab lished that 3H-norepi-nephrine could serve as a useful tool in studying the activity of brainadrenergic nerves (Axelrod, 1971).

After labeling adrenergic neurons in the brain (Glowinski and Axelrod,1964), we examined the effect of psychoactive drugs on brain biogenicamines. We found that only the clinically effective antidepressant drugsblock the r eupt ake of 3H-norepinephrine in adrenergic nerve terminals.This finding, together with the observation that monoamine oxidaseinhibitors have antidepressant actions and that reserpine, a depleter ofbiogenic amines, sometimes causes depression, led to the formulation ofthe catecholamine hypothesis of depress ion (Schi ldkraut, 1965). We alsofound that amphetami nes block the r eup take as well as the release of 3H-norepinephrine in the brain. Other investigators later showed the para-noid psychosis caused by excessive ingestion of amphetamines is due to therelease of the catecholamine dopamine. One of the reasons that LesIversen came to my lab as a postdoctoral fellow was to learn about thebrain and its chemistry. Iversen and Glowinski worked extensively togeth-er in my laboratory on the effects of drugs on the adrenergic system in dif-ferent areas of the brain. To conduct this study they devised a method ofdissection of various part s of the brain that has become a classic procedure.


24/34

68 Julius AxelrodF o r s e v e r a l y e a r s o u r l a b o r a t o ry w a s c o n c er n e d w i t h t h e a d a p t i v em e c h a n i s m o f t h e s y m p a t h o a d r e n a l a x is . O n e s u ch m e c h a n i s m , t h e i n d u c-

