Thomas A. Ban
Neuropsychopharmacology in Historical Perspective
Education in the Field in the Post-Neuropsychopharmacology Era
Leo E. Hollister’s Interview of Julius Axelrod – The Uptake of Neurotransmitters and Psychoactive Drugs
(Bulletin 16. Interviews 1)
Julius Axelrod career in research began in the early 1950s with studies in drug metabolism. It led by the mid-1950s to his discovery of microsomal enzymes (P450) responsible for the metabolism of drugs by methylation and deamination and to his demonstration that glucuronide conjugation was a major mechanism in detoxifying drugs (Axelrod 1955, 1982; Sulser 2011).
Subsequently Axelrod became intensively involved in catecholamine metabolism. In his autobiographic account, published in 1998, he gives an account how he became involved in this area of research:
“In the late 1950s adrenaline was believed to be metabolized and inactivated by deamination by monoamine oxidase (MAO). However, it seemed that after the administration of MAO-inhibitors (MAOI) the physiological actions of administered adrenaline and noradrenaline were rapidly terminated. This indicated that enzymes other than MAO are involved in the metabolism of adrenaline” (Axelrod 1998).
Instrumental to further development was Armstrong, McMillan and Shaw’s 1957 report which showed that patients with pheochromocytoma, a catecholamine-producing tumor (usually of the adrenal medulla), excreted not only large amounts of adrenalin and noradrenaline (NA) in their urine, but also large amounts of 3-methoxy-4-hydroxymandelic acid (VMA), its O-methylated product (Armstrong, McMillan and Shaw 1957). Guided by these findings, Axelrod (1957) discovered catechol – O - methyl – transferase (COMT), an enzyme that O-methylates catecholamines (adrenaline, NA, dopamine) in the presence of a methyl donor. By the end of 1958, he also identified S-adenosylmethionine, the methyl donor, and demonstrated that O-methylation was the principal route of metabolism for catecholamines (Axelrod and Tomchick 1958; Axelrod et al. 1958; LaBrosse, Axelrod and Kety 1958).
In the three years that followed, in collaboration Irwin Kopin (1929–2017), Axelrod identified the many metabolic products of catecholamines, such as metanephrine, normetanephrine, 3-methoxytyramine and 3-methoxy-4-hyrdoxypenylglycol (MHPG). He also demonstrated that the catecholamines are metabolized by O-methylation, deamination, glycol formation and conjugation with glucuronide and sulphate (Axelrod 1959; Axelrod, Kopin and Marin 1959; Kopin and Axelrod 1960; Kopin, Axelrod and Gordon 1961; Kopin and Gordon 1962).
In the early 1960s, Axelrod’s research entered a new phase. His reflections about entering this new phase more than 30 years later reads:
“(In the early 1960s) I assumed that the action of NA would be by the enzymes MAO and COMT. However, when these enzymes were inhibited by specific enzyme blockers and NA was injected into cats, NA’s blood pressure - elevating actions were rapidly abolished. These findings indicated that there were other mechanisms than MAO and COMT for terminating the action of NA. I thought that a study examining the distribution of injected NA might give me a clue. To do such a study, it was necessary to use a tracer 3HNA of high specific activity. When the labeled NA was injected into cats it was found to be highly localized after two minutes in tissues rich in sympathetic nerves, such as the heart and the spleen. Little 3HNA was detected in the brain, indicating that it did not cross the blood – brain barrier. An unusual observation was hat the 3HNA persisted in tissues long after its blood pressure – elevating action had ended. This suggested that it was taken up in sympathetic nerves and held there providing an effective way for terminating the actions of NA” (Axelrod 1998).
Axelrod’s hypothesis was tested in a series of experiments in collaboration with Georg Hertting (1925–2014) and L. G. Whitby, which consistently showed that NA’s activity was terminated by “uptake” (“reuptake”) into the sympathetic nerves wherefrom it was released. The compelling demonstration of the reuptake of NA by visualized autoradiography stimulated research which showed that serotonin, dopamine and γ–aminobutyric acid (GABA) were also taken up by nerves and established re-uptake as a major mechanism in the inactivation of neurotransmitters. Pursuing further the same line of research, Axelrod and his associates discovered that drugs like cocaine, amphetamine and other sympathomimetic amines, as well as tricyclic antidepressants, block the reuptake of NA; moreover, that only therapeutically effective tricyclic antidepressants block the reuptake of NA at nerve terminals (Axelrod, Whitby and Hertting 1961; Hertting et al. 1961; Hertting, Axelrod and Whitby 1961).
Simultaneously with these developments, in 1961 Axelrod discovered hydroxyindole – O - methyltransferase, the melatonin synthesizing enzyme and in 1962, phenylethanolamine – N - methyltransferase (PNMT), the enzyme that converts norepinephrine to epinephrine in the adrenal medulla (Axelrod 1961, 1962, 1964, 1974; Axelrod and Weisbach 1961; Wurtman, Axelrod and Fisher 1964).
In recognition of his contributions Julius Axelrod (1912–2004) shared the 1970 Nobel Prize with Ulf von Euler (1905–1983) and Bernard Katz (1911–2003) for his discoveries related to catecholamine metabolism and termination of the actions of norepinephrine by reuptake into the nerve terminals from which it was released (Snyder 2005). In his Nobel lecture Axelrod (1972), cited more than 50 of his papers that elucidated the regulation of norepinephrine biosynthesis, storage, release, metabolism and inactivation in brain, as well as at peripheral sympathetic nerve terminals. He also showed that drugs, such as amphetamine, cocaine and antidepressants, affect norepinephrine reuptake and pointed out that the finding that reuptake as a terminating action is not restricted to NA but applies also to other neurotransmitters, thus providing an important target for psychotropic drug development (Kopin 2014).
Armstrong MD, McMillan A, Sha KN. 3-Methoxy-4-hydroxy-D-mandelic acid, a urinary metabolite of norepinephrine. Biochim Biophys Acta 1957 25: 422–3.
Axelrod J The enzymatic demethylation of ephedrine. J Pharmacol Exp Ther 1955; 114: 430-438.
Axelrod J. O-methylation of epinephrine and other catechols in vitro and in vivo Science 1957; 126; 400–1.
