An Interview about Julius Axelrod: Rejected from every medical school- yet wins the Nobel Prize.

Aug 13, 2019 by

Julius Axelrod

Michael F. Shaughnessy –

1) Julius Axelrod – a pharmacologist, Nobel Prize winner and investigator- has researched more chemicals than anyone I can personally think of. Where was he born, and where did he get his foundational education?

Dr. Julius Axelrod, an American biomedical scientist and Nobel Laureate, was born on the 30th day of May in the year 1912 in New York City, New York, in the U.S. His parents were Polish immigrants of Jewish origin who had met after each of their respective arrivals to the U.S. His father Isadore and mother Molly lived in tenement housing, on East Houston Street, in the lower East Side district of Manhattan. The Axelrod family lived in near poverty. Isadore Axelrod was a grocer and basket-maker who sold groceries from a horse-drawn wagon in the streets of Manhattan.

Young Julius (“Julie”) attended public elementary school called PS22 (public school number 22), in Manhattan. It was an historical school that had been established prior to the onset of the Civil War and for which another student of prestige had been a pupil, namely, renowned physicist Dr. Isidor Isaac Rabi, a 1944 Nobel Laureate who helped build the first atomic bomb.

His high school, called Seward Park, located in New York City, had been his second choice, because his family could not afford the more prestigious Stuyvesant high school. While Seward Park High was a less than desirable educational institution, it had nonetheless certain other famous alumni, such as Tony Curtis, and Walter Matthau. Dr. Axelrod was later to remark that his true education during his high school years was on the account of having spent a great deal of time reading books voraciously on his own at the Hamilton Fish Park Library, which had been located nearby to his tenement home. He graduated from high school, in 1929, at the start of the Great Depression.

It had been during this time period that Axelrod had decided to become a physician.

Thus, after his high school graduation from Seward, Axelrod entered college at New York University, in 1929, focusing his studies on pre-medicine courses. However, his funding for university had been depleted after only one year of course study. Consequently, in 1930, he moved to City College of New York, where there had been no charges incurred to students for their tuition costs. Nevertheless, he still had to work as a laboratory assistant in the Bacteriology department at the New York University Medical School while also a student attending university, in order to have spending money for living expenses.

While in attendance at CCNY, Julius Axelrod focused his major studies primarily on pre-medicine related disciplines, such as Chemistry and Biology. His top academic performances, however, had been in other courses, such as history, philosophy and literature. This particular institution was located relatively further from his home, and it required that he commute back and forth by subway. The daily commute, a two-hour round trip, provided an opportunity for him to study his course materials on a moving subway during the transits. He later pointed out that this studying experience, in noisy city trains, had solidified his proficiencies for pure concentration during the learning process. He took his undergraduate degree, his Bachelors of Science, a B.S., in 1933, from City College. In addition to Dr. Axelrod, the institution, CCNY, is known for having graduated remarkable alumni, such luminaries as Nobel Laureates Drs. Robert Hofstadter (1961) and Leon Lederman (1988).

As a new university graduate, he had applied to a small number of medical schools, and every single one of them had rejected him outright. It had been apparent that these rejections had much to do with anti-Semitism that had been a conspicuous characteristic of the U.S. during that era, especially during the midst of the Great Depression years. Thus, Axelrod took on employment in a laboratory setting until, that is, the sources of funding had been exhausted, in 1935. Thus, he took on another job as a laboratory assistant at the New York Health Department, working as a chemist in their Laboratory of Industrial Hygiene, where he stayed until 1946. His duties were to assess the various methods for the measurements of vitamins contained within food and drink samples.

While working in the day at the New York laboratory during the Great Depression and World War II, Julius Axelrod had also enrolled in night school, taking graduate courses, in the master’s program, at New York University.

His M.S. thesis project at the University was based on work in the biochemical field of enzymology (esterases) and in the biology of tumors. It was during this time that an unfortunate laboratory accident resulted in the loss of his left eye, and he had to wear a patch over it for the rest of his life. In 1942, he took his M.S. graduate degree.

The route to the Ph.D. for Axelrod took more than additional 10 years. In the meantime, he was employed as a research associate at the Goldwater Memorial Hospital until 1949. Then, he worked as a chemist and later senior chemist at the National Heart Institute until 1953. Next, he moved to the Department of Health, where he became section chief of the Pharmacology division, until 1955. During this time, he had been continually encouraged by many of his colleagues to pursue the doctoral degree.

It had been at the National Heart Institute where he had learned that without a Ph.D. in hand, he could not proceed beyond his then current rank, and it was at that time that he decided to pursue the doctorate. Thus, he enrolled in graduate school at George Washington University, in Washington, D.C., working under Dr. Paul K. Smith, who became his graduate thesis advisor. Axelrod’s thesis project dealt with his previous work involved with metabolism of so-called sympathomimetic amine compounds like ephedrine and amphetamines. For this he had studied rabbit liver slices and delineated the various factors needed to metabolize the drugs. He had determined that the metabolic pathways encompassed various biochemical steps, like conjugation, deamination, demethylation, and hydroxylation.

Axelrod also was to locate the cellular location of the amphetamine-metabolizing enzymes, a preparation known as microsomes, now known to consist of parts of endoplasmic reticulum material laced with bound ribosomes, which are protein-making machineries. Later investigators were to confirm Axelrod’s work, referring to these drug metabolic systems as cytochrome-P450 monooxygenases. Submitting these publications to the graduate school and taking an additional year of graduate-level university courses, were all that were necessary for 42-year old Julius Axelrod to take his Ph.D. degree, from George Washington University, in 1955.