t i o n o f t h e c a t e c h o l a m i n e ' s b i o s y n t h e t i c e n z y m e , t y r o s i n e h y d r o x y l a s e , w a so b s e r v e d i n a n u n e x p e c t e d m a n n e r , a s o f t e n h a p p e n s i n r e s e a r c h . H a n sT h o e n e n , t h e n w o r k i n g i n B a s e l , a s k e d t o s p e n d a s a b b a t i c a l y e a r i n m yl a b o r a t o r y . H e a n d T r a n z e r h a d o b s e r v e d t h a t i n j e c t e d 6 - h y d r o x y d o p a m i n es e l e c t i v e l y d e s t r o y s c a t e c h o l a m i n e - c o n t a i n i n g n e r v e t e r m i n a l s ( T h o e n e na n d T r a n z e r , 1 9 6 8) . I i n v i t e d T h o e n e n t o j o i n m y l a b o r a t o r y a n d b r i n g 6 -h y d r o x y d o p a m i n e . T h e f i rs t e x p e r i m e n t t h a t T h o e n e n t r i e d w a s to e x a m i n et h e e ff ec ts o f t h e d e s t r u c t i o n o f p e r i p h e r a l s y m p a t h e t i c n e r v e s o n t y r o s i n ehyd roxy la se . As expec ted , a f t e r t he in j ec tion o f 6 -hyd roxyd opam ine , t y ro -s i n e h y d r o x y l a s e a l m o s t c o m p l e t e ly d i s a p p e a r e d f r o m s y m p a t h e t i c a l l yi n n e r v a t e d n e r v e s . A s u r p r i s i n g o b s e r v a t i o n w a s a m a r k e d e l e v a t i o n o f t y ro -s i n e h y d r o x y l a s e i n t h e a d r e n a l m e d u l l a . 6 - H y d r o x y d o p a m i n e w a s k n o w nt o c a u s e p e r s i s t e n t f i ri n g o f n e r v e s . W e s u s p e c t e d t h a t t y r o s i n e h y d r o x y l a s ew a s e l e v a t e d i n t h e a d r e n a l m e d u l l a b y c o n t i n u o u s f i r in g o f t h e s p l a n c h n i cn e r v e i n n e r v a t i n g t h e a d r e n a l s . T h i s s u p p o s i ti o n w a s c o n fi rm e d w h e n o t h e rd r u g s t h a t c a u s e d p r o l o n g e d n e r v e f i r i n g , s u c h a s r e s e r p i n e a n d a 2 - a d r e n -e rg i c b lock ing agen t s , a l so inc reased ty ros ine hydroxy la se (Thoenen e t a l . ,1 96 9) . S u b s e q u e n t e x p e r i m e n t s s h o w e d t h a t i n c r e a s e d n e r v e f ir i n g i n d u c e dt h e s y n t h e s i s o f n e w t y r o s i n e h y d r o x y l a s e m o l e c u le s i n n e r v e c ell b o d i esa n d t h e a d r e n a l m e d u l l a i n a t r a n s s y n a p t i c m a n n e r . S i m i l a r r e s u l t s w e r eo b t a i n e d w i t h a n o t h e r c a t e c h o l a m i n e b i o s y n t h e t i c e n z y m e , d o p a m i n e - B -h y d r o x y l a s e .A n o t h e r r e g u l a t o r y m e c h a n i s m f o r c a t e c h o l a m i n e s y n t h e s i s w a s f o u n dby a sk ing th e r igh t ques t ions r a th e r t h an by se rend ip ity . The r a t io o f ep i-n e p h r i n e t o n o r e p i n e p h r i n e i n th e a d r e n a l m e d u l l a w a s k n o w n t o be d e p e n-den t on how muc h o f t he m edu l l a was enve loped by the ad re na l co rt ex . Inspec ie s i n wh ich the co r t ex i s s ep a ra t e d f rom the m edu l l a , no rep in eph r ine i st h e p r e d o m i n a n t c a t e c h o l a m i n e . I n s p e c i e s i n w h i c h t h e m e d u l l a i s s u r -roun ded by the a d ren a l co rt ex , t he m e thy la t ed ca t echo lamine , ep inephr ine , isby f a r t he ma jo r amine . D ick Wur tman , a r e sea rch a s soc ia t e i n my l abo ra to -ry , sugge s t ed an e l egan t ex pe r im en t t o de t e rm ine the ro le o f t he ad re na l cor -t e x i n r e g u l a t i n g t h e s y n t h e s i s o f e p i n e p h r in e . H e r e m o v e d t h e r a t p i t u it a r y ,a p rocedure t ha t dep le t ed g lucocor ti co id in t he ad rena l co rt ex , and th en me a-s u r e d t h e e ff ec t o n t h e l e v e ls o f t h e e p i n e p h r i n e - f o r m i n g e n z y m e ,p h e n y l e t h a n o l a m i n e - N - m e t h y l t r a n s f e r a s e ( P N M T ) , i n t h e m e d u l l a . I h a dj u s t c h a r a c t e r i z e d P N M T a n d f o u n d t h a t i t w a s h i g h l y l o c a l i z e d i n t h ea d r e n a l m e d u l l a . T h e a b l a t i o n o f t h e p i t u i t a r y c a u s e d a p ro f o u n d d e c r e a s ei n P N M T i n t h e m e d u l l a a f t e r s e v e r a l d a y s ( W u r t m a n a n d A x e lr o d, 1 9 6 6) .T h e a d m i n i s t r a t i o n o f a d r e n o c o r ti c o t ro p i c h o r m o n e ( A C T H ), a p e p t i d e t h a tinc re ases t he fo rm a t ion o f g lucocort ico ids in t he ad ren a l co r tex , o r t he in jec -t i o n o f t h e s y n t h e t i c g l u co c o rt ic o id , d e x a m e t h a s o n e , i n c r e a s e d P N M T i nh y p o p h y s e c t o m i z e d r a t s a l m o s t t o n o r m a l v a l u e s .