Axelrod J. The metabolism of catecholamines in vivo and in vitro. Pharmacol Rev 1959; 11: 402 - 8.
Axelrod. J. Enzymatic formation of psychotoxic metabolites from normally occurring substances, Science 1961; 134: 343.
Axelrod J. The enzymatic N-methylation of serotonin and other amines. J Pharmaco Exp Terap 1962; 138: 28.
Axelrod J. Enzymatic oxidation of epinephrine to adrenochrome by the salivary gland . Biochem Biophys Acta 1964; 85; 247.
Axelrod J. The pineal gland. A neurochemical transducer. Science 1974; 184: 1341 -8.
Axelrod J, Reisine TD. Stress hormones. Their interaction and regulation. Science 1984; 224: 452 – 9.
Axelrod J. Purification and properties of phenylethanolamine-N-methyl transferase. J Biol Chem. 1962; 237:1657-60.
Axelrod J. Norepinephrine: Fate and control in the body. Nobel Lecture. In: Nobel Lectures Physiology or Medicine. Amsterdam: Elsevier; 1972.
Axelrod J. The discovery of the microsomal drug-metabolizing enzymes. Trends Pharmacol Sci 1982; 3: 383-386.
Axelrod J. The uptake of neurotransmitters and psychoactive drugs. In Ban TA, Healy D, Shorter E, editors. The Rise of Psychopharmacology and the Story of CINP. Budapest: Animula; 1998, p.116 – 9.
Axelrod J, Inscoe JK, Senoh S, Witkop B. O-methylation the principal pathway for the metabolism of epinephrine and norepinephrine in the rat. Biochim Biophys Acta 1958; 27:210 -1.
Axelrod J, Kopin JJ, Marin JD. 3-Methoxy – 4 – hydroxyphenylglycol sulfate a new metabolite of epinephrine and norepinephrine. Biochem Biophys Acta 1959; 36: 576-7.
Axelrod J, Tomchick R. Enzymatic O-methylation of epinephrine and other catechols. J Biol Chem. 1958; 233: 702–5.
Axelrod J, Weissbach H. Purification and properties of 5-hydroxyindole – O – methyl transferase. J Biol Chem 1961; 231: 211 - 3.
Axelrod J, Whitby LG, Hertting G. Effect of psychotropic drugs on the uptake of 3H – norepinephrine by tissues. Science 1961; 133: 383-4.
Hertting G, Axelrod J, Kopin IJ, Whitby LG. Lack of uptake of catecholamines after denervation of sympathetic nerves. Nature 1961; 189: 66-7.
Hertting G, Axelrod J, Whitby LC. Effect of drugs on the uptake and metabolism of 3H-norepinephrine. J Phramacol Exp Ther 1961; 134: 146-53.
Kopin JJ. Julius Axelrod. Miniprofile. inhn.or.Profiles. November 6, 2014
Kopin IJ, Axelrod J. 3, 4 – Dihydroxyphenylglycol a metabolite of epinephrine. Arch Biochem Biophys 1960; 89: 148-9.
Kopin IJ, Axelrod J, Gordon E. The metabolic fate on 3H-epinephrine and 14C metanephrine in the`rat. J Biol Chem 1961; 236: 2109-13.
Kopin IJ, Gordon E. Metabolism of norepinephrine released 3H-epinephrine by tyramine and reserpine. J Pharmacol Exp Ther 1962; 138: 351 – 9.
LaBrosse EH, Axelrod J, Kety SS. O-Methylation, the principal route of metabolism of epinephrine in man. Science. 1958; 128: 593–4.
Snyder SH. A Biographical Memoir of Julius Axelrod. Biographical Memoirs of the National Academy of Science; Volume 87: 2005.
Sulser F. Introduction and dramatis personae. In: Sulser F, editor. Neuropsychopharmacology. (In: Ban TA, editor. An Oral History of Neuropsychopharmacology The First Fifty Years Peer Interviews. Reviews.). Brentwood: American College of Neuropsychopharmacology; 2011.
Wurtman R, Axelrod J, Fisher JA. Melatonin synthesis in the pineal gland: effect of light mediated by the sympathetic nervous system. Sciene 1964; 143: 1328 – 9.
Julius Axelrod, interviewed by Leo E. Hollister
Washington, D.C., April 14, 1997
First published in Volume Five (Neuropharmacology, edited by Fridolin Sulser) of An Oral History of Neuropsychopharmacology The First Fifty Years, a series edited by Thomas A. Ban
(Brentwood: American College of Neuropsychopharmacology; 2011, p. 26 - 43)
LH: We are in Washington doing another tape in our series of the history of psychopharmacology. I’m Leo Hollister and our guest is a man who needs no introduction, Julius Axelrod.* Welcome Julius, and thank you for coming.
JA: It’s a pleasure.
LH: Your life began in New York.
JA: Yes, on the lower east side of New York. It couldn’t be more deeply in New York.
LH: A typical American saga.
JA: I suppose so. My parents came from Austrian Poland, at the beginning of the century. They met and married here.
LH: Were they fleeing a pogrom?
JA: No. In the Russian part of Poland there were pogroms, but not in the Austrian part. It was a bit more liberal. Franz Joseph was the emperor, and he was more tolerant towards Jews. It was mainly poverty.
LH: They wanted to get to the land of opportunity.
JA: Yes, the golden land.
LH: Unfortunately, they didn’t find the streets paved with gold.
JA: No, not at all. But they had talked to people who came from the same area of Poland and informed them what to expect.
LH: They networked. Were you the only child?
JA: I have two sisters. I was the oldest, born in 1912.
LH: You know there’s a current idea about birth order.
LH: David Healy tells me that most of the people he interviewed have been either first born or an only child.
JA: I don’t know whether there is anything to that, but it’s interesting.
LH: So, you have two sisters. Are they both alive?
JA: No, they both died this year. I’m the only surviving member. We lived in a part of New York that was almost all Jewish because otherwise we were either beaten up or called all kinds of names. But I enjoyed that life. We were very poor.
LH: That was common, wasn’t it?
JA: It was. We were very poor, but I didn’t know any better. That was life. Amongst Jewish people there was an intellectual ferment. There were theaters, libraries and a lot of talk and politics. Most of those living in the area were socialists and we had a socialist congressman, Pankin.