2)  Let’s talk glands first- tell us about his work on the pineal gland.

The pineal gland is an organ that is well-stocked with nerves of the so-called sympathetic nervous system. Dr. Axelrod’s interest in the pineal gland was first stoked, in 1958, by a scientific paper he read that had been written by Dr. Aaron Lerner regarding his isolation of a compound called 5-methoxy-N-acetyltryptamine, known now as melatonin, from the pineal glands of cows. Dr. Axelrod’s interest in the pineal gland work stemmed from fact that the melatonin structure was said to contain a serotonin platform with a methoxy group attached to it. The serotonin molecule was thought to be involved in generating psychosis in humans because of its structural similarity to lysergic acid diethylamide (LSD). The methoxy group was reminiscent of the metabolic work he had performed with the catecholamines called epinephrine and norepinephrine. Thus, Dr. Axelrod started work with the pineal gland in an effort to work out the metabolism of melatonin.

Another project dealing with study of the pineal gland was centered around assessing the effects of light on the biochemical activities of the organ. Thus, Dr. Axelrod and a medical student, Richard Wurtman, housed laboratory rats either in total darkness or in total light, instead of cycling the dark-light cycles in a conventional manner. The rats living in total darkness showed changes in the levels of certain enzymes that make melatonin—the melatonin-making enzymes were elevated, compared to the rats living in total light.

When certain nerves, called the superior cervical ganglia, which are connected to the pineal gland and innervate it, were surgically removed by Axelrod, the result was stunning. Cutting off the pineal gland’s electrical connection had abolished the effects of total darkness and the enzyme levels! It turned out to be a major breakthrough as the experiment demonstrated that the melatonin enzymes and its connection to light in the pineal gland was under the neuro-control of the sympathetic nervous system, as the phenomenon involved the superior cervical ganglia.

Another investigative focus by Dr. Axelrod upon the pineal gland was placed on the neurotransmitter called serotonin because it had served as a starting point for the biosynthesis of the melatonin. Dr. Axelrod and one of his postdoctoral fellows, Dr. Solomon Snyder, who would later become a world renowned neuroscientist in his own right, examined the relationship between light exposure and the levels of serotonin and melatonin. They discovered that in rats exposed to total darkness for a prolonged period of time the up and down rhythm of serotonin levels was unaffected, strongly suggesting an internal clock mechanism was responsible.

However, when Drs. Snyder and Axelrod housed the laboratory rats in total light for long periods, the circadian rhythm of serotonin (i.e., its alternating high and low levels) was gone! The biological clock was stopped in its tracks with continuous light exposure!

A follow up experiment conducted by Drs. Wurtman and Axelrod involved the surgical removal of the ganglia that innervated the pineal gland, a so-called ganglionectomy, and it resulted in the abolishment of the newly discovered serotonin circadian rhythm. When the control of the superior cervical ganglia was removed from the pineal gland, the cycling serotonin levels were observed to be lost. Thus, the work strongly suggested that the biological clock and the effects upon it by light exposure was located somewhere within the brain that was innervating the pineal gland. Based on this work Dr. Axelrod concluded that the pineal gland played a role as a sort of neuroendocrine transducer, where light signals were transformed into regulatory mechanisms that controlled levels of hormones. Furthermore, he concluded that the brain via its noradrenergic neurons were permitting the transducer functions.

3) Often some evenings, I take some melatonin to help me sleep. How was Axelrod involved in this?

In the early 1960s, Dr. Axelrod started work that was devoted to learning how melatonin was made in the pineal gland. In 1961, Dr. Axelrod’s melatonin work revolved around studying the effects of the compound on pineal gland activities. For instance, he and Wurtman discovered that the weights of rat ovaries were decreased by exposure to the melatonin. Next, Dr. Axelrod working with Wurtman and collaborator Dr. Harvey Shein, from McLean Hospital, tested the hypothesis that the pineal gland tissue that was cultured from rats could convert the amino acid tryptophan into the melatonin. The data strongly favored the hypothesis that tryptophan was a precursor for the biosynthesis of melatonin.

Next, they showed that the neurotransmitter called norepinephrine somehow regulated this conversion of tryptophan into melatonin in the pineal gland. Additional work by Drs. Wurtman and Shein demonstrated that the so-called β-adrenergic receptor was the conduit for its activation by noradrenaline.

Dr. Axelrod started working with Dr. Herbert Weissbach, and the collaborators set out to delineate the biosynthetic pathway for melatonin. They injected cow pineal gland extracts with a radioactively labeled precursor called SAM, for S-adenosyl-l-methionine, which was a good starting molecule that could introduce methyl groups into the biosynthetic pathway, plus a metabolite of serotonin called N-acetylserotonin. They found that the pineal gland extract produced the desired melatonin.

In another investigative push, they set out to purify the enzymes that responsible for making the melatonin in their pineal gland extracts. In so doing they discovered an enzyme they named as hydroxyindole-O-methyltransferase (HIOMT) now called acetyl-serotonin-O-methyltransferase (ASMT) which converts N-acetyl-serotonin to melatonin. They also discovered and purified the enzyme called serotonin-N-acetyltransferase (AANAT), which converts serotonin to N-acetyl-serotonin. Lastly, they proposed the entire melatonin biosynthetic pathway of biochemicals, starting with the amino acid tryptophan. To this day, the pathway is presented in textbooks of neuroscience and biochemistry.

