Thomas A. Ban
Neuropsychopharmacology in Historical Perspective
Education in the Field in the Post-Neuropsychopharmacology Era
Thomas A. Ban’s Interview of Irwin J. Kopin – Metabolic disposition of catecholamines
Irwin J. Kopin’s (1929-2017 career in research began in 1957 as a clinical associate at the National Institute of Health (NIH) of the United State. The first project he participated in was designed by Seymour Kety (1915–2000) during his tenure as Scientific Director of the National Institute of Mental Health for the detection of biochemical abnormalities in patients with schizophrenia. Kopin’s responsibility in the project was to select and care for “relatively healthy patients with schizophrenia” who had been screened for either a strong family history of schizophrenia or complete absence of any first degree relative with schizophrenia (Kopin 2006).
There were weekly seminars at the Institute in which recent findings and reviews relevant to the biology of mental illness were discussed. In one of these seminars the findings of Lauer, Inskip, Bernsohn and Zeller (1958) were presented which indicated that after the ingestion of a 5- gram “load” of tryptophan schizophrenic patients, in variance with normal subjects, did not show higher urinary concentrations of 5-hydroxyindole acetic acid (5-HIAA), indicating an abnormality in their tryptophan metabolism. The study was in line with the research Kopin was engaged in and on Kety’s suggestion he designed and conducted a study in order to verify the results. When measuring the collected urine Kopin found that the concentration of 5-HIAA from schizophrenic patients was lower than in normal subjects, in keeping with the findings of Lauer and his associates, but he also noted that the volumes of urine from schizophrenic patients were 3-fold higher than the volumes of urine from normal subjects. By taking into consideration both factors, Kopin (1959) recognized that the actual increases in the amount of 5-HIAA were the same in schizophrenic patients and normal subjects. The difference in urine volume was the result, as Kopin (2006) wrote in his autobiographical paper almost 40 years later, of “the enthusiasm of the nursing staff encouraging the patients to drink large amounts of water to ensure adequate urine flow to facilitate the collection of the specimens.”
In the late 1950s Seymour Kety arranged the custom synthesis of tritium-labelled catecholamines of sufficiently high specific activity to allow the administration of doses that would not elicit excessive physiologic responses. Soon after, in 1959, Kopin joined the laboratory of Julius Axelrod (1912-2004) to study catecholamine metabolism with some of the 3H labelled norepinephrine (NE) that Kety made available. In the three years that followed (1959–1962), in collaboration with Axelrod, Kopin identified the many metabolic products of catecholamines, such as metanephrine, normetanephrine (NM), 3-methoxytyramine and 3-methoxy-4-hyrdoxypenylglycol (MHPG). They 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) (See also Bulletin 16).
With the administration of 3H – NE to rats they recognized that the primary metabolite of NE was not vanillylmandelic acid (VMA), the product of oxidation and deamination by monoamine oxidase (MAO), and in collaboration with Mann, they demonstrated the presence of 3-methoxy-4-hydroxyphenylglycol (MHPG), an O-methylation product at sympathetic nerve ending (Axelrod, Kopin and Mann 1959). They also detected, in collaboration with Labrosse and Kety, the presence of MHPG sulfate in the human urine (Labross et al. 1961) and 3, 4 -Dihydroxyphenylglycol (DHPG), the substance from which MHPG is formed, in the urine of rats that had been treated with pyrogallol to inhibit-methylation (Kopin and Axelrod 1960). Pursuing further the same line of research, in collaboration with Gordon, they discovered that NE metabolizes into DHPG that converts through MHPG into VMA (Kopin Axelrod, Gordon 1961).
To study the magnitude of O-methylation versus deamination in the initial phase of inactivation of epinephrine (EPI), Kopin (1960) administered EPI in humans. He used a double-label protocol that required 14C – metanephrine (MN) as well as 3H - EPI. He synthesized 14C – methyl – S –adenosylmethionine (14C - SAME) to use for the enzymatic preparation of the labeled O-methylated EPI and used the product for studies in both animals (Kopin, Axelrod and Gordon 1961), as well as in humans. 14C - SAME was to be used by Axelrod to discover a number of other methylating enzymes, including phenylethanolamine – N - methyl transferase (PNMT) which converts NE to EPI in the adrenal medulla (Axelrod 1962). In 1961 Axelrod and Weissbach discovered the melatonin synthesizing enzyme hydoxyindole - O – methyltransferase and in the same year Kopin, Pare, Axelrod and Weissbach (1961) used the enzyme and 14C – SAME to make 14C – melatonin and established that 6-hydroxymelatonin was the major melatonin metabolite.
Kopin was also a member of Axelrod’s team which conclusively demonstrated that the action of catecholamines is terminated by reuptake into the neurons wherefrom they were released (Hertting et al. 1961).
Irwin Kopin was interviewed by Thomas A. Ban for ACNP’s Oral History series and his interview was published in Volume Three of the series (Ban 2011; Kopin 2011; Sulser 2011).
Axelrod J. The metabolism of catecholamines in vivo and in vitro. Pharmacol Rev 1959; 11: 402 - 8.
Axelrod J. The enzymatic N-methylation of serotonin and other amines. J Pharmaco Exp Therap 1962; 138: 28.
Axelrod J. Purification and properties of phenylethanolamine-N-methyl transferase. J Biol Chem. 1962; 237:1657-60.
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, Weissbach H. Purification and properties of 5-hydroxyindole – O – methyl transferase. J Biol Chem 1961; 231: 211 - 3.
Ban TA. Preface. In: Sulser F, editor. Neuropharmacology. (In: Ban TA, editor. An Oral History of Neuropsychopharmacology The First Fifty Years Peer Interviews. Volume Three). Budapest: Animula; 2011, p. ix – xxx.
Hertting G, Axelrod J, Kopin IJ, Whitby LG. Lack of uptake of catecholamines after denervation of sympathetic nerves. Nature 1961; 189: 66-7.
Kopin JJ. Tryptophan loading and excretion of 5-hydroxyindoleaetic acid in normal and schizophrenic subjects. Science 1959; 129: 835 – 6.
Kopin JJ. Technique for the study of alternate metabolic pathways. Epinephrine metabolism in man. Science 1960; 131: 1372 – 4.
Kopin JJ. My ten years at NIH: The dawn of psychopharmacology. In Ban TA, Ucha Udabe R, editors. The Neurotransmitter Era in Neuropsychopharmacology. Buenos Aires: Polemos; 2006, 40 -50.
Kopin IJ. Interviewed by Thomas A. Ban. In: Sulser F, editor. Neuropharmacology. (In: Ban TA, editor. An Oral History of Neuropsychopharmacology The First Fifty Years Peer Interviews. Volume Three). Budapest: Animula; 2011, pp. 329-54.
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 JJ, Pare CM, Axelrod J, Weissbach H. The fate of melatonin in animals. J Biol Chem 1961; 236: 3072 – 5.
Labrosse EH, Axelrod J, Kopin JJ, Kety SS. Metabolism of 7 3H epinephrine d-bitartrate in normal young men. J Clin Invest 1961; 40: 253 – 60.
Lauer JW, Inskip WM, Bersohn J, Zeller EA. Observations of schizophrenic patients after iproniazid and tryptophan. AMA Arch Neurol Psychiatry 1958; 80: 122 -30.
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, p. xl – l.
Irwin J. Kopin, interviewed by Thomas A. Ban
Acapulco, Mexico, December 12, 1999
First published in Volume Three (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.329-54)
TB: We are at the Acapulco Princess Hotel in Mexico. It is December 12, 1999. I will be interviewing Dr. Irwin Kopin for the Archives of the American College of Neuropsychopharmacology. I am Thomas Ban. Let’s just start from the very beginning. Where are you from?
IK: I was born in New York.
TB: Where were you brought up?