LH: I remember him. That was not a bad idea in those days.
JA: No, it wasn’t. The Russian revolution occurred around 1917 and people were split on the basis of whether they read the socialist or communist newspaper.
LH: Socialism in a democracy, as in the Scandinavian countries, is pretty benign.
JA: Yes, but the discussions in our area were sometime very emotional.
LH: Political discussions can get pretty emotional.
JA: For me they were very interesting.
LH: You went to the New York public schools?
JA: The first public school I went to was built before the civil war. There was one famous alumnus: Isadore Robbie, a physicist. He graduated long before me. And in high school, I went to Seward Park on Hester Street. I wanted to go to Stuyvesant, a school close by where all the smart kids went, but I couldn’t get in. I wasn’t that smart.
LH: What a paradox!
JA: I wasn’t a bad student, but I wasn’t in the top of my class and I enjoyed going to Seward Park. We had a lot of interesting alumni. Most were entertainers: Walter Matthau, Zero Mostel, and Tony Curtis were all graduates of Seward Park, and also the songwriter, Hip Haburg. Over the Rainbow was one of his songs.
LH: A lot of talent came from that area.
JA: Oh, yes.
LH: Where did you go to college?
JA: I went to City College; that was tuition-free, a sort of poor man’s Harvard. It was not easy to get in. It was fortunate for me because if it were tuition-free, I never would have gone to college, we couldn’t afford it. I received a high quality education there and we had some world-class teachers. In philosophy we had Morris Rayfield Cohen.
LH: He wrote a textbook.
JA: Yes, he was a famous philosopher. We had good teachers in chemistry, biology and some other subjects. I wanted to get into medical school and majored in biology and chemistry. When I graduated I applied to several medical schools, but could not get in.
LH: You think that was due to the quota system?
JA: Well, to the quotas they had at the time. The only graduate I know who got into medical school was Arthur Kornberg. He was about three years behind me and a smart kid.
LH: He was an MD, wasn’t he?
JA: He got an MD, yes. I graduated from college in 1933.
LH: Ooh, bad time.
JA: It was a bad time to graduate, especially from City College. Fortunately, a stroke of luck determined my whole career. I heard of a position to work in a laboratory as a volunteer for $25 a month and I applied. I could have worked in the post office for more than $25 a month, but I accepted the position at Harriman Research Laboratory of NYU. Making that choice was crucial to my career. I was a technician in the laboratory of Dr. K.G. Falk, a biochemist. He was fairly well known because he wrote a textbook on the mechanism of enzyme action. He worked on enzymes in malignant tissues, and I got my first taste of research by assisting Dr. Falk.
LH: So that was the door to biochemistry in your career.
JA: Yes. I became very interested but after two years I decided to get married. My wife was a student at Hunter College and couldn’t live on $25 a month.
LH: That old saying two can live as cheaply as one is not true.
JA: Fortunately, the city of New York opened up a laboratory to test vitamins and food supplements. It was a non-profit laboratory. This was in the 1930s; vitamins were just being developed and became a big thing. They still are to a degree. They added vitamin A and D to milk, and various supplements to bread. My job was to set up assays to measure vitamins in milk, bread and pills. I didn’t develop my own methods but had to modify the existing ones. For this I read the original literature. It was a very good experience because methods are so crucial to research. If you have a hypothesis or an idea, you wouldn’t get very far, if you can’t develop methods for testing it. So, I learned about devising methods, and not only chemical or microbiological methods. They were using a spectrophotometer, and I got a great deal of experience working with it that was very useful. I thought I would stay in that lab for the rest of my life. The salary wasn’t bad and the work was fairly interesting. And I kept up with the literature. The laboratory subscribed to The Journal of Biological Chemistry that I read, so I had a feel for what was going on, mainly in enzyme research, vitamins and nutrition. I was working there for 11 years. In 1945, the head of this vitamin-testing laboratory was George Wallace, the former chairman of pharmacology at NYU. He was editor of The Journal of Pharmacology. One day a group of people from an institute for the study of analgesic drugs, a consortium of manufacturers involved in selling drugs like acetanilide, came to Dr. Wallace with the problem that some people became habituated to bromoseltzer.
LH: That had bromine in it.
JA: Yes. But it also contained acetanilide and many people taking the drug got methemoglobinemia. They were very concerned about this and wanted to find out why people get methemoglobinemia on acetanilide. They came to Dr. Wallace for advice, and Dr. Wallace asked me whether I would like to work on the problem. I said yes but told him I had no experience in research. So, he said I can send you to one of my associates, Dr. Bernard Brodie, at NYU.
JA: You probably know him. They called him Steve Brodie.
LH: Your name has been intimately connected with his ever since.
JA: I called Brodie and he asked me to visit him. He was at Goldwater Memorial Hospital, on an island now called Roosevelt Island. It was in 1946, a very fateful day for me. It was Lincoln’s Birthday, February 12. Brodie was a magnetic man with a great presence. We talked about the problem I was supposed to address. I was fascinated just talking to somebody like him. He had a way of talking I found stimulating. The first thing he told me was that anytime one takes a chemical or drug, the substance changes in the body, it’s metabolized and transformed. He asked me to put the structure of acetanilide on his blackboard. And I did. Then he said, let’s see what changes this molecule can undergo. Acetanilide consists of an aminobenzene ring with an acetyl group. One possible change is the removal of the acetyl group that should result in aniline. And I vaguely remembered that aniline could cause methemoglobinemia. So, I learned immediately the importance of asking the right questions. The second question to be answered was whether aniline was really formed from acetanilide. In order to answer that one has to develop methods to measure aniline in the blood and urine. Brodie was a great methods man, and we developed a specific and sensitive method to measure aniline in the urine, plasma, and blood. And I took acetanilide and found aniline in my urine. So, we knew we were off to a good start.
LH: Self-administration, huh?
JA: Yes. There were patients at Goldwater Memorial Hospital. We gave them acetanilide and found aniline in their urine. I don’t remember whether they gave informed consent but we definitely told them that the powder they were given was harmless and used for treating headache. Then I took some aniline myself. I thought I’d turn blue.