The works of Shein and Wurtman with the β-adrenergic receptor system sparked further interest by Dr. Axelrod into examining the relationship between the receptor regulation and its hormone. In 1970, Dr. Axelrod studied levels of the serotonin N-acetyltransferase enzyme using agents that blocked the β-adrenergic system. Such blocking agents included reserpine, protein synthesis inhibitors, and surgical removal of the ganglia—the so-called ganglionectomy. The result was that the β-adrenergic blockers also blocked the increase in the enzyme normally observed after exposure to a continuous dark environment. These results suggested that the noradrenaline which was secreted by elements of the sympathetic nervous system, actually activated the β-adrenergic receptor, thus causing the production of the enzymes that are involved in the synthesis of the melatonin.

4) We often joke about Thanksgiving- and sleeping after eating turkey- which contains tryptophan- how do these things relate to each other and the pineal gland?

Humans cannot make tryptophan on their own. We need this molecule in order to make our own proteins. We lack, however, the basic biochemical machinery for its synthesis. Therefore, we must intake tryptophan in our basic diet. There are other potential effects of tryptophan.

The basic idea is that because the amino acid tryptophan is a biochemical precursor to the synthesis of melatonin, a compound that is associated with the sleep and wake cycles, then dietary intake of tryptophan during meals should make an effective sleep-aid for insomniacs. The melatonin is produced by the pineal gland, and it uses tryptophan as a starting point to make it. The tryptophan is one of 20 amino acids that are found in protein, and meats like turkey, beef, and pork all have a high protein content. Thus, the content of tryptophan would be correspondingly higher as one increases the intake of relatively high protein diets.

The tryptophan-sleep connotation is problematic, however, because a heavy meal, such as that accomplished with enjoying a Thanksgiving dinner, turkey with all of the trimmings, can also direct an enhanced blood flow to the gut in order to handle the heavy meal, for digestion purposes. It is believed that the altered blood flow can make a person sleepy, as well. Another putative problem with tryptophan is that in addition to being a metabolite, it is also a neurotransmitter precursor, serving as a foundation point for the synthesis of serotonin. So, large doses of tryptophan may result in unintended side effects after it partakes in downstream biochemical and neurological processes.

5) Catecholamines- what exactly are they and how was Axelrod involved?

The catecholamines are a group of hormones called neurotransmitters. The three most-studied of the catecholamines include epinephrine (used to be called adrenaline), norepinephrine (was noradrenaline) and dopamine. The main source of the catecholamines is the amino acid called tyrosine. In the catecholamine biosynthetic process, tyrosine is converted to L-DOPA, which is then converted to dopamine, which then goes to norepinephrine and then to epinephrine as the endpoint of the pathway. All three neurotransmitters act on the α- and β-adrenergic receptors to various extents.

While the body of work dealing with catecholamine studies by this investigator is extensive, Dr. Axelrod’s prime involvement with these biochemicals has to do with their disappearing acts after they are released by neurons and perform their various neuronal functions. Dr. Axelrod discovered that rather than their degradation, they were merely taken back by the neurons, a process now known as neurotransmitter reuptake. It was to become his major Nobel Prize winning work.

Epinephrine has also been colloquially referred to the “flight or fight” hormone. This is due to the fact that during times of acute stress, epinephrine is secreted by one of the body’s organs, such as the adrenal medulla. The secreted epinephrine mediates a series of physiological consequences, such as a faster than normal rate of heart beating and the mobilization of the body’s energy storage forms in order to generate the increased need for energy during the stress by breaking down the body’s stores of glycogen and lipids.

Norepinephrine works on the body’s noradrenergic system. This neurotransmitter is secreted from the so-called postganglionic synapses of the sympathetic portion of the overall autonomic nervous system. Norepinephrine serves a suppression type of role in the sympathetic system, resulting in a corresponding suppression in the activities of the gut and in the reduced flow of blood.

Dopamine is a rather potent neurotransmitter that regulates various functions of the brain. This neurotransmitter is produced by so-called dopaminergic neurons that reside in the inside of the brain. The absence of dopamine in patients with Parkinson disease results in severely debilitating neurological consequences. Film actor Michael J. Fox has the aliment and has been a longtime advocate for investigative studies. While there is no effective cure for Parkinson disease, certain treatments can alleviate the symptoms. One such treatment includes L-DOPA, which is a precursor along the synthetic pathway for norepinephrine and epinephrine. The motion picture film Awakenings features the accidental discovery by Dr. Oliver Sacks, portrayed by actor Robin Williams as the fictional character Dr. Malcom Sayer, of the benefits of L-DOPA, for the treatment a catatonic disease called encephalitis lethargica, sometimes called “sleeping illness” or “sleepy sickness.”

6) Some of his work led to what we abbreviate as SSRI- Selective Serotonin Reuptake Inhibitors (most notably Prozac) which apparently stops the reuptake of the neurotransmitter serotonin.   (Great book for future reading- Listening to Prozac- a medication still used today) 

Dr. Axelrod’s Nobel Prize winning work involved discovering the reuptake mechanism of neurotransmitters, such as serotonin. This molecule is a precursor to the synthesis of melatonin in the pineal gland. Dr. Axelrod’s earlier work in the area started with elucidating the synthesis pathway for melatonin, starting with serotonin as a precursor metabolite. His laboratory also learned that in the pineal gland the serotonin concentrations varied depending on the light / dark cycle, observing elevated serotonin concentrations during the daytime and lowered concentrations during the nighttime. The work was profound as it pointed to the presence of an internal cellular molecular clock system, a so-called circadian rhythm for alternating the levels of serotonin!