25/34

J u l i u s A x e l r o d 69M e t h y l t r a n s f e r a s e R e s e a r c hA f t e r t h e d e s c r i p t i o n o f c a t e c h o l - O - m e t h y l t r a n s f e r a s e ( C O M T ) , I b e c a m ev e r y m u c h i n v o l v e d w i t h m e t h y l t r a n s f e r a s e e n z y m e s ( A x e l r o d , 1 9 8 1 ) . Is p e n t m o s t o f m y t i m e a t t h e l ab b e n c h w o r k i n g o n m e t h y l a t i n g e n z y m e sf o r m a n y y e a r s . S o o n a f t e r d e s c r i b i n g C O M T , I t u r n e d m y a t t e n t i o n t ot h e e n z y m a t i c N - m e t h y l a t i o n o f h i s t a m i n e . A m a j o r p a t h w a y fo r h i s ta -m i n e m e t a b o l i s m o c cu rs v ia N - m e t h y l a ti o n . T h i s f in d i n g p r o m p t e d as e a r c h f o r a p o t e n t i a l h i s t a m i n e - m e t h y l a t i n g e n z y m e . A s is t h e c a s e w i t ho t h e r m e t h y l t r a n s f e r a s e s , I s u s p e c t ed t h a t t h e m o s t l ik e ly m e t h y l d o n o rw o u l d b e S - a d e n o s y l m e t h i o n i n e . To m a k e t h e i d e n t i t y o f t h e h i s t a m i n e -m e t h y l a t i n g e n z y m e p o s s i b le , D o n a l d B r o w n , a p o st d o c i n t h e l a b o f ac o l l e a g u e , a n d I s y n t h e s i z e d [ 1 4 C - m e t h y l ] - S - a d e n o s y l m e t h i o n i n e e n z y -m a t i c a l l y f ro m r a b b i t l i v e r w i t h 1 4 C - m e t h y l m e t h i o n i n e a n d A TP .B e c a u s e o f i ts a b i l it y t o la b e l t h e O o r N g r o u p s o f p o t e n t i a l s u b s t r a t e sb y t h e t r a n s f e r o f 3 H - m e t h y l m e t h i o n i n e , t h e a v a i l a b i l i ty o f 1 4 C -S - ad e n o-s y l m e t h i o n in e l ed t o th e d is c ov e ry o f a n u m b e r o f m e t h y l t r a n s f e r a s ee n z y m e s . H i s t a m i n e N - m e t h y l t r a n s f e r a s e w a s s oo n f o u nd a n d p u r if ie da n d i t s p r o p e r t i e s d e s c r ib e d . T h e e n z y m e i s h i g h l y l o c al iz e d i n t h e b r a i n ,a n d i t al so h a s a n a b s o l u t e s p e ci fi ci ty fo r h i s t a m i n e . O t h e r m e t h y l t r a n s -f e r a s e s s o o n d i s c o v e r e d u s i n g [ 1 4 C - m e t h y l ] - S - a d e n o s y l m e t h i o n i n e w e r eP N M T , h y d r o x y i n d o l e O - m e t h y l t r a n s f e r a s e , t h e m e l a t o n i n - f o r m i n ge n z y m e , a p r o t e i n c a r b o x y m e t h y l t r a n s f e r a s e e n z y m e , a n d a n o n s p e c i f i cN - m e t h y l t r a n s f e r a s e . T h i s l a t t e r e n z y m e w a s f o u n d t o c o n v e r t t r y p t a -m i n e , a co m p o u n d n o r m a l l y p r e s e n t i n t h e b r a i n , to N - N - d i m e t h y l t r y p t -a m i n e , a p s y c h o t o m i m e t i c a g e n t .T h e s e m e t h y l t r a n s f e r a s e e n z y m e s , t o g e t h e r w i t h [ 3 H - m e t h y l ] - S -a d e n o s y l m e t h i o n i n e o f h i g h s p ec if ic a c t i v i ty w e r e u s e d i n d e v e l o p i n g v e r ys e n s it iv e m e t h o d s f or t h e m e a s u r e m e n t o f t r a c e b i og e n ic a m i n e s . W e w e r ea b l e t o d e t e c t , l o c a l i z e , a n d m e a s u r e o c t o p a m i n e , t r y p t a m i n e , p h e n y l e t h y -l a m i n e , p h e n y l e t h a n o l a m i n e , a n d t y r a m i n e i n t h e b r a i n a n d o t h e r t is s u e s .T h e m e t h y l t r a n s f e r a s e s a n d [ 3 H - m e t h y l ] -S - a d e n o s y l m e t h i o n i n e a ls om a d e i t p o s si bl e t o m e a s u r e n o r e p i n e p h r i n e , d o p a m i n e , h i s t a m i n e , a n ds e r o t o n i n i n 1 3 0 s e p a r a t e b r a i n n u c l e i . B e c a u s e o f t h e s e n s i t i v i t y o f t h ee n z y m a t i c m i c r o m e t h o d s , m y c o l l e a g u e s a n d I w e r e a b l e t o s h o w t h e c o ex -i s te n c e o f s e v e r a l n e u r o t r a n s m i t t e r s i n s in g l e i d e n ti fi e d n e u r o n s o fA plys i a (B row ns te in e t a l . , 1974) . L a te r , T hom as H okfe l t e t a l . (1980) ,u s i n g i m m u n o h i s t o f lu o r e s c e n t t e c h n i q u e s , d e m o n s t r a t e d t h e c o e x is te n c eo f n e u r o t r a n s m i t t e r s i n m a n y n e r v e t ra c t s.T h e P i n e a l G l a n dI w a s s t r u c k b y a n a r t ic l e f ro m A a r o n L e r n e r ' s l a b o r a to r y , p u b l i s h e d i n1 9 58 , t h a t d e s c r i b e d t h e i so l a t io n o f 5 - m e t h o x y - N - a c e t y l t r y p t a m i n e ( m e l a-