LH: And prove it beyond any question?
JA: It was really crazy.
LH: Did they have the methylene blue treatment for it then?
JA: No. I didn’t take that much. I became a little woozy but found a lot of methemoglobin in my blood. We did show there was a direct relationship between methemoglobinemia and aniline in the blood. So, we solved that problem.
LH: This was the first demonstration that the toxic effect of a drug could be due to the metabolism of the compound.
JA: One of the first demonstrations.
LH: Did you do this work at Goldwater?
JA: Yes. I forgot to tell you Brodie asked me to come and work with him, although the laboratory at NYU paid my salary. We also found that when one took acid anilide, aniline represented only about 4%, a very small amount of the entire drug. So, there was some other pathway for metabolism of the drug. Within three months we identified acetanilide’s major metabolic product. It was acetyl-para-aminophenol. Dr. Brodie checked it for analgesic activity and it was just as good an analgesic for headache as acetanilide but had the advantage it wasn’t toxic and did not cause methemoglobinemia. We suggested it should be used instead of acetanilide. It was used mainly by pediatricians, because it was soluble. This work led to the publication of my first paper.
LH: This was phenacetin?
JA: No. Acetanilide metabolized by hydroxylation to acetyl-para-aminophenol and phenacetin, and phenacetin metabolized by de-ethylation to acetyl-para-aminophenol. I think that Squibb had a concoction that consisted of Aspirin, phenacetin and acetyl-para-aminophenol. They called it acetaminophen because of the acetyl-para-aminophenol it contained. But then the company sold the compound to McNeil. Acetaminophen puttered along until Johnson & Johnson bought McNeil in 1970 and had a very powerful marketing campaign for Tylenol. It was their name for acetaminophen.
LH: A very successful drug.
JA: Very successful. All we got was a $10,000 grant. But I got much more, the beginning of a research career. I was pretty good at research, and I loved it. At the time all I had was a master’s degree in chemistry from New York University which I had earned by taking night courses while I worked in the vitamin testing laboratory. So that was the beginning of my career as an investigator.
LH: So, you found that acetanilide metabolized to phenacetin and phenacetin metabolized to acetaminophen?
JA: Both acetanilide and phenacetin are metabolized to acetyl-para-aminophenol. We didn’t call it acetaminophen.
LH: I think that was probably the first time that sequence had ever been used.
JA: Yes, it was. We showed that a drug could be metabolized to a toxic as well as to a nontoxic metabolite. Actually, there was a precedent for this when, in the early 1930s, Gerhard Domagk developed prontosil (for which he received the Nobel Prize), a very toxic substance that metabolized to sulfonamide.
LH: Sulfonamide was the first really effective antibacterial drug.
JA: Yes, and it revolutionized medicine. Antibiotics, penicillin came later. People think that drug metabolism is not in the mainstream of science. But it certainly was, at least in these cases. Let me talk to you about Goldwater Memorial Hospital. During World War II malaria was very prevalent in troops fighting in the Pacific and the Japanese cut off the supply of quinine. There was a need for new anti-malarial drugs and Shannon, a renal physiologist, was asked to test clinically some synthetic anti-malarial drugs at Goldwater. This happened before Shannon went to Bethesda to become the founding director of the NIH. Shannon had a good nose for picking people and he had at Goldwater a group of young people who, instead of fighting in the Pacific, worked with him on the clinical testing of anti-malarial drugs. The group included Bob Berliner, Bob Bowman, who was to develop the spectrophotofluorometer, Sidney Udenfriend, Stu Broad, the cancer man, Tom Kennedy, David Earl Steele, an internist, and several others. It was a stimulating group of people. They had a great influence on my thinking. After working for four years at Goldwater, I knew I didn’t have a chance for an academic appointment without a PhD but I had no inclination at the time to obtain one. Then I saw an advertisement in The New York Times that Shannon was appointed director of the NIH. I wrote to him and he hired me. Well, the NIH was not like it is now.
LH: That was 1949?
JA: Yes, that was when congress established the National Institutes of Health. It was not just the Heart Institute but also the Cancer Institute, the Arthritis Institute, and various other institutes. The Mental Health Institute was started with Bob Felix as the director. And Shannon persuaded Steve Brodie, Bob Berliner and Sid Udenfriend to join him. He recruited a remarkable group of people. In Building 3, there were three people who ultimately became Nobel Prize winners, Kornberg, Anderson and myself, and there were 20 people who became members of the National Academy of Sciences. It was a small building of three stories. Well, a secure job meant more than anything else to me, and particularly a job doing research. When I joined NIH, I worked first under Brodie. He recruited a lot of people and had a very large team and I wasn’t happy after awhile working in a large group. I was offered a position by one of the drug companies, and I told Brodie I would like to leave. But he asked me “What would it take for you to stay?” I answered: “If I could be completely independent to do my work I would stay.” I didn’t have a PhD yet. Still, he said: “Fine.” So, my first project was to study the fate of caffeine in man. There was no study on that despite the fact caffeine was the most widely used drug.
LH: Still is.
JA: Yes, it is. I did that work myself but got only one senior-authorship in 15 to 20 papers we had written. I became interested in sympathomimetic amines, amphetamine, and ephedrine. They interested me primarily because they affected behavior. They also raised blood pressure and being in the Heart Institute, I thought it would be a good idea to work on the metabolism of sympathomimetic amines. I worked out the metabolism of amphetamine and became very curious about why the body can metabolize thousands of synthetic compounds it never saw before. I thought I would like to tackle that problem. My lab mate, the man who occupied the bench next to mine, was Gordon Tompkins, a post-doc with Brodie.
LH: He died early, didn’t he?