Known also in scientific circles as 5-hydroxytryptamine (5-HT), serotonin has long been thought to be involved in certain aspects of depression and schizophrenia. There are two main hypotheses for the involvement of serotonin with depression. The first one states that low serotonin concentrations lead to depression, and the other hypothesis maintains that depression itself leads to lowered serotonin concentrations.

Similarly, there are two seemingly disparate hypotheses for serotonin’s involvement in schizophrenia. The first hypothesis holds that, in post-mortem studies, patients with schizophrenia exhibit higher than normal concentrations of specific integral membrane proteins called 5-HT1A receptors that bind to the serotonin, in the frontal cortex of the brain. The other hypothesis states that in patients with schizophrenia the serotonin binds less well to their 5-HT1A receptors that reside in another portion of the brain called the amygdala.

Regardless of the cellular mechanism for the role that serotonin plays in depression or schizophrenia, the reuptake system for the re-use of serotonin appears to be a prime target for anti-depression and anti-psychotic drugs. Hence, the SSRI drugs that you mentioned are of great importance. Let’s briefly discuss the serotonin reuptake system.

First, tryptophan is metabolized to synthesize serotonin. The newly synthesized serotonin is stored in vesicles, in pre-synaptic neurons, waiting for their release into the synapse, the space between adjoining neurons. The newly released serotonin in the synapse then binds to the post-synaptic receptor and mediates its normal electrophysiological effect, depending on the receptor’s location in the brain. After serotonin performs its post-synaptic function, it is taken back into the pre-synaptic neuron that had released it in the first place. The serotonin uptake (re-uptake) is performed by a dedicated serotonin transporter protein that resides in the membrane of the pre-synaptic neuron. The re-acquired serotonin can then be stored in pre-synaptic vesicles, until they are needed again.

The SSRIs interfere with this serotonin reuptake system, often at the level of the pre-synaptic serotonin transporter, blocking the transporter, and thus leading to relatively higher concentrations of serotonin within the synapse, the so-called synaptic cleft between two given associated neurons. The increased serotonin levels now permit its function go ahead. The SSRIs, such as Prozac (chemical name fluoxetine) and others are increasingly used by clinicians to treat a variety of conditions, such as depression, psychosis, and other disorders, like anxiety.

7) Epinephrine and norepinephrine (Also known as adrenaline and noradrenaline) are really very foundational drugs/chemicals) (and epi-pens are widely used today. Can you explain his work in these areas?

As I mentioned earlier, Dr. Axelrod was awarded the Nobel Prize for his work in determining that catecholamine neurotransmitters like epinephrine and norepinephrine are taken back (“re-acquired”) by neurons after participating in conducting electrical nerve transmission. These neurotransmitters that are regained back by neurons are then saved as inactive forms within these nerve cells while residing inside tiny intracellular packets called vesicles. The re-captured neurotransmitters can then be re-used for the next nerve conduction process.

His first experiment in this area involved the metabolism of epinephrine. Dr. Axelrod had been interested in this biochemical because of its purported involvement in schizophrenia. Two prominent psychiatrists had hypothesized that epinephrine was broken down into another compound called adrenochrome, which then produced certain schizophrenia-like behavioral and hallucinogenic effects, after exposure to the adrenochrome.

Thus, Dr. Axelrod was interested in understanding the events that occurred after epinephrine performed its nerve transmission duties. He wanted to know whether epinephrine was degraded. Its effects, after all, were known to disappear almost as quickly as it appeared, after conducting its neuronal transmission. We now know he discovered that epinephrine was re-taken back by the pre-synaptic neuron, a Nobel-worthy finding, but Dr. Axelrod first considered whether and how it was degraded metabolically.

Thus, Dr. Axelrod first examined whether epinephrine was deaminated, a process in which an amino chemical group is removed from (in this case) an epinephrine structure. So, he measured epinephrine’s effects in the presence of inhibitors of enzymes known to deaminate compounds, such as the monoamine oxidases, a family of related enzymes. He and his laboratory colleagues found that the epinephrine still disappeared even in the presence of monoamine oxidase inhibitors. It was a major scientific finding.

Dr. Axelrod considered next whether other oxidative enzymes metabolized epinephrine to make it disappear after mediating its neuronal effects. These enzymes did not, either, and it was deemed another disappointing dead end. Next, he and co-workers examined whether epinephrine was methylated. Exploring this methylation avenue further, they failed to find how epinephrine was inactivated but did manage to elucidate new enzymes that metabolized it, calling one of these enzymes catechol-O-methyltransferase. After publishing these works, in 1957, Dr. Axelrod considered himself a bona fide connoisseur of neurochemistry, making contributions to the biochemistry of catecholamine catabolism.

He next turned to the question of epinephrine’s deactivation, rather than its metabolic degradation. Some of his findings pointed to an inactivation mechanism for epinephrine because inhibitors of catabolism did not produce the desired increase in its effects. The inactivation proceeded even in the presence of degradation inhibitors. These next efforts led to the Nobel.