26/34

70 Julius Axelrodt o n i n ) f r o m t h e b o v i n e p i n e a l g l a n d , a c o m p o u n d t h a t h a d p o w e r f u la c t i o n s i n b l a n c h i n g t h e s k i n o f t a d p o l e s ( L e r n e r e t a l. , 1 95 8 ). T h i s c o m -p o u n d a t t r a c t e d m y a t t e n t i o n f o r t w o r e a s o n s : it h a d a m e t h o x y g r o u p a n da s e r o t o n i n n u c l e u s . T h e m e t h o x y g r o u p o f m e l a t o n i n h a d a s p ec ia l a t t ra c -t i o n f o r m e . A l s o , a t t h a t t i m e , s e r o t o n i n w a s b e l i e v e d t o b e i n v o l v e d i np s y c h o s e s b e c a u s e o f i ts s t r u c t u r a l r e s e m b l a n c e t o L SD . I t h o u g h t i t w o u l db e fu n t o s p e n d s o m e t im e w o r k i n g o n t h e p i n e a l g la n d , a n o r g a n t h a t w a sa m y s t e r y t o m e . T h e b e s t w a y t o s t a r t w a s t o c o n c e n t r a t e m y e f f o r t s o na s p e c t s o f t h e p r o b l e m t h a t I w a s f a m i l i a r w i t h , s u c h a s O - m e t h y l a t i o n .