JA: Yes. He was a brilliant fellow. I used to have wonderful times with him. He was a great raconteur who also used to play the clarinet in the evenings at a nightclub. Knowing my interest in drug metabolism Gordy asked, “Julie, why don’t you find out what enzymes there are?” When I told him I had no experience in enzymology he said all you need is a liver and a razor blade. One used to make slices of the liver in those days to study metabolism. By that time I had a method for measuring amphetamine and learned that amphetamine was not deaminated by monoamine oxidase, because it did not have the right structure, but by another enzyme. I was curious to find out what part of the cell carried out amphetamine’s metabolic deamination. Around that time Pauletti described methods to separate sub-cellular fractions, such as the mitochondria in the liver by differential centrifugation in sucrose. I learned these methods and found that, when the various sub-fractions were separated, amphetamine couldn’t be metabolized. It was metabolized only when I used cofactors like TPN or APN. At the same time Bert La Du, working in Brodie’s laboratory on a similar problem, found that TPN could cause the metabolism of one of the drugs I was working on. I think it was antipyrine or something that required ATP so when I added TPN to the mitochondria, amphetamine was metabolized. But I wasn’t careful and didn’t wash the mitochondria. Fortunately, Bernard Harke, a very good biochemist, who was working on the pentose phosphate shunt in the laboratory below mine, loaned me the substrates he used, and when I added a substrate like isocitric acid or gluconic acid to the unwashed mitochondria, amphetamine was deaminated. And when I added isocitric acid and TPN to the mitochondria, it generated reduced TPN. So is I washed the mitochondria and added reduced TPN, amphetamine was metabolized. I knew I had something. I was also working on ephedrine and when I added ephedrine to the mitochondria it was demethylated. Here were two different metabolic pathways using common cofactors, reduced TPN and oxygen. One led to the deamination of amphetamine, and the other to the demethylation of ephedrine. We named the enzyme responsible for both pathway the microsome. This discovery led to parting with Brodie; I wrote two abstracts based on my findings for the pharmacology meeting in 1953 and when Brodie saw these he became very upset.
LH: Was he upset about the order of authorship?
JA: No, he wasn’t a co-author at all. He didn’t do anything. He was upset that I solved the problem because there were other people in the lab trying to solve the same problem. He had put the whole laboratory to work on almost any drug they tried and wouldn’t allow me to publish until the rest completed all of their work. And he called us together and said: “Let’s publish this in Science with the authorship alphabetically.” I realized I would be cursed; they would just put my name first along with everybody else. I knew then I had to leave, I had to get my PhD.
LH: By that time, you had more than enough work for a PhD.
JA: Of course I did. I applied to George Washington University, a local school. I knew the chairman. He told me: “Since you have a master’s degree, you will not need take courses, but you will have to pass tough exams in five subjects: physiology, biochemistry, drug metabolism, and some other fields. And as far as your thesis is concerned, you can use the work on the sympathomimetic amines and enzymes.” I had already published four papers so I put them together in my thesis. I was also asked to teach a course on drug metabolism while working for my PhD. Although I didn’t have to, I decided to take the courses for medical students on the various subjects. Shannon, the director of NIH, was very generous. He said I could take a year off for my PhD and still get my salary.
LH: It seems paradoxical you would take courses on drug metabolism.
JA: I had to take the exams on drug metabolism after I gave the course because it was required. I didn’t set the questions, somebody else did.
LH: When you started work on the sympathomimetic amines, had epinephrine been discovered?
JA: Epinephrine was discovered way back in 1897 by John Abel. He isolated it from the adrenal gland.
LH: But it wasn’t identified as a transmitter?
JA: There was a big controversy about the neurotransmitter of the sympathetic nervous system. Walter Cannon thought it was epinephrine and named it sympathin A. But then von Euler isolated the substance and showed it was norepinephrine.
LH: Was he the one who called it sympathin first?
JA: No, that was Cannon. It’s a pity Cannon didn’t get the Nobel Prize, he certainly deserved it.
LH: He was a giant.
JA: Yes, he did so much work on stress and behavior and how stress affected various organs. Anyway, I left the Heart Institute and sent my application to the Cancer Institute and the Mental Health Institute. At the time Seymour Kety was the director of the intramural program of the Mental Health Institute. He called me for an interview and seemed to be very pleased with it. He thought I had a good chance for a position at the Institute and sent my application to the heads of several laboratories. One of the people was Ed Evarts.
LH: He was a physiologist, wasn’t he?
JA: Yes, but he was also a psychiatrist and neurologist working on LSD. He saw my application and asked me if I would join his laboratory. So, after my PhD I worked in his lab, developing a method for detection of LSD. LSD at that time was a big thing in psychiatry. They thought it was a good tool to study.
LH: For model psychoses.
JA: Actually a nurse can recognize the difference between LSD and amphetamine.
LH: That’s what we found.
JA: I know, I remember when you did that work. Anyway, I developed a method for the detection of LSD. Bob Bowman was developing a fluorometer, and I asked if I could use it. He gave me one of his experimental models, and I developed a method for detection of LSD. So Ed Evarts and I studied the metabolism and distribution of the substance. We found it went into the brain in incredibly small amounts and must have been very potent. I got my own laboratory and was working alone by 1955. I had no experience in neuroscience and knew very little about the brain. I thought neuroscientists had to be very gifted theoreticians and experimentalists working on this very complicated electronic apparatus. I was worried Kety would want me to work on schizophrenia or depression but instead he said, “Julie, you can work on anything you want as long as it’s important and original.” So I started to work on the metabolism of drugs I knew best, on morphine and its conjugation. I collaborated with Jack Strominger, a very good biochemist and immunologist, on glucuronide conjugation as a major mechanism for detoxifying drugs. When Jack and I met at NIH, there was a paper published showing glucuronides were formed by a cofactor, uridine diphosphate glucuronic acid; since I had a good method for measuring glucuronides, Jack suggested we should study glucuronide conjugation. To do our research we required uridine diphosphate glucose we could convert to glucuronic acid either by TPN or DPN. Herman Colcott happened to be at the NIH. He was a very distinguished Danish biochemist who had uridine diphosphate glucose. So, we all collaborated and showed that DPN, NADP plus uridine diphosphate glucose, would form morphine glucuronide. At that time I had to leave the laboratory to get my PhD but Strominger purified the enzyme and published it. When I returned to the Mental Health Institute, I noticed a paper by Rudy Schmidt, the former Dean of the San Francisco medical school, who found that bilirubin, was detoxified by forming a glucuronide and if it didn’t conjugate one became jaundice. I called and told him I could find the enzyme. We collaborated and found the enzyme that forms bilirubin glucucronide. Then Rudy Schmidt told me about a mutant strain of rats, the Gunn rat, studied by Castle at Harvard that has jaundice. He thought it would be a good idea to see whether they developed jaundice because they couldn’t form bilirubin glucuronide. Sure enough, we found a defect in the liver, an inability to form glucuronides. When I told Rudy Schmidt we also found acetaminophen was formed from phenacetin by glucuronidation we got patients with Crigler-Najjar disease, and gave them acetaminophen.