At about this time he was fortunate enough to get his hands on newly radioactively-labeled epinephrine! Using epinephrine labeled with tritium (a radioactive form of the hydrogen atom, referred to commonly as 3H), Dr. Axelrod could find out what happened to it once it finished its neuro-electrical duties. A short time later, he was able to get ahold of tritium-labeled norepinephrine, as well. He examined various tissues in cats to learn the fates of the catecholamines 3H-epinephrine and 3H-norepinephrine.

To his shock, Dr. Axelrod found that after injecting cats with the 3H-epinephrine and 3H-norepinephrine, the labeled catecholamines remained stable within their tissues long after the catecholamines conducted their neurophysiological effects! Interestingly, Dr. Axelrod and his laboratory personnel noticed that the tritium-labeled epinephrine was especially stable in cat tissues which were innervated by nerves belonging to the sympathetic nervous system. Next, they formulated their hypothesis: catecholamines could be re-taken back up by pre-synaptic neurons and then stored there. They referred to this catecholamine fate as uptake and retention of epinephrine and norepinephrine by neurons. Then, they tested their Nobel Prize hypothesis.

The critical Nobel Prize experiment involved cat surgeries. Dr. Axelrod and co-workers surgically removed certain nerves called superior cervical ganglia from one side of the cat eye muscles and of the saliva-producing gland, leaving the other side intact, complete with their innervations uncut. Then, they injected the tritium-labeled catecholamine, the 3H-norepinephrine, into the surgically-treated cats, and the results were dramatic!

The norepinephrine appeared to be re-absorbed and stored only on the uncut (intact) innervated sides of the eye muscles and salivary glands. However, the norepinephrine seemed to be almost completely missing from the denervated side, with its nerve supply having been surgically removed! These data were interpreted to mean that sympathetic neurons selectively took up and kept catecholamines, rather than degraded and re-made them biochemically, a process of which would use up so much energy to perform repeatedly.

In a follow-up experiment, Dr. Axelrod found that when the intact nerves were stimulated to conduct an electrical pulse, the labeled catecholamine 3H-norepinephrine was taken up by neurons and secreted into the synapse when turned on. These data were then interpreted to mean that after working at the post-synaptic neuron, with its function complete, the neurotransmitter was then quickly inactivated by being re-absorbed back into the pre-synaptic sympathetic neuron, rather than destroyed and re-made later on. It was a fundamental breakthrough in the functioning of neurotransmitters and led directly to the Nobel.

8) Codeine, morphine and methamphetamine (all end with the “ine” ending). But what are their uses, and how are they alike, yet different? And how was Axelrod involved?

Codeine, known also by its chemical name 3-methoxymorphine, is an opiate chemical that is used for the medical treatment of pain. Morphine is another member of the opiate chemicals (known in modern times colloquially as opioids) that’s been frequently used for pain on a historical basis. Codeine and morphine have similar chemical structures, differing primarily by only a few atoms, with codeine harboring a methoxide (CH3O) substituent at one location on the basic structure while morphine harbors only a simple hydroxide (OH) moiety at the same position. Methamphetamine, known also as N-methylamphetamine, is structurally dissimilar to either codeine or morphine, but is on its own is a potent neurological stimulant of the central nervous system and an illicit drug to which many people are highly addicted.

Each of these drugs seem to have various levels of addictive qualities. In 1956, Dr. Axelrod provided an early indication for why this seemed to be the case. After having discovered, in 1953, how these drugs were metabolized biochemically (see below), he made an astute observation. He found that repeated exposure of these narcotic drugs, such as morphine, to laboratory animals resulted in a certain level of tolerance to the agents and, importantly, to a decreased ability to be degraded, as well. That is, after repeated administration to these drugs, they worked less well (needing more drug to do the same work) and showed less metabolic degradation each time. Furthermore, opiate antagonists prevented the tolerance induction but also seemed to inhibit the degradative enzymes.

Dr. Axelrod astutely provided an explanation. He hypothesized that the enzymes had morphine binding sites that were similar in structure to the morphine receptor sites. Thus, with morphine continually binding to both enzymes and receptors, it resulted in the inactivation of both proteins. That is, the degrading enzymes and morphine receptors became inactive, requiring more of the drug to mediate the same level of effects as before.

Dr. Axelrod’s first foray into studying these compounds, however, started with his interest, in 1953, in certain so-called sympathomimetic amines, such as ephedrine and amphetamine, which had been previously shown to stimulate neurons belonging to the sympathetic nervous system. He then closely examined their various metabolic pathways. For instance, Dr. Axelrod provided some evidence that ephedrine could be degraded metabolically by two distinctive pathways, one by demethylation (removal of methyl groups) and the other by hydroxylation (addition of hydroxyl groups). He then demonstrated that difference species of laboratory animals had favored one or the other of these two pathways.

Next, Dr. Axelrod closely studied methylamphetamine and amphetamine. He demonstrated their various metabolic fates when acted upon by the body’s liver enzymes. Biochemically, Dr. Axelrod showed that these two agents possessed an assortment of metabolic fates, depending on whether the drugs were deaminated (amine removal), demethylated, hydroxylated, or conjugated together. In much the same way as before, Dr. Axelrod then showed that various animal species treated these compounds differently, depending on the types of metabolic enzymes present in the animals. He exposed laboratory rabbits to amphetamine. What he found surprised him. The drug simply disappeared with nary a trace.