IK: My first memories are of the Bronx and we used to go away during the summer to Long Island. We had a small place in Rockaway on the beach where I learned to swim. Swimming has been part of my life. My wife says that there are four S’s in my life: swimming, science, stamps and spouse, and she says, the most important better be spouse. In any case, science started a long time ago. When I was about nine years old, I got a chemistry set, and this is why my wife married me; the connection you’ll see in a minute. I played with the chemistry set and told my father, when I was about ten or eleven years old, that I wanted to be a chemist when I grew up. He responded: “You’ll never be a chemist unless you know how to make a mirror.” Well, my father had a factory that made mirrors. He was in the “mirror business” and I was intrigued by the idea that you could
make a mirror with chemicals. I went to the public library and read up on making mirrors. I found out that forming a mirror is a test for the identification of aldehyde. You take a silver nitrate solution and add ammonia to it. At first, you get a precipitate. Then the precipitate dissolves. When a reducing agent, such as an aldehyde, is added, you get a mirror. I tried what I read and got a black precipitate with a little silver streak of a mirror along the side. If you see that streak, you know silver has been deposited and that’s the way you make a mirror. Well, I showed this test tube with the black precipitate and the silver streak to my father and he said, “Do you think I could sell that for a mirror”? And, even I, an eleven year old, knew that you could never sell this black thing with a little silver streak as a mirror! So, I went back to the books and read some more. This was over years. By the time I was fourteen, I had learned a good deal more chemistry. I went to the Bronx High School of Science and during that period I s persisted and tried to make a mirror about thirty different ways. I wrote to the Department of Commerce and asked, “How do you make a mirror?” They sent me a brochure that listed about a hundred ways of making a mirror. Over the years, before and after receiving the brochure, I tried about sixty of them. They would all give the black precipitate and a little bit of silver streak. My father said, “At this rate you’ll never be a chemist. But at least you should have a trade.” I was about sixteen at the time and was allowed to work. On weekends he took me to the factory and I learned how to drill holes in glass. At that time they used a tungsten carbide drill with water dripping on it to keep it cool and prevent glass powder from being inhaled. If you pressed too hard, the glass broke. If you didn’t press hard enough, you could sit there all day and wouldn’t get a hole. After a while, it took about seven days, my father said I had “the touch.” I could drill one hundred and eighty holes an hour in the glass, but I hated to do it. I didn’t know what to do during the boring task. I used to skip lunch so I could go home early. When I told my father how I felt, he replied, “Well, you’ll have to learn how to make a mirror.” So, one day, I went up to the person who was in charge of silvering glass to make mirrors and told him about my experience. He said, “You have to wash the glass! Grease or a little bit of dirt, act as a nidus for the black precipitate. You have to clean the glass thoroughly!” So he taught me how to clean glassware. First, use sodium hydroxide solution, then use distilled water to wash that out, then scrub with red cuprous oxide, then wash with more distilled water. Only after this do you add the silver nitrate solution. When I did this at home I was able to bring a beautiful silver finger, like the inside of a thermos bottle, like a Dewar flask, to show my father, who said, “Now you can go to college!” So, that was my entry to college. I went to City College for two years with the idea I would do something in chemistry. At about that time, however, I decided, I wanted to go to medical school. In those years, it was very difficult to get into medical school from City College so I had to transfer to a different college.
TB: What year was that?
IK: It was in 1948. About that time my father wanted to bring my aunt to the United States who had been in a concentration camp, the only one of my father’s five siblings who survived the Holocaust. She was unable to get a visa to enter the United States, but Canada was more receptive. So, my father arranged for her to go to Montreal and settle there. I was an only child and he knew I needed to get out of the house and go off to college. But he didn’t want me to be alone, cold and hungry in a strange city without any relatives. So he convinced me to go, with my good friend, Rubin Bressler, to McGill University in Montreal. Rube and I are friends since second year high school and went to City College together. So that’s how I got to McGill. In the organic chemistry course at McGill, Rube Bressler and I were lab partners. There were girls taking the same course and in one of the laboratory exercises we had to make an aldehyde and test for its presence. Although we had done everything together, when it came time to testing for the aldehyde, I said, “Rube, you must step aside. I will do this”. Of course, I cleaned the glassware thoroughly and got this beautiful silver finger of a test tube. We brought this to the instructor and he’d never seen one like it. He was used to seeing the black precipitate with a silver streak on the side of the tube. When we gave him our test tube, he put it on exhibit in front of the class. The girl I was dating, Rita, was so impressed she finally agreed to marry me! That was my first introduction of how important it is to wash glassware; the details of laboratory work were impressed on me very early. Years later, Julie Axelrod, with whom I worked at NIH, claimed he used to get his best ideas washing glassware. His laboratory work was mainly enzymology and it was important to have clean glass. If I had known that when I was younger, I would have been able to convince my father earlier that I was college material. But, it was a very useful experience. At McGill, Rube and I majored in Biochemistry. Professor David L. Thompson, who was Chairman of the Department of Biochemistry, was a wonderful inspiring lecturer. He used to come to class with a small card and taught us everything from Nutrition to advanced Protein Physical Chemistry. Professors Orville Denstedt, Murray Saffran and Joshua H. Quastel constituted a great group of biochemists. After graduating from the Honors Course in Biochemistry, I went to McGill’s Medical School. It was a very good school, and it was there I first found out there could be a rational approach to drug treatment of psychiatric disorders. Professor Heinz Lehmann was a gem of a teacher. He could bring a patient into the room with a group of about fifteen of us. He would introduce the patient, and he tell us to examine him or her. We were to watch the patient’s behavior and discuss later what we saw. I can still remember, vividly, after over fifty years, many of the patients. To show us mania, in 1953, before drug treatment had been introduced, Heinz Lehmann brought in a female patient for us to examine. She was unable to remain still. She danced around the room, flirting from one student to the next saying, “Oh, what a beautiful tie you have! Oh, my, look at your jacket! It’s gorgeous. Your shoes are so polished,” We were all laughing with her, not at her; we enjoyed her presence. When she left, Dr. Lehmann said, “This is mania. This is a pure manic patient. You feel happy with the patient, you enjoy the patient”. Of course, when she did what she was doing in class, for twenty-four hours a day, it became anything but enjoyable to her husband. Nevertheless, that is the feeling mania induces. Another time he brought in someone who was depressed. The patient told us that he committed an unpardonable sin and we all felt the depression the patient experienced. The hebephrenic schizophrenic patient he showed us had received a PhD at McGill in biochemistry before he became sick. One day he was found wandering around in the nude on a mountain behind McGill. When he entered the room, he said, “Ah, ha, what a wonderful idea, what a happy, happy day.” He spoke nonsense in a high tone, although he was a big guy and we expected he would have a deep voice. Professor Lehmann explained this was typical of hebephrenic schizophrenia. The patient is silly. Unlike the manic patient, you find the patient uncomfortably laughable. Another time he brought in a person who came well dressed in a jacket and tie with the daily newspaper under his arm. He sat down comfortably and when we interviewed him we found absolutely nothing wrong with him. He was oriented in time and place. He knew current events. He seemed aware of everything. We finally asked the patient why he was in the hospital. Then, he explained, “Well, you know, these people don’t understand me. My wife had this X-ray machine, and she keeps looking into my brain and telling me things to do. It became impossible, and because of that, I had to kill her”. Dr. Lehmann said, “This is an example of the island of abnormality in the mind of a paranoid schizophrenic”. He warned us, “Don’t turn your back on a paranoid schizophrenic. You’ll have a nice conversation with him and, suddenly, he’ll pick up the ash tray and hit you over the head with it”. This was Heinz Lehmann, only he could carry this off. I don’t know where he found these typical patients. I’ve never seen them again. The patients I encountered always had mixed, unclear diagnoses but he had these rare, “typical” patients from the large population at Verdun Protestant Hospital, which was the McGill teaching hospital for psychiatry. There was another psychiatric hospital that was closer to the Medical School, the Allan Memorial Institute, which was up on the hill. There I saw shock treatments given to schizophrenics, but we never really got the same feel for the disease that Heinz Lehmann was able to impart.
TB: What did you do after graduation from McGill?