LH: And they couldn’t conjugate that either?
JA: Exactly. They could, but very, very weakly. By now, I felt a little guilty not working on the brain. Around 1956, Ed Evarts stepped down from his position of lab chief, because he didn’t like to be an administrator, and Seymour Kety stepped down from the Directorship of the Institute, to become the head of the Laboratory of Clinical Science. During Kety’s tenure we had seminars every week and on one of these we heard a report from two Canadian psychiatrists who found when they left adrenaline in the air it turned pink.
LH: The famous pink spot!
JA: That comes later. They claimed they hallucinated when they took the pink adrenaline.
LH: Adrenochrome. Was this Hoffer and Osmond?
JA: Yes. They had a great impact on my life. They claimed schizophrenia might be caused by the abnormal metabolism of adrenaline. I was fascinated and looked through the literature, but all I could find was an enzyme, monoamine oxidase, discovered many years before by Blaschko that deaminated adrenaline.
LH: Would that be the same enzyme you were using for deaminating amphetamine?
JA: No, that was the microsomal or P450 enzyme, one of the most studied enzymes in the world. Anyway, I thought I might as well work on the metabolism of adrenaline since it is so closely related to amphetamine. First, I looked for the enzyme that converted adrenaline to adrenochrome and spent four frustrating months, but couldn’t find it . Then, one day there was an abstract published by McMillan and Marvin Armstrong showing patients with pheochromocytoma excreted a lot of vanillylmandelic acid. It was a methylated compound and, looking at its structure, I knew it must come from adrenaline or noradrenaline. I suspected it was formed first by methylation of adrenaline or noradrenaline and then by deamination of the resulting substance by monoamine oxidase. I thought the methyl donor was adenosylmethionine. I didn’t want to ask Cantoni, who discovered the methyl donor was adenosylmethionine, so I added a cofactor that contained adenosylmethionine, magnesium, liver extract, methionine and ATP. When I added all these ingredients, adrenaline disappeared. It was metabolized, so I knew I had an enzyme that transferred the methyl group of adenosylmethionine to one of the hydroxy groups of adrenaline. We called the methylated substance metanephrine.
LH: To do all this the Bowman spectrophotofluorometer was indispensable?
JA: Yes, that’s what I used. We didn’t have radioactive isotopes but I had a new enzyme. We called it catechol-methyl-transferase. And at the time there were only two neurotransmitters recognized; one was acetylcholine and the other was noradrenaline, discovered by von Euler a few years before. But there were a lot of other putative neurotransmitters such as serotonin and dopamine. Nachmansohn and Leary had discovered acetylcholine was inactivated by choline acetyltransferase so I thought the catecholamines, noradrenaline and adrenaline, would be inactivated by catechol methyltransferase. But just around that time, Zeller discovered an inhibitor of monoamine oxidase.
JA: Yes, but when they injected iproniazid to inhibit the activity of monoamine oxidase, it didn’t affect the metabolism of norepinephrine sufficiently to be reflected in blood pressure changes. At the same time, we found an inhibitor for catechol methyltransferase, called copaline or something like that. But when Dick Crout, who worked at the Heart Institute, inhibited both monoamine oxidase and catechol methyltransferase, and then injected norepinephrine, its action on blood pressure was still rapidly terminated, in spite of the fact that the functioning of both of the enzymes responsible for the metabolic breakdown of norepinephrine were blocked. So, we knew they were not the only enzymes that inactivated norepinephrine.
LH: So, you didn’t stop at the enzymes?
JA: Then it became an intriguing problem. About the time I was conducting these experiments, Kety ordered some tritium-labeled adrenaline to study the metabolism of adrenaline in schizophrenics to test the adrenochrome hypothesis so I asked him for some. By then Irv Kopin and I had already identified several metabolites of adrenaline and noradrenaline including normetanephrine and MHPG so Kety could study the metabolism of adrenaline in schizophrenics. So, we studied the tissue distribution of tritium-labeled adrenaline, and found that it persisted in tissues unchanged, long after the physiological actions of the substance were over. The highest concentrations were found in organs that contained a lot of sympathetic nerves, such as the heart and the spleen. So, we suspected it must be sequestered in sympathetic nerves, an important finding.
LH: That was a revolution.
JA: Yes, what it led to was…
LH: The reuptake mechanism!
JA: Exactly! Let me tell you how we did the rest of it. Around that time I was attracting post-docs. One was George Hertting. He was a real classical Viennese pharmacologist, and when I was discussing how we could prove norepinephrine is taken up in sympathetic nerves, he came up with a very brilliant idea. He said what we can do is take out the superior cervical ganglia unilaterally. When we do that, the nerves will degenerate on one side and we will have a unilaterally denervated animal. Then, when he injected radioactive noradrenaline he found the radioactivity was localized on the inervated side and we knew it was going into the nerves. We realized we had something very important and began thinking of other experiments. In one of these we perfused norepinephrine in the spleen, and when we stimulated the nerves to the spleen there was a release of noradrenaline. Then we knew noradrenaline was not only taken up but was also released from the sympathetic nerves in the spleen. We called this process “reuptake”. In the next experiment we did autoradiography. It was carried out by Lincoln Potter, one of my first post-docs, who worked with Keith Richardson and David Wolf, both autoradiographers. I happened to be working on the pineal which is very rich in sympathetic, noradrenergic nerves. When we injected radioactive noradrenaline to do autoradiography, Wolf told me it should take weeks before to get the films ready. I was very impatient so asked to have it in two days. And we did! All radio-activity was in the sympathetic nerves, localized over dense core granules in little vesicles. We suspected these little vesicles were the storage place for noradrenaline. We also studied the distribution of noradrenaline with Weil-Malherbe, a German biochemist who did a lot of work on the biochemistry of mental illness. He left Germany during the Nazi regime and he developed methods in England for measuring adrenaline. Well, I thought, let’s measure the effect of drugs on uptake. We couldn’t do it in the brain because noradrenaline didn’t cross the blood-brain barrier. The first drug Hertting and I tried was cocaine and found it blocked the uptake of +noradrenaline into the tissues of the heart and spleen. Then we tried a whole bunch of drugs. Amphetamine did the same as cocaine. But we wanted to get into the brain. At the time I had another post-doc, Jacques Glowinski, who is now vice-president of the College of France. Most of my young people turned out very well.