Not being a biochemist who knew about enzymes at the time, in the early 1950s, Dr. Axelrod nevertheless became interested in how the amphetamine was degraded. He inquired of his colleagues who were biochemists, such as Dr. Gordon Tomkins, who told Dr. Axelrod that all he needed to study amphetamine breakdown was a laboratory technique for detection of the drug, some liver from a laboratory animal, and a razor blade!

So, Dr. Axelrod took rabbit livers out of the animals, sliced the extracted livers with his razor blade, incubated the liver slices in buffers, added the drug, and measured levels of the amphetamine in order to determine its fate. He then saw the amphetamine simply disappear!

Next, he blended the animal livers, making a so-called rabbit liver homogenate and repeated his amphetamine addition to the newly blended animal organ. This time the drug failed to disappear! In order to make the liver homogenate degrade the amphetamine, Dr. Axelrod needed to add back certain biochemicals, such as enzymatic co-factors like ATP, NAD+ and NADP. Quickly becoming a competent biochemist, Dr. Axelrod decided to find out what cellular component was responsible for making the amphetamine disappear. Thus he then broke up the liver into certain cellular parts, like the nuclei, ribosomes attached to membranes of endoplasmic reticulum (called microsomes), mitochondria, and the cytoplasm (called cytosol). Disappointingly, he found that none of the cellular parts acted upon the amphetamine, even if he added back all of the various enzymatic co-factors. However, if Dr. Axelrod combined each of the previously separated microsomes with the purified cytosol and added the necessary co-factors, the amphetamine readily conducted its disappearing act.

Follow-up experiments by Dr. Axelrod and colleagues showed that amphetamine’s vanishing act involved a deamination biochemical step, making phenylacetone and ammonia end-products in its wake. At about this same time, he discovered that ephedrine was demethylated to make norephedrine, plus formaldehyde! Since the enzymes responsible for these biochemical degradations were localized to the endoplasmic reticulum-ribosome mixture, the microsomal fraction of liver cells, many other investigators entered the burgeoning field started by Dr. Axelrod. The liver microsome enzymes are nowadays referred to as the cytochrome-P450 monooxygenases. This particular liver biochemical system is important for metabolizing a great variety of chemical and biochemical agents, some of which may be potentially toxic to humans, and to this day the system still represents an important biochemical field in the biomedical sciences.

9) Certain mornings, I stop at McDonalds to chat with my good friends and colleagues in the Biology department- but Axelrod also looked at the mechanisms and effects of caffeine. Tough question- but can you tell us about caffeine, its impact, addictive qualities, stimulant qualities and Axelrod’s work in this realm?

I fondly recall your frequent visits with us scientists during our morning coffee breaks! I believe our visits over a cup of coffee ultimately led to our collaborations on these wonderful books. It has been great fun working together on these projects, and it was our common interest in consuming caffeine that started it all!

Caffeine is a fantastically popular substance, being consumed since ancient times in staggeringly large amounts world-wide in the form of coffee, cocoa, and certain teas. It is a powerful stimulant of the central nervous system. Its historical popularity is evident in playing important roles in commerce, economies, human migration, politics, and even in cultural and societal norms. In modern times, many of us enjoy the occasional visits to bookstores with their adjacent coffee houses.

While caffeine, an alkaloid biochemical agent, is derived from over several dozen different species of plants, the most common source seems to be the beans of cocoa trees from the Coffea genus, such as Coffea arabica and Coffea canephora.

Biochemically speaking, the structural nature of caffeine, a member of the methylxanthine group of chemicals, is similar to those of theobromine (found in cocoa), theophylline (found in teas) and adenosine (found in DNA). The adenosine molecule, a nitrogenous base, is also a neuromodulator, serving to reduce the numbers of spontaneous neuronal firings and thus to decrease the release of other neurotransmitters. The overall effect of the adenosine is to permit us to sleep.

Caffeine, on the other hand, is said to counteract the sleepy effect of the adenosine. Thus, instead of waking us up, caffeine actually hinders the normal sleep-inducing role of the adenosine. The mechanism of this anti-sleep effect by caffeine is by way of its binding to the adenosine receptor, occupying the adenosine-binding site and preventing the adenosine from binding it its dedicated receptor. The caffeine prevents the adenosine from binding to its receptor. The physiological effects of adenosine receptor blocking by the occupying caffeine molecules are to induce the so-called “caffeine buzz.” Among these effects include increased heartrate, increased blood vessel constriction, and in permitting muscles to flex more readily.

For medical purposes, caffeine has been used traditionally to treat migraine headaches, asthma, and low blood pressure. It has also been used as a diuretic. Many scientific studies attest to its relatively safe effects and in certain cases being a generally healthy substance.

However, caffeine can be toxic, if consumed in staggeringly large concentrations. For instance, in a regular cup of coffee, on average, the amount of caffeine can range between 80 and 200 milligrams per cup, depending on how the coffee is brewed. A lethal dose of caffeine, however, is about 10 grams. Thus, in order for a human being to consume a lethal dose of caffeine by drinking cups of regular coffee, over 50 cups of the strong coffee (or over 120 cups of weaker coffee) must consumed in one sitting! That’s obviously a tremendously impossible number of coffee cups that need consuming in order to be deadly!