IK: I took my internship and residency at Boston City Hospital. At that time, in the U.S., there was the “Berry Plan”. If you enlisted in the Army during Medical School, they allowed you to take your residency and, then, you went into the Army after you completed your training. Entry into the Army was postponed. Since I went to McGill in Canada I had not been part of the Berry Plan. My draft board wrote to me in March 1957, that I would be drafted into the Army unless I enlisted by July1. I decided I would enlist but would finish my internship and my second year of residency and then go into the Army. But the Army told me I couldn’t enlist until September. At that time I had a wife and two children, having married at the end of my first year in medical school. I was very lucky to have a supportive wife. Our first child was born in Montreal during my last year of medical school and the second child was born during my internship in Boston. We had these two babies and I couldn’t afford not to have a job for three months. So, I decided I would call the Navy, but the Navy gave me the same story, as did the Air Force. Then, I heard about the US Public Health Service. They were accepting enlistments on the 30th of June. So, I decided I would apply. I was accepted and received a letter saying I was assigned to the Tuberculosis Research Section because of my “background in mathematics”. I had been a good mathematician in college. I won a prize at CCNY (College of the City of New York) for “Pure and Applied Calculus” and when I graduated from McGill, I had won the Hiram Mills Gold Medal in Biological Science along with Honors in Biochemistry. I thought they had assigned me to a real research project. But I soon found out this assignment was all statistics. At that time, I was caring for patients and I didn’t want to lose my touch so I went to Washington and explained, “I’m delighted I’m with the US Public Health Service; however, I would like to be in a hospital where I see patients”. The personnel department was very accommodating, “Well, there are two jobs open in this new hospital on the outskirts of town called Bethesda and there’s a new clinical center”. I’d been at the old Boston City Hospital and when I walked into the beautiful new marble hallway of the Clinical Center at the NIH I thought, “I would take a job sweeping floors here”. It was a gorgeous place. I was interviewed by two groups of people. One was in the Dental Institute. They were studying dental agenesis in patients with albinism. The other was a study of schizophrenics at NIMH. They wanted a physician to take care of the normal controls and a very select group of schizophrenic patients. That job was in the Clinical Center, whereas the other involved living in a trailer in a south portion of Maryland. It was June and very hot. The trailer had no air conditioning so the choice was an easy one, “I’ll be in the Clinical Center”. So, by accident, I choose to work with the project on schizophrenia.
TB: What was your task in the project?
IK: My first task was to go to the mental hospitals and examine the patients to determine whether they were appropriate for admission to the schizophrenia project. Seymour Kety helped in designing this project. He wanted to find out whether or not there was a familial tendency in schizophrenia and whether there was a biological difference between schizophrenics that had a strong family history and those that didn’t. My job was to make sure the schizophrenics were healthy except for their psychiatric disorder. So, I examined them and made sure they didn’t have any Parkinsonian symptoms from their drugs, liver disease, etc. We brought into NIH fourteen schizophrenic patients; seven of whom had a family history. Some families were loaded with the illness with one parent, an uncle, a cousin, three or four people in the family blatantly schizophrenic. The others had absolutely no history of schizophrenia. Four hundred man-hours went into the examination of these patients to select them. It took about three months but after that I would go on ward rounds, which took about fifteen minutes in the morning, and I had free time all day. At that time, serotonin had just been found in brain. So, I wanted to find out whether or not serotonin had anything to do with brain function in schizophrenia. I went to Marion Keyes, who was head of the Section on Biochemistry in Seymour Kety’s laboratory, and told her I wanted to look at spinal fluid for 5-hydroxy-indole-acetic acid, 5-HIAA, the metabolite of serotonin. Paper chromatography was at that time the method for detecting such a substance and I proposed looking for 5-HIAA in spinal fluid using paper chromatography. To do this, Dr. Keyes told me, I had to get rid of the salts first and then do paper chromatography to find if 5-HIAA was present. She also told me, “I’m writing a book on allergic encephalomyelitis, so I’m not using my bench. Feel free to use it”. I went to the library to find out how to remove salts from spinal fluid, and made a large desalting apparatus with mercury bubbling up. It was one of those complex glass things; it reminded me of a cartoon I once saw, where ladies are cleaning the laboratory and inspecting a huge complex glass apparatus with a boiling solution. One cleaning lady says to the other, “I don’t know what they use it for, but I use it for making coffee”. Well, that’s what this thing looked like, but it worked. I was able to get the desalting apparatus to function and was already doing lumbar punctures on the schizophrenic patients to be sure they didn’t have syphilis so I froze some of the spinal fluid to try to detect 5-HIAA in it.
TB: Are we in the late 1950s?
IK: This was in 1957, shortly after serotonin was discovered in brain by Park Shore. About the same time, in 1957, it was decided to have a conference on catecholamines at NIH. The reason was that catecholamines had become very important. Ulf von Euler had, in the early 1950s, discovered norepinephrine was the neurotransmitter of the sympathetic nervous system and a great deal of research followed his discovery, seeking the role of catecholamines in disease states. Also, there was an hypothesis that adrenochrome, derived from the oxidation of epinephrine, might cause schizophrenia. The adrenochrome hypothesis was based on anecdotes that, during World War II, when outdated adrenaline, which had become pink from formation of adrenochrome, was injected into people they became psychotic. The hypothesis that catecholamines might be involved in causing schizophrenia was sufficiently important it had to be investigated. Seymour Kety, who was Chief of the Laboratory of Clinical Science, had spawned interest in biological factors in mental disorders. He is regarded by many of us as the father of Biological Psychiatry. We were encouraged to investigate various biological factors related to brain function and psychiatry. Kety encouraged Julie Axelrod to follow his interests in catecholamine metabolism and I was encouraged to examine tryptophan and serotonin metabolism.
Zeller had described that after giving a tryptophan load orally to schizophrenics and normal subjects, the increase in the urinary concentration of 5-HIAA was significantly lower in schizophrenic patients than in normal volunteers. Seymour suggested that, perhaps, since I was involved in measuring 5-HIAA anyway, I should look at this problem. So we loaded the patients and controls with tryptophan and collected their urines. Well, schizophrenics aren’t very cooperative and the conscientious nurses would follow the schizophrenics around the ward to make sure they got a complete urine collection. To encourage them to urinate, patients were urged to drink a lot of water. As a result, the concentration of 5-HIAA in the two to three liters of urine collected from schizophrenics was low compared to that in the one liter of urine that came from normal controls. Zeller had reported concentrations and not the absolute amount. Well, his findings of lower concentrations were confirmed; we obtained the same results that he reported. Yet, although the concentrations were low, the total amount of HIAA excreted was the same for the schizophrenic and normal subjects.
About that time, Julie Axelrod had become deeply involved in the study of O-methylation as the route of epinephrine metabolism. This was an interesting story, because Julie, who was an expert biochemical pharmacologist and had for many years, worked with Bernard Brodie, just recently obtained his PhD, but was already a Section Chief in Kety’s Laboratory. Julie attended the Federation meetings in 1957 in Atlantic City, where Armstrong described vanyllilmandelic acid, VMA, in urine as a product of epinephrine in patients with pheochromocytoma. Since, on the basis of earlier experiments with 14C-labelled epinephrine reported by Schayer, it was generally believed that epinephrine was deaminated, Armstrong proposed that epinephrine was deaminated and then O-methylated to form VMA. But Julie thought this might not be the order of events and had the novel idea that maybe O-methylation was first and more important than deamination. It was fortunate that Julie’s laboratory was just down the hall from Julio Cantoni’s lab. Cantoni had previously discovered S-adenosylmethionine (SAMe), the methyl donor for such methylation. So Julie got some S-adenosylmethionine from Cantoni’s lab, used it to incubate epinephrine with a homogenate of liver and found a new spot on chromatography. But he could not prove that this spot was O-methylated epinephrine. It’s stained like a phenol and it seemed by its extraction properties to be an amine, but to prove that it was an O-methylated product he had to have the authentic compound. Julie phoned Bernardt Witkop, who was head of the Laboratory of Chemistry at another institute, NIDDK, and asked if he could synthesize the hypothetical O-methylated product of epinephrine. The Visiting Scientist Program at NIH was just initiated, and Bernardt assigned the task to Shiro Sanoh, the first Japanese Visiting Scientist to come to NIH. Shiro synthesized metanephrine for Julie in three days and by chromatography they showed that it had the same retention, Rf value, and had the same staining characteristics as the substance formed from epinephrine and SAMe in the liver homogenate. They published this in Science. Julie showed that formation of metanephrine was important; but he could not find any adrenochrome formation from adrenaline in animals.