LH: You’ve had so many distinguished graduates.
JA: Glowinski developed a technique for introducing radioactive noradrenaline right into the third ventricle. Then we tried antidepressant drugs, a whole series of tricyclics compounds we got from Geigy. We gave these first and then injected radioactive noradrenaline into the brain and measured the amount in the nerves before and after the drug. We found a reduced level of radioactivity in the nerves only after we gave a clinically effective tricyclic drug. Later on, one of my post-docs, Joe Coyle, found not only were the antidepressants blocking reuptake of noradrenaline, but they also blocked the reuptake of dopamine. Then Sol Snyder found the antidepressants blocked reuptake of serotonin as well. Antidepressant development was based on the employment of simple methods of reuptake inhibition. Thousands of synthetic drugs were screened with these simple methods rather than giving them to humans. That’s why it was so easy to develop antidepressant drugs.
LH: Those methods are probably still used.
JA: Of course. In fact, they call these drugs serotonin reuptake inhibitors.
LH: After you discovered the action of neurotransmitters was terminated by reuptake, did you ever have an idea this was important enough to win a Nobel Prize?
JA: Well, we all think we’ll win a Nobel Prize! At the time the catecholamines, norepinephrine and dopamine were a hot-subject and there was von Euler, and there was Carlsson.
LH: Did Carlsson work in your lab?
JA: No, he worked with Brodie. Carlsson, Blaschko, Butterworth and I all worked with Brodie. I thought I might have a chance to get the Nobel Prize, but there were other deserving people.
LH: A crowded field?
JA: Yes. I got it with von Euler and Bernard Katz. There were a lot of other things I did. One was discovering catechol methyltransferase. We also found the enzyme that makes adrenaline, noradrenaline and phenylethylamine. The PNMT story is an interesting one; Dick Wurtman got his MD from Harvard and when he came to my lab as a post-doc, he pointed out that in the adrenal gland of the rabbit, the cortex is separate from the medulla, and the catecholamine in the medulla is noradrenaline exclusively. Since in animals in which the cortex and medulla are not separated, the medulla also contains adrenaline, we suspected the cortex has something to do with the formation of adrenaline from noradrenaline by methylation. Evidently glucocorticoids were affecting the synthesis of adrenaline. To study this further we hypophysectomized rats and found it caused a decrease in the synthesis of cortisol and in the activity of PNMT. But we also found that when we gave dexamethasone to hypophysectomized animals, PNMT activity was increased.
LH: Nature made sense putting the adrenals where they were.
JA: Exactly. We also showed the brain can stimulate tyrosine hydroxylase, the enzyme required to make dopamine and also the rest of the catecholamines, trans-synaptically. We’ve done a lot of experiments with Hans Thoenen and Bob Muller in this area, but when Dick Wurtman came I was working on the pineal gland. I don’t know whether you want to hear that story?
LH: Sure. I had a little adventure with the pineal gland myself.
JA: I know. And I think Altschule thought the pineal gland was involved in schizophrenia. I came across that story in 1958 in an article by Aaron Lerner, a dermatologist and biochemist at Yale, who found when he added an extract of the pineal gland to a tank where tadpoles were swimming, it blanched their skin and affected their melanophores.
LH: Did Lerner use the term melatonin?
JA: That’s what he called it. He isolated the active principle responsible for blanching the skin of tadpoles and that was melatonin, a methylated serotonin. When I saw the abstract, I became very interested in how melatonin was made because of the methyl group. Herb Weisbach together with Sid Udenfriend worked out the metabolism of serotonin. Since melatonin was a serotonin analogue, I asked Herb whether he wanted to collaborate with me, finding the enzyme that makes melatonin. We found two enzymes; acetyl transferase that acetylated serotonin, which later became a very important enzyme, and another that methylated acetyl serotonin to melatonin. Dick Wurtman and I found light would affect the synthesis of melatonin; in the dark there was more melatonin synthesized than in light.
I love working with the pineal gland. Usually when I was working with catecholamines, many experiments didn’t work and that made me feel a little depressed. But every time I did an experiment on the pineal gland, it worked, and it lifted my spirit. It was a good antidepressant! It was a wonderful gland to work with. Dick and I called the pineal gland the neuroendocrine transducer. It was in 1963 or 1964 and we couldn’t measure melatonin directly then. What we could measure was serotonin, its precursor. Then, when Sol Snyder came to work in my lab around that time we developed a very sensitive method to measure serotonin in the pineal gland of the rat; we found in the dark serotonin was very low and in the light it was very high. The reason for the low serotonin and high melatonin in the dark was that in the dark serotonin was acetylated and methylated. We thought that would be a measure of melatonin synthesis. Then Bob Moore came to work on this project. He brilliantly identified the biological clock responsible for formation of melatonin from serotonin at night. It was in the suprachiasmatic nucleus and the pineal gland, which was an arm of that clock.
LH: Did you ever think melatonin would become such a big thing as it is now?
JA: I think it’s a lot of hype although it may have something to do with sleep.
LH: I think so.
JA: But cancer, aging and all that; it’s a lot of baloney!
LH: It makes some sense; it may be related to sleep and perhaps the fragmentation of sleep in older people.
JA: I know Dick Wurtman uses melatonin for all kinds of indications. They sell it over the counter now because it’s a natural compound; it’s a big seller.
LH: I didn’t think there were many things that would put me sound asleep until I tried melatonin. But melatonin sure could put me to sleep.
JA: I tried it but it didn’t help me. Anyway, that’s the short history of melatonin. We also found it stimulated the β-adrenergic receptor that in turn stimulated the enzyme acetyl transferase. It was acetylation, as David Klein had shown, that drove the biological clock.