The biochemical effects of caffeine are extensive. In addition to serving an important role in antagonizing the adenosine receptor, caffeine has also the ability to regulate the cellular mobilization of calcium stores inside muscles. The result of this is to lower the amount of signals needed to induce muscle contraction and to prolong these muscular contractions. Caffeine also inhibits the degradative activities of so-called cyclic nucleotide phosphodiesterase enzymes, resulting in increases in the concentrations of cAMP (adenosine 3’,5’-cyclic monophosphate) and in permitting increased effects of the catecholamines.

Relatively high concentrations of caffeine are reported to affect the concentrations of certain neurotransmitters. For instance, caffeine increases the biosynthesis and breakdown of both noradrenaline and acetylcholine. However, caffeine is known to increase the cellular concentrations of dopamine and serotonin in the brain. Caffeine can also induce the amounts of glutamine, an amino acid with an inhibitory neurotransmitter role.

Like other neuromodulatory agents, caffeine addiction involves certain withdrawal symptoms, such as headache, tiredness, and drowsiness. In cases where an individual has consumed an excessive amount of caffeine, additional withdrawal symptoms may include nausea and vomiting. Caffeine that is consumed continually or chronically is said to increase the numbers of the adenosine receptors and in decreases in the numbers of both cholinergic- and β-adrenergic receptors in the brain. While caffeine is also reported to bind to so-called benzodiazepine receptors, its effects on this system is less clear with respect to caffeine tolerance and addiction (dependence).

Dr. Axelrod’s work with caffeine involved his interest in the physiological and biochemical dispositions of the agent within human beings. First, he and co-workers developed a technique for measuring caffeine levels in blood plasma. With this method at hand, they then determined the tissue distribution of caffeine, publishing the results in 1953. The work led to his interest in studying amine-containing compounds.

10) We hear it all the time- Tylenol and acetaminophen- how was Axelrod involved and what are the issues here?

Dr. Axelrod’s efforts in studying acetaminophen (the active principle in Tylenol) was sparked after his discussions on the topic with his good friend and collaborator Dr. Bernard Brodie, known affectionately as “Steve” by Dr. Axelrod. The topic was relevant in the sense that it was Dr. Axelrod’s first serious experience in conducting research after having earned his M.S. degree. He writes in his memoir that it was this topic that enhanced his interest in biomedical research as a career.

The inspiring topic involved the question about why or how the analgesics called acetanilide and phenacetin produced a serious consequence in patients in which they exhibited met-hemoglobinemia. It was a condition characterized by the production of an abnormal concentration of a hemoglobin metabolite called met-hemoglobin, hence the name met-hemoglobinemia. They set out to determine whether and how acetanilide might result in the abnormal condition.

Their first hypothesis to explain the serious side effect of the met-hemoglobinemia caused by the two analgesics was that acetanilide made aniline, an agent known to produce the abnormal condition. Their study showed that as the blood concentration of aniline increased so did the production of the met-hemoglobinemia! Dr. Axelrod was hooked—on biomedicine-based research, that is.

A follow-up experiment examined what happened to the acetanilide in the body. Drs. Axelrod and Brodie found that the acetanilide was missing from the urine, suggesting that it was metabolized to other forms. So, they searched for possible metabolites that may have resulted from the degradation of the acetanilide. One of these metabolic candidates was identified as N-acetyl-p-aminophenol which was produced from a hydroxylation of acetanilide and is also known as acetaminophen!

Another follow-up experiment by Drs. Brodie and Axelrod delineated the direct conversion of the acetanilide into the acetaminophen. Furthermore, they showed that the acetaminophen itself was, surprisingly, more potent as an analgesic, compared to the more notorious acetanilide, and as an added benefit, the acetaminophen failed to produce the confounding met-hemoglobinemia! They had solved both a mystery and a problem, at the same time! The first project was published in 1948. It was Dr. Axelrod’s first scientific paper. A second publication followed, in 1949.

11) Apparently Axelrod was also interested in vitamin supplements (such as Linus Pauling was interested in Vitamin C). What were his contributions?

Dr. Axelrod’s involvement with vitamin C started with one of his first jobs after graduation from university, in 1933. At the New York Health Department he was tasked with evaluating the vitamin supplement content in foods and drink. The list of vitamins to be tested by Axelrod included, in addition to vitamin C, vitamins A, B, B2, and D. The purpose of these studies was to ensure the accuracies of the vitamins in randomly selected food and milk samples, as the importance of vitamins was just beginning to be recognized in human food and drink production circles.

The discovery of vitamin C, also known as ascorbic acid, by Dr. Albert Szent-Györgyi was of major importance. Because vitamins, by definition, cannot be biosynthesized by a living being, it becomes important that such individuals are supplied with the necessary substances by dietary means in order to ensure that their biological effects are conducted. Thus, the field was so important that Szent-Györgyi was bestowed the Nobel Prize, in 1937, for having performed such an important discovery for the proper metabolism of living beings. Thus, a major push was on to include vitamin C and other vitamins in foods and drink that were provided to humans. In later years, Dr. Pauling was to make the vitamin C even more relevant as a putative health substance and potential preventer of cancer.

Dr. Axelrod’s duties as an examiner of vitamin content in foods and milk involved learning new methods. This laboratory approach necessitated that he acquire an expertise of sorts in disparate fields, such as chemistry, biology, and microbiology, in order to conduct the various assays for vitamin compositions in foods and drink. It also necessitated that he delve into the scientific literature in order to make sense of the new methods. Following the published literature permitted him to modify any given method when necessary. It also provided a certain self-confidence in the laboratory setting. Furthermore, the experience of testing vitamins in foods taught him how to approach the scientific method of conducting biomedical research in later years.