Kety organized a group of us to present reviews in a symposium on newly emerging findings in biological psychiatry. Lou Sokoloff, Seymour Kety, Julie Axelrod, Elwood LaBrosse and I presented summaries about various biological aspects of mental disease, and also about some of the pitfalls of studies in biological research in psychiatry. Much of this was about the mistakes that had been made. An example was one based on the use of paper chromatography, a popular technique at the time. Based on urine samples from patients and normal subjects subjected to chromatography there were reports of a spot that always showed up in urine from schizophrenics, but didn’t appear in the urine of the normal subjects. LaBrosse was studying at the time schizophrenics and had normal controls, most of whom were volunteer Mennonites. These normal Mennonite men came to NIH to be volunteers in medical research, instead of serving in the armed forces, because they didn’t believe in violence or war. Kety had arranged to have fourteen schizophrenics and fourteen normal controls in the study. All of the normal subjects were Mennonites except one, and he was a little bit peculiar. When their urine was compared to that of the schizophrenics there was a clear difference in the samples. The urine of all of the schizophrenics except of one, who had no family history of schizophrenia and who was a little bit different than the others, produced a specific spot on chromatography. Only one of the normal subjects had the spot and he was not a Mennonite. He was also an older fellow, a little bit different from the other control subjects. After searching to find out the nature of the “schizophrenia spot” in the urine, they found it was caffeic acid, a constituent of coffee. The young Mennonites didn’t drink coffee and they didn’t smoke, but the older fellow, who was a little different, drank coffee. All the schizophrenics drank coffee, lots of it, but the one patient who was a little bit different, avoided it. So, it was the “coffee spot” that was different. Elwood LaBrosse was the person who was responsible for this work. This was a good example of the errors and pitfalls that were being made in schizophrenia research in early years.
Another important development was the introduction of reserpine, which was initiated by a pharmacologist from India, who went to various drug companies with evidence that a folk medicine, Rauwolfia alkaloid, calmed animals and excited patients. Finally, Ciba picked it up and isolated reserpine, which turned out to be a useful drug and was brought to market. Park Shore, in Brodie’s laboratory, showed that reserpine depletes brain serotonin and noradrenaline. When reserpine came into use to treat hypertension, it was found that it sometimes caused depression. The hypothesis that depression was related to the depletion of brain norepinephrine was partially based on this finding.
Julie Axelrod had been working with catecholamine metabolism and disposition in those years. He used to sit in an open laboratory. His desk was in the laboratory with a sink right next to the seating area and his workbench next to that, with a blackboard behind the desk. On that blackboard had been written all of the questions, all of the formulas and all the outlines of the experiments being planned.
Seymour Kety had introduced radioactive adrenaline and noradrenaline into the laboratory. Seymour made an arrangement with New England Nuclear Company to make radioactive noradrenaline so that we could follow it through the body. He did this for clinical purposes. Julie used it to study the metabolism and disposition of these amines. I remember the time when George Hertting, a pharmacologist from Vienna came to the NIH, and Julie and I were standing around discussing some findings. Julie said, “You know, after we inject 3H-adrenaline intravenously into animals, we find half of it is retained in the tissue”. He had done this research in intact animals in the mouse, and in cats. A large fraction of 3H-adrenaline remained in the heart and Julie said, “It seems that adrenaline goes to where noradrenaline is and maybe there’s something special about this. Maybe uptake is important in some way”. George Hertting, listening to the conversation, recalled that after denervation, after cutting sympathetic nerves, they degenerate and the tissue becomes supersensitive to adrenaline. So, he said, “If the nerves are where 3H-adrenaline remains, that won’t happen on the side where the nerves degenerate”. Hence, George and I removed the right superior cervical ganglion from cats and waited for a week for the nerves to degenerate. We then injected 3H-noradrenaline and an hour later removed the tissues from the nictitating membranes and salivary glands on both sides. We found the tissues on the side from which the superior cervical ganglion had been removed didn’t take up the 3H-noradrenaline whereas the tissues on the intact side did. The basis of supersensitivity became apparent. Since there was no uptake on the side where the superior cervical ganglion was removed, uptake was perceived as the mechanism for inactivation.
At that time there was a disagreement about whether O-methylation or deamination was the important mechanism for inactivation of noradrenaline. A Belgian pharmacologist, Zacq, had found that pyrogallol, a catechol, slightly potentiated the actions of adrenaline, whereas inhibition of monoamine oxidase had almost no effect. Of course, we now know that it is uptake that is the mechanism of inactivation of catecholamines released from the nerves. But, the fate of injected adrenaline is somewhat different. The question was whether O-methylation or deamination was important? I had suggested we use double radioactive labeling to find this out. It required the labeling of metanephrine with 14C, which we could make with radioactive S-adenosylmethionine. We used this 14C- metanephrine simultaneously with tritiated adrenaline. I did the experiment in patients, and together with Julie I started to study the metabolism of catecholamines in rats. From the ratio of tritium to carbon in the urinary metanephrine, it became clear that O-methylation was the predominant route of metabolism of the administered catecholamine in rats and in humans. Yet, inhibition of O-methylation didn’t potentiate the effects of nerve stimulation. George and Julie showed that cocaine, which was known to potentiate the effects of sympathetic nerve stimulation, prevented the accumulation of injected 3H-noradrenaline in tissues. The concept that neuronal reuptake is important for the inactivation of a neurotransmitter stemmed from that early work, done around 1959, and published in 1960 and 1961.
During the studies of urinary 3H-catecholamine metabolites, a new metabolite had appeared in the urine of rats. It was neither VMA nor metanephrine. The metabolite could not be obtained from N-14CH3-labeled metanephrine, but did form after administration of the side chain labeled 3H-catecholamines. It turned out to be 3-hydroxy-4-hydroxyphenylglycol (MHPG). In rats, MHPG was the major urinary catecholamine metabolite. In humans, MHPG is also excreted in urine, but VMA is the major urinary metabolite. At that time, we thought that this was a species difference in the metabolism of the intermediate aldehyde metabolite, but this was not the case.
TB: What year was MHPG identified?
IK: In July 1960, I went off to complete my residency in internal medicine and returned to NIH after one year. Seymour Kety had left NIH by then to become Chairman of the Department of Psychiatry at Hopkins. Seymour invited me to go to Hopkins with a joint appointment in the Departments of Medicine and Psychiatry, but in order to “pay back” NIH for the period of time they allowed me to take my residency, I had to remain at NIH for at least one more year. While I continued doing research on noradrenaline, another compound, melatonin, became of interest.
Melatonin, which is 5-methoxy-N-acetyl of serotonin was discovered by Aaron Lerner at Yale. He presented a seminar on melatonin at NIH, and suggested that the substance was metabolized to 5-HIAA. Lerner thought that after the N-acetyl and the methyl groups are removed from melatonin, the resulting serotonin is converted to 5-HIAA, a metabolite of serotonin. I had been working with double labels at the time and suggested to Julie that we label the whole molecule of melatonin. We labeled the O-methyl group with carbon and the acetyl group with tritium. If Lerner was right, we should not find any radioactive compounds related to indoles in the urine. If we would find radioactive compounds related to indoles, we had the capability to determine whether one or both ends of the administered melatonin remained intact. Michael Pare, a psychiatrist from England, joined us at that time and participated in this project. It turned out that the ratio of carbon, that labeled the O-methyl group, and tritium, that labeled the acetyl group, was identical in the urine to the ratio in the melatonin that was injected. Clearly, there was no deamination or deacetylation. When we gave large amounts of unlabeled melatonin, paper chromatography of the urine sprayed with Ehrlich’s reagent, which stains indoles, showed a sky blue spot. We found that the same type of spot was present in the urine of a woman that Aaron Lerner sent us, who had been given large doses of melatonin to treat her melanoma. Of course, it didn’t help melanoma, but we had the urine and she had this sky blue spot, also. Well, I didn’t know much about that type of chemistry, but NIH is a wonderful place, because there’s an expert in almost any field. Among them, was an expert in the field of indoles, Evan Horning. I took the material to him, and he recognized, from the shy-blue color reaction with Ehrlich’s reagent, that it was a 6-hydroxy-indole. Thus, 6-hydroxymelatonin, and 6-hydroxymelatonin sulphate were found to be the major metabolites of melatonin.