LH: The cycling of melatonin.
JA: We missed that one. Let’s see, where am I now?
LH: You must be close to about 1970.
JA: Then I worked on methylation reactions, on histamine methyl transferase, which is the major enzyme for inactivation of histamine. Then we found a curious enzyme that methylated tryptamine in the lung and the brain. It became a big thing. Some people thought it might be one of the compounds that would cause…
LH: Endogenous psychosis.
JA: I didn’t buy that, it was too simple an explanation. Our brain is not that simple. But it was fun working on it, and it gave other people something to work on. You remember the pink spot and the Ackerfeld test?
LH: Yes. Once Ackerfeld and I were on a panel together, and he was reporting on his negative results.
JA: He wrote a very influential article for Science about the kind of sloppy work being done.
JA: They found the reason schizophrenics reacted differently from normals on the Ackerfeld test was they didn’t drink orange juice.
LH: There was a wonderful article published back in the 1950s. A biochemist from Illinois wrote “Fact and Artifact in the Biology of Schizophrenia,” and it should be on everybody’s wall.
JA: I remember a story that happened at the Mental Health Institute. They were doing studies on paper chromatography in the 1950s and found that schizophrenics always had two spots, which controls didn’t. Kety was very skeptical about the finding. He said something must be wrong. When the findings were scrutinized it turned the controls were Mennonites who didn’t drink coffee. So you have to be very critical about this sort of thing.
LH: You didn’t rest on your laurels after 1970 but have done a hell of a lot of things since.
JA: After I retired officially in 1984 I wasn’t even called emeritus, but a guest researcher. I was interested in transduction reactions, and one reaction we were especially interested in was the receptor-mediated activation of phospholipase A2. We found it formed arachidonic acid, a very active carcinoid substance.
LH: So, you began to get in the 3rd messenger field.
JA: 2nd messenger. I didn’t get to the 3rd messenger, it got too complicated. But I was involved in research with Carol Gelsma on G proteins that became very important in signal transduction.
LH: Oh, yes.
JA: The Nobel Prize went to Marty Rodbell and Al Gilman for that discovery. These G proteins were heterotrimers. It was thought the alpha subunit activates phospholipase C or A, when the first messenger, a transmitter or a hormone, recognizes a receptor. But, later it was shown that it was the β,γ-subunit that activates phospholipase A2. We sent that paper to Nature. They rejected it. And just four months later another paper came out saying that the β,γ-subunit activates one of the potassium channels. The β,γ-subunit became a big thing. Of course, we didn’t get much credit for it. If Nature would have accepted our paper, we would have had more recognition. But it was fun working in this area of research. One problem I’m working on now should have importance in neuropharmacology. It is cannabis.
LH: The cannabinoid receptor.
JA: It was cloned in my laboratory by Lisa Matsuda and Mike Brownstein.
LH: You know Raphael Meshulam?
JA: Of course. Once the cannabinoid receptor was identified we knew there had to be a natural ligand for it. And Bill Devane, who worked in Meshulam’s laboratory at Hebrew University, isolated the natural ligand. It is arachidonoylethanolamide, which they named anandamide. Bill Devane came to my lab and we found one of the enzymes that make it. It’s an important enzyme because its receptor is distributed in very interesting places: the hippocampus, the striatum, the cortex, and the cerebellum. It must be doing important things. I think it has a great future.
LH: This raises an interesting philosophical question. Why in the world would the body have receptors for drugs it never heard of?
JA: These receptors were there for the normal ligand. Evidently, they lack specificity but have survival value. I have a feeling the animide receptor is not there to give you a high. It must be for very important reasons because of its distribution.
LH: What we need is a theory similar to what the Japanese fellow did with antibodies.
JA: I think like antibodies, we can recognize and detoxify any compound the chemists can synthesize. Anyway, we have been at this for an hour and a half. You should have a general idea of what I have been doing.
LH: I think it has been a remarkable career. You have had more influence in psychopharmacology than any person I can think of, largely because of the eminence of your graduate students and fellows.
JA: Thank you, you’re very kind. But these post-docs were so bright to begin with. And when they came to my lab, I realized most of them were much smarter than I am. I could never have gone to Harvard Medical School, to Hopkins or wherever they went. They picked up things fast. They developed things. The interaction between their good brains and my ability to see connections made a good combination. I tried to pick a problem we were both interested in, and got them +enthusiastic enough to succeed initially, so they could go off on their own, as most of them did.
LH: Now it goes into the second generation. There is this wonderful book, called Apprentice toGenius, in which you figure very prominently.
JA: I came out very well in that.
LH: You tell me you are going to be 85. It’s so true, you know; you and Brodie had a tremendous influence.
JA: Brodie had a tremendous influence. I mentioned in the book that the greatest thing that happened to me in research was working with Brodie. The second greatest thing was leaving Brodie. It’s been beyond my wildest dreams to think I would last so long do the things I did. It was very satisfying.
LH: I think the whole story of your life is inspirational.
JA: You know, I wasn’t a brilliant student. I was a good student. I will be 85 years old next month, on May 30.
LH: And you still have a laboratory.
JA: Actually, I have a new post-doc. I can’t tell you much about what we are doing because it is still in the process of development, but if it does develop it’s going to be interesting.
LH: I see you are still publishing.
JA: I publish, but not like I used to. I used to publish 15 to 20 papers a year. Now it is good if I publish one or two a year. I’ve been lucky; research wasn’t always a happy experience. There were lots of disappointments; most of the experiments didn’t work out. I had very high expectations, and when experiments didn’t work I felt pretty depressed. But once an experiment worked, there was nothing like it.
LH: You certainly have been an inspiration and I want to thank you so much for taking time out and coming here.
JA: It’s a pleasure. I don’t know whether you want to ask me any more questions.
LH: I just wish you could be around for the next 50 years.
JA: I’ll be happy to hang around until the year 2000.
LH: And see all the great developments in the future.
JA: Things are happening so fast; just in the last five years the reuptake molecule has been cloned. We call it a transporter.
LH: It’s an exciting period.
JA: I know. I think neuropharmacology has a great future.
LH: Thank you so much. It has been a great pleasure.
JA: Well, thank you.
May 3, 2018