H e r b e r t W e i s s b a c h e x p r e s s e d a n i n t e r e s t i n c o l l a b o r a t i n g w i t h m e t ow o r k o u t t h e b i o s y n t h e t i c p a t h w a y f o r m e l a t o n i n . W e i s s b a c h h a d a l r e a d ym a d e i m p o r t a n t c o n t r ib u t i o n s o n t h e m e t a b o l i s m o f s e ro t o n in . T h e a v ai l-a b i l i ty o f S - a d e n o s y l - l - m e t h i o n i n e w i t h a r a d i o a c t i v e m e t h y l g r o u p p r o v id -e d a n o p p o r t u n i ty t o e x a m i n e w h e t h e r t h e p i n e a l g l an d c ou ld f o rm l a b e le dm e l a t o n i n f r o m p o t e n ti a l p r e c u rs o r c o m p o u n d s . W h e n w e in c u b a t e d b o v in ep i n e a l e x t r a c t s w i t h N - a c e t y l s e r o t o n i n a n d [ 1 4 C - m e t h y l ] - S - a d e n o s y l - 1 -m e t h i o n i n e , a r a d i o a c t i v e p r o d u c t t h a t w e s o o n i d e n t i f i e d a s m e l a t o n i n w a sf o u n d ( A x e l r o d a n d W e i s s b a c h , 1 9 6 1 ) . W e i s s b a c h a n d I t h e n p u r i f i e d t h em e l a t o n i n - f o r m i n g e n z y m e , w h i c h w e n a m e d h y d r o x y i n d o l e - O - m e t h y l -t r a n s f e r a s e ( H I O M T ) , f r o m t h e b o v i n e p i n e a l g l a n d . W e a l s o f o u n d a n o t h e re n z y m e t h a t c o n v e r t e d s e r o t o n i n t o N - a c e t y l s e r o t o n i n i n t h e r a t p i n e a lg l a n d . F r o m t h e s e o b s e r v a ti o n s , w e p ro p o s e d t h a t t h e s y n t h e s i s o f m e l a -t o n i n i n t h e p i n e a l p ro c e e d s a s fo llo w s: t r y p t o p h a n -~ 5 - h y d r o x y t r y p t o p h a n-~ s e r o t o n i n -~ N - a c e t y l s e r o t o n i n -~ m e l a t o n i n ( Axe lr od , 1974). I r w i nK o p i n , W e i s s b a c h , a n d I a l s o f o u n d t h a t m e l a t o n i n w a s m e t a b o l i z e d m a i n -l y b y a m i c r o s o m a l e n z y m e v i a 6 - h y d r o x y l a ti o n . I n a s t u d y o f t h e t i s s u ed i s t r i b u t i o n o f H I O M T w e o b se r v e d t h a t t h e e n z y m e w a s h i g h l y lo c a li ze di n t h e p i n e a l . T h i s f i n d i n g c o n v i n c e d m e t h a t t h e p i n e a l w a s a b i o c h e m i -c a l l y a c t i v e o r g a n c o n t a i n i n g a n u n u s u a l e n z y m e a n d p r o d u c t a n d w a sw o r t h f u r t h e r s t ud y .

Dur i ng 1960 t o 1962 I s pe n t l i t t l e t i me do i ng p i ne a l r e s e a r c h . Mos t o fm y e ff o rt s w e r e d i r ec t e d t o w a r d t h e b i o c h e m i s t r y o f c a t e c h o l a m i n e s a n d t h ee ff ec t o f p s y c h o a c t iv e d r u g s . I n 1 9 62 , w h e n W u r t m a n j o i n e d m y l a b o ra t o ry ,I t h o u g h t t h a t h e s h o u l d d e v o t e m o s t o f h i s ti m e t o c a t e c h o l a m i n e r e s e a r c h .A s a m e d i c a l s t u d e n t W u r t m a n h a d a l r e a d y m a d e a n i m p o r t a n t f i n d i n gt h a t b o v i n e p i n e a l e x t r a c t s b lo c k e d g o n a d a l g r o w t h in r a t s i n d u c e d b y li g h t .A l t h o u g h p i n e a l r e s e a r c h w a s n o t a f a s h i o n a b l e s u b j e c t f o r r e s e a r c h t h e n ,W u r t m a n a n d I w e r e c a u g h t u p b y th e r o m a n c e o f t h i s o r g a n , s o w e d e cid -e d t o s p e n d o u r s p a r e t i m e w o r k i n g o n t h e p i n e a l . W e t h o u g h t t h a t a g o o dp l a ce t o s t a r t w a s t h e i s o la t io n o f t h e g o n a d - i n h i b i t o r y f a c to r o f t h e p i n ea l .N e i t h e r o f u s w a n t e d t o go t h r o u g h a t ir e s o m e i s o l a ti o n a n d b i o a s s a y pr o -c e d u r e , a n d w e d e c id e d to t a k e a c h a n c e a n d e x a m i n e t h e e f fe c ts o f m e l a -t o n i n . W e f o u n d t h a t m e l a t o n i n r e d u c e d o v a r i a n w e i g h t a n d d e c r e a s e d t h

larry r. squire- the history of neuroscience in autobiography: volume 1

Documents