12)  Those who knew him, and knew about him- were aware of the eye patch he wore- what is the story about the loss of one of his eyes?

The incident with his eye occurred during the time just prior to the start of World War II, when Axelrod was a graduate student in a master’s program at New York University. A laboratory accident involving an exploding bottle of ammonia caused Axelrod to lose one of his eyes.

He was forever to wear a patch on the affected left eye, sometimes wearing glasses with one of the glass lenses darkened. The permanent eye injury had prevented his participation in the military during the Second World War.

In later years, in 1986, two years after retirement, he studied cow eyes, examining the rod outer segments. With an interest in molecules called phospholipase A2 which could turn on GTP-binding proteins known also as G-proteins or transducing, he and colleagues found that these molecules consisted of sub-units called alpha, beta and gamma. Furthermore, he learned that they could combine into various dimers, such as beta-gamma, and that these dimers could turn on the phospholipase A2 molecules. The dimer could then form a trimer, alpha-beta-gamma to turn off the receptor. The work was submitted to Nature and rejected, but not before having first undergone a rather prolonged review period. Thus, their work was sent to the Proceedings of the National Academy of Sciences (PNAS) and duly published. Afterwards, many other investigators repeated the work, not only confirming Dr. Axelrod’s findings but also extending them. Dr. Axelrod had firmly established a new biomedical field.

13) What kind of summary can we provide-? Perhaps all those medical schools that rejected him- did us, and the field a service? What do you think?

Given his rather astonishing array of biomedically-based discoveries, having been performed both without and later with a Ph.D., I can understand your inquisitiveness about Dr. Axelrod having been largely rejected from medical schools. His rejections, however dreadful for Julius Axelrod, have been tremendously beneficial to humankind.

Indeed, Dr. Axelrod’s scientific excursion into a vast array of scientific biomedical fields is astonishing. His scientific body of work extends into many aspects of our own everyday lives.

Many human beings take vitamins on a daily basis, as a matter of routine. Many foods are fortified with them, and many others supplement their diets with vitamins. Vitamins are, after all, vital. We need them as proper co-factors for many of our enzymes. We may also need them for better health, maybe even to circumvent cancer, as has been touted by certain proponents, like the famous Dr. Linus Pauling has proposed with the vitamin C.

Countless millions of people take analgesics for pain; the extent of this practice for pain treatment is monumental. It is a daily practice for physicians to prescribe analgesics to their patients, and over-the-counter equivalents are extensive on a vast scale.

The neurotransmitter reuptake mechanisms discovered by Dr. Axelrod has had an enormous contribution to our understanding of nervous system functioning, both on a purely fundamental basis and on a practical applied basis. This Nobel Prize-winning work is a prime topic in any basic neuroscience course, taught to any undergraduate with an interest and to medical students as a matter of course.

The work involving the handling of certain chemicals by liver enzymes of the cytochrome-P450 monooxygenases is a major mechanism for controlling the levels of toxicity for potentially dangerous metabolites. This work has greatly contributed to our understanding of basic metabolism and of cancer biochemistry. Biomedical science has been made an important field of study because of this discovery. It is surprising to me that Dr. Axelrod did not receive a second Nobel Prize for this work, as well.

The work with the effects of narcotic drugs by Dr. Axelrod continues to be an important topic, even to the present day, with the problem of addiction to opiates. Scientifically, the issues are clear. Sociologically, politically, and economically, however, drug tolerance and dependence (addiction) is a seemingly intractable problem.

Caffeine seems to be a universally enjoyed substance, providing many a grateful coffee drinker with great joy! The health benefits of pure caffeine (without confounding cream and sugar) can be profound, helping both the heart and the mind. This one substance has had and will continue to have profound influence on the daily lives of its followers, many of whom are its strongest advocates.

The discovery of the serotonin circadian clock and its light / dark cycle has relevance to every sleeper; and Dr. Axelrod’s studies of melatonin synthetic pathway has relevance to every insomniac. These biomedical findings have universal relevance.

Although it was not mentioned above, Dr. Axelrod also studied a series of compounds called glucuronides, discovering that morphine could be converted into glucuronide conjugates using rat liver enzymes. It was thought that these enzymes could play a role in reducing the bilirubin. The work had a direct role in the importance of bilirubin which is pronounced in its levels in patients with certain genetic disease patients, premature babies and neonates with jaundice. The high bilirubin levels inherent in the jaundice condition could perhaps be reduced by glucuronide-specific enzymes.

Dr. Axelrod’s enduring catecholamine research will no doubt be of great importance in the biomedical sciences for a great many years to come. His work is a permanent contribution to our understanding of neurotransmitter metabolism, nerve conduction and synapse functioning. Along these lines, the pineal gland work with neurotransmitter metabolism will forever remain a major component to our understanding of the brain within the neuroscience field of the biomedical sciences.

After retirement at the age of 72 years, he was to make more important scientific contributions. Working with his postdoctoral fellows, he studied several neurotransmitter receptors, namely, the so-called bradykinin receptor and three new muscarinic receptors. He also studied the cytokine called interleukin-1 in which he found that it activated phospholipase A2 and that it released the second messenger molecule called arachidonic acid by working through one of several G-proteins. The G-protein activation had effects in terms of regulating the activities of other molecules such as adenylate cyclase, phospholipases A2 and C, and in the functioning of ion channels.

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