It was about this time, that, Dick Wurtman joined the laboratory. There were also a number of other young scientists coming in from all over the world. George Hertting had already been there. Leslie Iversen and Jacques Glowinsky came to the NIH to work in Julie’s lab, and these people became the founders of a major portion of the biochemical aspects of pharmacology, particularly in the nervous system. Many of the stars in neuropharmacology, particularly in the amine area, grew up in the laboratory that was established by Seymour Kety. Seymour, after one year at Hopkins, decided that Hopkins was not for him. He told a story, that when he first went to his new office at Hopkins and sat down in the chair of the department, the chair broke. He claimed he felt this was an indication he might not last. After a year, he decided to return to NIH; when he came back he told me I should stay. He wanted me not to go to Hopkins. So I agreed to stay. At that time, I had the good fortune of being able to hire a wonderful technician. Edna Gordon was a woman who had worked with Jarvis on phenylketonuria in New York. After she had married and had a child, she left work for about eight or ten years. But at this point in time she was ready to return to the laboratory. When I went home and told my wife, Rita, about this woman whom I had interviewed, she said, “You should hire her, because that’s the type of person who would have gone on to get a PhD.” And she was right. Edna Gordon was a gem. She did all of the work I couldn’t do with the precision that she brought. She taught me how to keep notebooks. She kept all of the data, beautifully organized. Also, a normal volunteer, Dale Horst, a Mennonite, started to work in the laboratory. Dale was bored on the ward where he worked and offered to help out in the lab. After a while, Dale decided that he had some interest in biology. He left NIH, went back to school and majored in biology. After receiving his degree he applied to NIH, looking for a position as a technician. I gave him a job in the laboratory. As part of the research I was doing, I had learned how to inject the tail vein of mice and rats to get urine flowing so we could get clean samples. I asked Dale to learn how to do this and explained it would not be easy to do initially, it would take time to get the hang of it, so he must be very patient. After explaining all of this and how to put the needle underneath the skin in the tail where the vein can be seen, he easily did it the first time he tried! He had done it beautifully; much better than I would have. He then constructed a rack, so we had eighteen animals with intravenous infusions of fluid going in their veins while their urine was collected. It seemed as if the fluid input were connected to the penis, because as fast as the fluid was infused, the rats begun to urinate at almost the same rate. We were able to get half-hour urine samples from these animals and could study the kinetics of the excretion of metabolites of the labeled catecholamines we injected. We started to study the effects of drugs on the excretion of the products of 3H-catecholamines. We could distinguish which were the immediate metabolites in the urine excreted in the first hour. They were mostly O-methylated. After several hours however, the major metabolites were deaminated metabolites. After tyramine was administered there was a large increase in sympathetic responses, and the urine contained increased amounts of O-methylated products. But after reserpine administration, which depleted catecholamines from their stores and interfered with sympathetic function, we found marked increases in the deaminated metabolites in the urine. That led us to the conclusion that the reserpine-induced depletion of amines is accomplished by interference with their storage. If the catecholamine is released into an active form outside the nerve it is O-methylated. But O-methylation is relatively unimportant for inactivation of most of the released amines, because most of the amines are inactivated by reuptake.
TB: When did you become a section chief at NIH?
IK: By 1963 I had become a Section Chief. There were a series of outstanding postdoctoral fellows who came to work with Julie Axelrod and me during the next decade. I already mentioned Leslie Iversen, Jacques Glowinski and Dick Wurtman by name. Others included Ross Baldessarini, Sol Snyder, Dick Wurtman, Jose Mussachio, Joe Fischer, Saul Schanberg, Joe Schildkraut, Goran Sedvall, Lou Lemberger, Tom Chase, George Breese, Richard Kvetansky, Perry Molinoff and Dick Weinshilboum all of whom later made their mark as outstanding investigators and leaders in academia and the pharmaceutical industry.
This was the time of the Korean War and there was a draft to serve in the military. Those who joined the US Public Health Service could satisfy their military obligation by serving at NIH, rather than go to Korea. This was a popular option and, at one year, I had six young physicians, each of whom were first in their class in medical school, apply to come to our Laboratory as a Research Associate. They would serve for two years and we had star applicants. One of them was Joe Schildkraut, a young psychiatrist who joined me in 1966 after having extensive discussions with Seymour Kety. Together, building on the earlier observation that reserpine sometimes induced depression, they gathered the evidence which supported the hypothesis that catecholamine depletion was the basis of depression. Goran Sedvall, another psychiatrist who joined our laboratory, subsequently became Chair of the Department of Psychiatry at the Karolinska Institute. Joe Schildkraut, became a professor of psychiatry in Boston. Ross Baldessarini, then a young medical student at Hopkins was referred to our lab by Dr. Kety who phoned me, saying, “This fellow is very bright. Why don’t you take him as a summer student”? At that time, we were interested in S-adenosyl methionine and we were employing the double label technique again, using melatonin as the product. Melatonin could be separated from both the added 14C–methyl-labeled S-adenosyl methionine, and from 3H- N-acetyl serotonin. The added 14C –methyl-labeled S-adenosyl methionine was diluted by the tissue S-adenosyl methionine and enzymatically converted to melatonin. From the ratio of the carbon/ to tritium we could calculate how much endogenous S-adenosyl methionine had been in the tissue. This was the project that Ross did over the summer of 1963 and we published it first as an assay for S-adenosyl methionine. Several years later, Ross came back to our laboratory as a Research Associate. Seymour Kety by that time had returned to NIH and I had become a Section Chief. Catecholamines had become important, not only in psychiatry, but to all those who studied the sympathetic nervous system. Hence future neurologists, anesthesiologists, and internists, came through our laboratory at one time or another. Mike Roizen, who became Chairman of the Department of Anesthesiology at the University of Chicago, had his first experience with catecholamines in our Laboratory. In the late 1960s we started to study the release of noradrenaline and related compounds from the sympathetic nerve endings. Joe Fischer, a surgeon, in our Laboratory, became expert at perfusing cat spleens with intact sympathetic nerves. This was a very useful means of studying amine release when nerves were stimulated. The people that came to our laboratory and left who have had a major impact on developments in the drug industry as well as in academia, included Perry Molinoff , Steve Paul, Gus Watanabe and Bill Potter. Because of the responses of the sympathetic nervous system in emergencies, we became interested in stress. Stress elicits responses in the sympathetic nervous system and the adrenal medulla. Richard Kvetnansky, from Bratislava, came as a visiting scientist, and, brought a model for studying “immobilization stress” in rats that he’d been working with in Bratislava. But in Bratislava, they didn’t have the techniques that we had to examine catecholamines. We started to study the effects of stress on the adrenal medulla and on the sympathetic nervous system, and particularly enzyme induction. Goran Sedvall had developed a technique for stimulating, in a rat, the sympathetic nerves in the neck on one side of the head, so we could compare changes in the two sides. Using DOPA labeled with one isotope and tyrosine labeled with a different isotope, he was able to show that conversion of tyrosine to DOPA was the rate-limiting step in norepinephrine synthesis and that this conversion was enhanced by nerve stimulation. DOPA was easily converted to noradrenaline, but if you stimulated the nerve, more tyrosine was converted to noradrenaline; so the carbon/tritium ratio in the salivary gland was increased on the side the nerve had been stimulated. This was the first indication that sympathetic nerve stimulation increases tyrosine hydroxylase activity. We subsequently found that DHPG (di-hydroxy-phenylethylene-glycol) is the major initial metabolite of noradrenaline and is converted by O-methylation to MHPG in the tissues. The MHPG enters the blood stream and is converted in the liver to VMA, which is the product that is excreted and can be measured in the urine in humans. We could then use blood levels of MHPG as a basis for studying sympathetic activity in humans in many studies. Graham Eisenhofer, Dave Goldstein and I continued to develop much of the MHPG story.
TB: Are we now in the mid-1960s?
IK: We’re spanning the mid-1960s. We conducted a series of studies on false transmitters in the early 1970s. The concept of false transmitters began with the introduction of α-methyldopa. When α-methyldopa was given, α-methylnoradrenaline was formed and largely replaced norepinephrine. α-Methyldopa is used as an anti-hypertensive agent, because the α-methylnoradrenaline formed doesn’t stimulate the α-receptor and norepinephrine is more active at ß-receptors, which causes vasodilation. In the brain, after alpha-methyldopa administration, alpha-methyldopamine and alpha-methylnorepinephrine are formed.
TB: You have made major contributions in setting the neuroscience foundation of neuropsychopharmacology. Am I correct that you were the recipient twice of the prestigious Anna Monica Award?
IK: Yes, once with Joe Schildkraut, and once alone, for the MHPG story. Joe led a group of us that won the first Anna Monica Award for work that led to the concept of noradrenaline being involved in depression. Later on, when the MHPG story developed, almost ten years later, I won the Anna Monica Award. Our research clarified the important role MHPG plays as an index of sympathetic activity and as a means for using plasma MHPG and CSF-MHPG to evaluate norepinephrine metabolism in brain. Many people contributed to these studies, and I was lucky to have been singled out for the award. At the time MHPG was a central area of our research.
TB: Tell us about some of the other young people you didn’t mention as yet, who spent time in your laboratory.
IK: We had a number of young physicians who began their research careers in our laboratory. One of them, Steve Silberstein, joined us to study tissue cultures. He’s currently a neurologist, and is studying headache. Another one was Justin Zivin, who was doing research with us on stroke and trauma, promulgating the idea that catecholamines have an important role in development of pathological changes after spinal cord injury. People like Silberstein and Zivin got their early training with us and, then branched off into their own areas of research. It’s given me great pleasure to see how they developed and continued to do research using the conceptual framework they learned in our Laboratory for their investigations, as, in Walter Cannon’s words, “the way of an investigator.” I learned from Julie and from Seymour how to think and how to manage a laboratory, and I see the things I learned I’ve been able to pass on to them, like to my children.
TB: Isn’t your son a molecular biologist?
IK: My son started as a gastroenterologist, but has evolved into a molecular biologist. As part of the requirement to participate in research to obtain Boards in gastroenterology, he learned to clone a gene. He was new to this area but became good at it so I learn a lot about molecular genetics from him! We live now in a new world of research and I’ve had the good fortune of bridging the time when we knew little about the molecule, and current times when we know so much about it. In this new world, information comes faster than we can possibly digest it. We need computers to keep track of everything that’s going on; it’s difficult to see how we managed before 1965, when we didn’t have Medline. In the 1980s you would have to have gray hairs to remember what happened before Medline, and this is the time I bridged.
TB: Weren’t you involved in the 1-methyl-4-phenyl-1,2,3,6-tetrahydro pyridine (MPTP) story?
IK: There was a turning pointing in my research in the late 1970s. In 1978, I got a call from a neurologist, who said that he had a very peculiar type of patient, a twenty-four year old boy, who appeared to suddenly develop catatonic schizophrenia. His mother found him in his room, lying in bed in his feces, unable to move, and took him to the local hospital. The boy grew up in the shadow of NIH, he was taken to the local Suburban Hospital where, after diagnosing his disorder as typical catatonic schizophrenia, they sent him to a mental hospital. During the next month or two, he became more rigid and they called in a neurologist. The neurologist recognized that the boy appeared to have severe Parkinson’s disease rather than catatonic schizophrenia. When he was treated with L-DOPA, lo and behold, he suddenly loosened up and said, “What have you guys been doing to me?” About four hours after he got the L-DOPA, he was back into his prior state. They had given him ammonia to smell, trying to get a response, but he was unable to move. Later he said, “I just couldn’t carry out any actions”. So, they had started to treat him with L-DOPA. The neurologist asked if I was interested in studying this young man and I said, “He sounds fascinating, bring him to NIH.” After he was admitted, Dr. Davis, a young NIMH psychiatrist found out the patient was abusing drugs and to increase their effectiveness he started to take Demerol with cocaine. He felt this was a marvelous mixture, but had a great deal of difficulty in getting Demerol. He was a bright young man and went to the library where he found there were other compounds like Demerol he could synthesize himself. So, he set up a laboratory in his basement to synthesize a derivative of an isomer of Demerol in which the carbon and the oxygen atoms were reversed on the molecule. He had all the equipment for doing this, and when he tried the compound he synthesized, he thought it was wonderful. He obtained the crystalline compound and was taking about twenty-five milligrams of it at a time. He decided, during one summer, that the amounts he was preparing were too small, so he tried to make a big batch. While preparing a big batch he realized he would lose a lot of the compound if he recrystallized it so he took some of the uncrystallized material. After two doses, he suddenly developed the syndrome for which he was hospitalized. This was the first case of MPTP toxicity. Sandy Markey, who joined our laboratory to head the mass-spectroscopic facility, went to the patient’s house to try to get some of the substance the patient had made, but his mother had cleaned up her son’s laboratory and threw out most of the stuff. The only thing left was one desiccator. That desiccator had a little bit of the powder left in it, that Sandy was able to analyze by mass-spectroscopy and found it contained MPTP along with two other compounds. We thought that we should publish this interesting case, but it was very difficult to get it into print. Finally, we did succeed. About a year or two after this, in California, there was an outbreak of Parkinsonism among drug addicts and Bill Langston traced down the compound that had been used and sent it to Sandy Markey, who found that it was MPTP. At the time we had tried this compound in rats, guinea pigs and rabbits, with little effect. However, it did cause a Parkinsonian syndrome in monkeys, and the MPTP story started a new era in neurology. As you know, DOPA had been suggested as a potential treatment for parkinsonism after dopamine had been discovered by Carlsson in the late 1950s, and, in the early 1960s, Hornykiewicz reported that dopamine was depleted in the brain of Parkinsonian patients. Early attempts at using DOPA in the treatment of Parkinson’s disease failed because of its side effects, but in the late 1960s, George Cotzias was brave enough to give the large doses of DOPA that were needed and proved its efficacy in alleviating the symptoms of Parkinson’s disease. Soon after, the side effects of DOPA, which were largely due to formation of dopamine outside the brain, were found to be preventable by the use of peripheral DOPA decarboxylase inhibitors. Gus Watanabe studied the effects of DOPA after the administration of peripheral decarboxylase inhibitors on the vascular system and later became vice president of Eli Lilly. Tom Chase was also with us as a Fellow in those years. He had been studying the release of compounds from brain slices with electrical stimulation. Later Tom was promoted to Section Chief at the Institute. Subsequently, he became scientific director of the Neurology Institute and, in 1983, I succeeded him.
TB: Any further developments in the MPTP story?
IK: After the MPTP story became well known, I received a telephone call from Denmark about a young chemist who had suddenly developed Parkinson’s disease several years earlier. He had been working in the drug industry and made a large batch of MPTP used as an intermediate in the synthesis and manufacture of some drugs. After re-crystallizing it, he spread it out on paper with his hands to dry it. Although he went home sick that day, he did the same thing a week later. He got sick again, but never returned to work, because he developed severe Parkinsonism. I traveled to Denmark and, after confirming the diagnosis, I asked him to come to NIH with Dr. Pakkenberg, his neurologist. At NIH, this brave patient agreed to be taken off L-DOPA and Dr. Pakkenberg noted the patient’s Parkinson’s disease became as severe as it was ten years earlier at the time he started medication. Ten years of treatment with L-DOPA had not affected the severity of his Parkinson’s disease and he was still responding to his medication without developing dyskinesia that is often a problem after long term treatment.
In parallel, Stan Burns, who had been studying the effects of MPTP in monkeys, developed the first animal model of Parkinson’s disease. This was followed a short while later by Kris Bankiewitz with whom we were able to produce a hemiparkinsonian animal. We could make half the brain Parkinsonian by infusing MPTP in one carotid artery. Like rats that had received 6-hydroxydopamine into one side of the brain, the monkey circled towards the affected side. An affected animal would be able to reach for food with his hand on the opposite side and he was very smart. If you offered him two pieces of food, he wouldn’t reach with two hands. He would take the first piece and put it in his mouth and, then, reach for the second piece with the unaffected hand, whereas before MPTP, they would reach with both hands. It was clear that he wasn’t able to use the hand on the affected side. We could measure the circling effect, as it had been done in rodents made hemi-Parkinsonian with the administration of 6-hydroxydopamine for studying dopaminergic systems. The MPTP treated hemiparkinsonian monkey became a useful tool for studying treatments of Parkinsonism. Kris moved on to study the effects of fetal tissue implants, but US government policy prevented NIH scientists from using fetal human tissue implants. In fact, they frowned on use of fetal tissue in research of any type at that time. Private funds allowed such research outside of NIH. Later the same animal model of Parkinson’s disease was used to study the effects of transfer genes into the brain of animals, whether it be rats or monkeys.
We continued to study stress in our laboratory, another area of research that has been highly productive. The sympathetic nervous system, of course, controls blood pressure. In our studies of patients with orthostatic hypotension, many years ago, Mike Ziegler and I found that patients could be divided into two groups. One group had central nervous system disease, with their sympathetic nervous system essentially intact. They had normal plasma catecholamines at rest, but when they stood up they didn’t have the normal elevation of plasma catecholamines. These patients had multiple system atrophies in their brain. In the other group of patients, the central nervous system was intact, but the sympathetic nerves were almost absent.. This group had peripheral autonomic neuropathy, with absolutely no symptoms of central nervous system disease. Their orthostatic hypotension was associated with abnormally low plasma catecholamines as well as failure to increase the plasma catecholamine levels when standing. These patients have a better prognosis because only their sympathetic nervous system has failed.
We had been studying false transmitters and one of the early false transmitters that interested us was labeled with fluorine, fluorodopamine. I anticipated it would be useful for imaging the sympathetic nervous system. If fluorodopamine is injected intravenously, it is taken up into sympathetic nerves where it can be converted to fluoronorepinephrine. I thought that if 18F labeled fluorodopamine is used, we could detect it in the peripheral sympathetic nervous system and determine its distribution by PET scanning. We saw some patients with Parkinson’s disease who developed orthostatic hypotension that was attributed to the accumulation of dopamine instead of noradrenaline in their sympathetic nerves after being given high doses of L-DOPA. Dave Goldstein showed these patients had degeneration of their peripheral sympathetic nerves, which could be demonstrated using 18F-fluorodopamine and PET scanning of the heart. This was a new observation we published in the New England Journal of Medicine. Later, it was amply confirmed. Dave Goldstein was doing the PET studies in humans; it took about ten years to develop his method from the time that we used fluorodopamine accumulation in tissues to label noradrenergic nerves in animals. The work to develop the method began with “Mike” Chiueh with unlabeled fluorodopamine. Graham Eisenhofer followed it up using 18F-fluorodopamine made from the excess of 18F-fluoro-dopa that was being prepared for imaging dopaminergic neurons in the brain. We did the same experiment we had done many years before, using unilateral sympathetic denervation. In a dog, we removed the superior cervical ganglion on one side, gave 18F-fluoro-dopamine, and used PET imaging to examine effects on the accumulation and retention of the 18F-fluorodopamine. We found that the denervated side didn’t have any radioactivity, whereas the salivary gland on the intact innervated side did. We repeated this experiment in humans and found we could not visualize, with 18F-fluoro-dopamine, sympathetic nerves in the hearts of patients with orthostatic hypotension with primary autonomic failure. The other group of patients with orthostatic hypotension, suffering from multiple system atrophy, appeared to have intact cardiac sympathetic innervations but they couldn’t appropriately activate their sympathetic nervous system because of central nervous system disorder. These patients sometimes have Parkinsonian symptoms. So it appears we have a spectrum of patients who display Parkinsonian features with orthostatic hypotension as their primary symptom. Many patients with Parkinson’s disease have orthostatic hypotension; although originally it was thought that was secondary to L-DOPA, it has become evident it is due to degeneration of the sympathetic nerves in the heart and probably elsewhere. Both internists and neurologists were interested in orthostatic hypotension, so several have come through the lab who’ve been interested in such studies. One of the first was Ron Polinsky, a neurologist who has gone on to a career with drug companies. Another was Dave Goldstein who was to become a leading figure in this area of research. Over the years, I’ve been fortunate in having people with broad expertise join our laboratory. They benefited from the excitement about science that pervades the NIH. To repeat, Leslie Iversen, Jacques Glowinsky, Sol Snyder, Dick Wurtman, and Perry Molinoff all spent their early years in the Laboratory of Clinical Science with Kety, Axelrod and me. In psychiatry, Joe Coyle, Steve Bunney, his brother, Biff Bunney, Mike Ebert, Fred Goodwin and Dennis Murphy, as well as others, began as young post-docs in our laboratory. Dick Weinshilboum, who went to the Mayo Clinic, started his work on the genetics of different enzymes with studies of S-catechol-O-methyltransferase in our laboratory. Dave Dunner, Walter Kaye and Bill Potter also came through the lab. Martha Weinstock, who is chairman of Pharmacology at Hadassah, came to work with us as a visiting scientist. So did Giora Feuerstein, originally from Israel, who stayed here in the pharmaceutical industry, Joe Fisher, who was a surgeon, and is now chairman of the Department of Surgery. He and Ross Baldessarini carried out studies of S-adenosylmethionine to try and explain the deficits in hepatic encephalopathy. Joe, as a surgeon, made portal vein shunts in animals, and studied the effects of this on methylating processes in brain. The people that came through the NIH are a source of pride and we keep track of their progress and accomplishments. They’re “family.”
The NIH has been very good to me and it’s given me a great deal of pleasure over the years to have worked and been taught by such stellar people. I’m grateful to the teaching of people like Heinz Lehmann, who, when I was a medical student at McGill, introduced me to psychiatry, and of Seymour Kety and Julie Axelrod, my supervisors and collaborators, as well as the many young post-docs that came through our Laboratory. I also benefited from several outstanding technicians, like Edna Gordon and Virginia Wiese. These are people who spent thirty or forty years working with me, ensuring the quality of our studies. Edna Gordon, unfortunately, has died. Virginia Wise is retired. She lives near NIH and I see her every once in a while, and some of my secretaries have been with me for twenty years. Virginia has visited scientists that spent time at NIH, like George Hertting in Vienna. They have been friends for over forty years. There is a unique perspective in seeing the carryover from the old pharmacology to the new molecular genetics and looking ahead to see that molecular genetics is not going to be the total answer. It’s going to raise more questions than we can answer and the pendulum is going to swing back towards the intact animal research, the polymorphisms, the genomics, the informatics that we have now. The future direction of the College is going to be fun to follow. Many of the people I’ve mentioned are members of the ACNP and some are foreign corresponding members. There are also those who are in other professional organizations, such as in neurology, anesthesiology, internal medicine, and some who are working in drug companies. All these people contributed immensely to the intellectual environment of NIH and have had a major impact their disciplines in the United States and abroad. It’s been such a great pleasure to work with them, and many friends that I’ve made at ACNP. I am a Past President of ACNP, so I keep going to the Past Presidents luncheons. I have also continued for many years as Treasurer.
TB: When did you become a member of ACNP?
IK: In 1968, Sid Udenfriend and Seymour Kety urged me to join this group. It was very fortunate for me that I did.
TB: When did you become president?
IK: In 1992. The theme that year was to put the “Neuro” back into Neuropsychopharmacology. As president, I tried to do that. It may have been premature, but I think that it is also the theme of the current president, Steve Paul. Steve is another Laboratory of Clinical Science (LCS), alumnus, as was his predecessor at Eli Lilly, Gus Watanabe.
TB: All of them were in the LCS?
IK: Yes, all of them. They’ve grown up. They are analogous to children and grandchildren, if you like. They have expanded beyond the areas we’ve been studying, whether it was depression or Parkinson’s disease or orthostatic hypotension. But there remains some overlap with the main theme being brain function, not only psychiatry, but for neurology, anesthesiology, internal medicine, etc. For example, Alzheimer’s disease is being studied in many Institutes; by the Institute of Aging, by the Neurology Institute, by the NIMH. No one institute can claim it’s the only one to study the brain. The Child Health Institute has a tremendous influence on what’s becoming neuroscience and neuroscience encompasses so many disciplines. This is being recognized more and more widely.
TB: You have been in research in neuroscience since the 1950s, the time of paper-chromatography and the discovery of monoamines in the brain.
IK: Yes. I’ve seen the field develop and it’s been a real privilege to work with the people who had so much impact.
TB: Are you still involved in the training of young researchers?
IK: Yes, I’m still involved. I’m officially retired, but a Scientist Emeritus at NIH, so I have my office and, most importantly, parking space. Although I do not have a lab bench, I still have discussions with post-docs and I’m able to bounce ideas around or have people come to me and use me as a sort of memory. Having the gray hair, I am supposed to remember what happened a long time ago.
TB: Your bibliography reflects the development of neuropsychopharmacology.
IK: Well, partially, yes. I’ve published over seven hundred papers, and that’s largely due to the people who worked in the lab. I had these talented people that came through that really spark your interest and keep your enthusiasm going.
TB: But it was you who trained them.
IK: It’s mutual. They trained me; I trained them. When they come to a new lab, they bring new ways of thinking. They raise problems and the solving of these problems is a joint effort. I like to interact, draw out and be drawn out. It’s never a one-way street.
TB: Your research had a great impact on psychiatry but you’re not a psychiatrist.
IK: Neither was Seymour Kety. The Laboratory of Clinical Science, which he started and I inherited, trying to carry on the tradition, was a founder of biological psychiatry based on what has now become the discipline of neuroscience. Kety’s lab probably trained half the people who were in on the beginning of biological psychiatry in this country. The people we trained continued to train others and we’re now on the second and third generations of people who are trained by them. But, it all stems from Seymour, who got the Lasker Award for his lifetime contributions. He started as a physiologist and developed the first method for measuring cerebral blood flow, for which he became famous. Then Lou Sokoloff, who was initially interested in psychiatry, and Seymour exchanged ideas, and, then changed their courses of research interest. Lou went on to become more of the physiologist and developed the deoxyglucose method that is now used for imaging brain blood flow with PET scanning, whereas Seymour picked up the psychiatry and he’s considered by many to be the father of biological psychiatry in this country.
TB: You have made, in addition to your research, a major contribution by training many of the people who became leaders in the field. It’s a most important contribution.
IK: The most important contributions were made by the people that came through the lab and what they’ve done afterwards. It’s been a pleasure and a source of great satisfaction to me. I’ve worked with melatonin, with MPTP, with false transmitters, with brain imaging and with heart imaging. All of these things are relatively minor compared to the people that have come through and have gone on to do research, both at the clinical and the basic level, and the impact they’ve had on the drug industry, on thinking in the field, on the whole of neuroscience and on neuropharmacology. That is what I consider my greatest source of satisfaction.
TB: You should feel very pleased with the results. Look at the changes that have taken place in the field and not just in the United States.
IK: Yes, Marta Weinstock in Israel, Sedvall in Sweden, Glowinski in Paris, Hertting in Freyburg…they are all over the world. There’s also Corsini, who is now in Pisa. Many of these people became heads of departments, and they send their young people to NIH. There must be over a hundred who’ve come at various times and spent up to two or three years with us.
TB: During the years have you been affiliated with any university?
IK: Just with local universities. I have an appointment as an Adjunct Professor at Georgetown and at the Armed Forces Medical School across the street from NIH and I lectured at four minority colleges when I was president of the ACNP. I also had the good fortune to attend many international meetings and catecholamine conferences that have been held every few years to bring things up to date. I was at all the International Catecholamine Symposia held every few years throughout the world. Dave Goldstein was President of the one held in California in 1996. It included a wide variety of interests, from very basic neuroscience to the clinical studies of cardiovascular disease, pain, neurologic disorders, psychiatry and everything in between.
TB: Didn’t the people who worked at the LCS organize a gathering at one of these conferences and have a Festschrift in your honor?
IK: That was the Eighth International Catecholamine Symposium at the Asilimar Conference Center. Many of my old post-docs contributed to the Festschrift in my honor. It brought back old memories such as the work I did with Sophia Zukowska, who is professor now at Georgetown. She first came to our laboratory about 25 years ago. I first met her in Bratislava at a meeting on Stress organized by Richard Kvetnansky. When she presented her work at the meeting, she expressed interest in coming to NIH. She stayed with us for three or four years. In fact, she and Dave Goldstein did the work on monoamine uptake. When Dave came to me with an idea for trying to find out what the concentration of noradrenaline is at the synapse he suggested the administration of tyramine. For a number of reasons that couldn’t be done, but that started me thinking. We compared the effects on blood pressure of stimulating the spinal cord of a pithed rat with the effects of infused noradrenaline. This was done by comparing the pressor response curves to the plasma noradrenaline levels in relation to blood pressure. We found that you have to raise plasma catecholamines to much higher levels with exogenous norepinephrine that the levels in plasma attained with an equivalent pressure response elicited by stimulating the spinal sympathetic outflow. The reason for this is that there is uptake in between the plasma and the synapse. But the reuptake is the same whether the norepinephrine is coming out or going in. So the concentration that you obtain during stimulating the nerve is less than is at the synapse, but when you give it exogenously the concentration in the blood is higher than at the synapse; the synaptic concentration is in between. So, by comparing the log of the plasma catecholamine-blood pressure response curves, the concentration in the synapse is halfway between; the logarithmic mean of the concentrations at any given pressor level. To prove that this was the case, we gave desipramine and the curves moved closer together because the uptake was blocked. The exogenous catecholamine gets more effective the less the endogenous NE is removed and the curves move together towards the synaptic concentration. That’s another story that has been applied clinically to study patients with orthostatic hypotension.
TB: Your research embraced a wide range of different areas. You were involved first with research in schizophrenia, or even before with research on making a mirror. That was probably crucial.
IK: It may well have been. I sometimes think my father was very wise in the way he stimulated me.
TB: Obviously you are a dedicated teacher.
IK: Everything is taught earlier now than before. When my son went to high school, they also told him how to test for aldehyde by making a mirror, but they also told him to clean the glass. I guess I’ve told this story so often that everybody now knows you had to clean the glassware to get a good mirror. He was thrilled because he had reproduced what I had done at about his age.
TB: Is there anything we left out and you would like to add?
IK: No. It’s been such a privilege to be a member of this college, to be part of the NIH and to have lived during this marvelous transition. In the future even greater contributions will be coming from molecular biology to provide a better understanding of brain function, that will lead to better treatments of neurological and psychiatric disorders. Thank you very much.
TB: Thank you very much for sharing this information with us, and, for contributing to the training of many of the participants at this meeting.
IK: Well, it was just being there at the right time and it was, as I said, a privilege and a pleasure.
TB: You were the right man at the right place at the right time.
IK: Thank you.
May 10, 2018