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The State University of New York at Buffalo, School of Medicine and Biomedical Sciences, Buffalo, New York
Foreword I. Prologue II. Major Discoveries in Pharmacology A. Thomas Renton Elliott: Elaboration of the Concept of Chemical Neurotransmission B. Sir Henry Dale and Otto Loewi: Chemical Transmission of Nerve Impulses 1. Sir Henry Dale. 2. Otto Loewi. C. Daniel Bovet: Synthetic Compounds That Inhibit the Action of Certain Body Substances, and Especially Their Action on the Vascular System and Skeletal Muscle D. Ulf von Euler, Julius Axelrod, and Sir Bernard Katz: Humoral Transmitters in the Nerve Terminals and the Mechanism for Their Storage, Release, and Inactivation 1. Ulf von Euler. 2. Julius Axelrod. 3. Sir Bernard Katz. E. Arvid Carlsson: Signal Transduction in the Nervous System F. Sir James Black, Gertrude Elion, and George Hitchings: Important Principles for Drug Treatment 1. Sir James Black. 2. Gertrude Elion and George Hitchings. G. Paul Ehrlich: The Magic Bullet H. Gerhard Domagk: Antibacterial Effects of Prontosil I. Sir Alexander Fleming, Cecil Paine, Harold Raistrick, Ernst Chain, and Sir Howard Florey: Penicillin and Its Curative Effects in Various Infectious Diseases 1. Sir Alexander Fleming. 2. Cecil Paine. 3. Harold Raistrick. 4. Ernst Chain and Sir Howard Florey. 5. Jack Strominger. J. Selman Waksman: Streptomycin: The First Antibiotic Effective against Tuberculosis 1. Albert Schatz. K. Sir Frederick Banting, Charles Best, John Macleod, and James Collip L. Philip Hench, Edward Kendall, and Tadeus Reichstein: Hormones of the Adrenal Cortex, Their Structure, and Biological Effects 1. Philip Hench. 2. Edward Kendall and Tadeus Reichstein. M. Sune Bergström, Bengt Samuelsson, and John Vane: Prostaglandins and Related Biologically Active Substances 1. Ulf von Euler and Sune Bergström. 2. Bengt Samuelsson. 3. John Vane. N. Earl W. Sutherland: Cyclic AMP O. Paul Greengard: Signal Transduction in the Nervous System P. Martin Rodbell and Alfred G. Gilman: G Proteins and Their Role in Signal Transduction in Cells 1. Martin Rodbell. 2. Alfred G. Gilman. Q. Robert Furchgott, Ferid Murad, and Louis Ignarro: Nitric Oxide as a Signaling Molecule in the Cardiovascular System 1. Robert Furchgott. 2. Ferid Murad. 3. Louis Ignarro. III. Epilogue
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Several problems were perceived. First, it would take an enormous effort, one which few authors would be willing to undertake. Second, the likelihood that a quality publication of that magnitude could be produced by 2008 was slight. Third, no matter how thorough an author might be, the work of many excellent pharmacologists would be omitted and could lead to conflicts. Finally, possibly the most important problem would be that the shear mass of material would not attract many young pharmacologists as readers. More than anything else, the Centennial Committee wants this publication to be interesting to young scientists.
It came to the attention of the Committee that Dr. Ronald Rubin had been independently considering writing about key discoveries in the history of pharmacology. The Committee offered to sponsor the project. What follows is the outcome of that effort by Dr. Rubin. In the view of the Committee, what Dr. Rubin has written avoids the major problems noted above.
The history is written in a highly interesting vein and is of a length that can be read in a relatively short period of time. The theses chosen are of such importance and are developed in such a style that it would be difficult to fault their selection. The lead investigators that Dr. Rubin highlights were (or are) remarkable individuals. Although each discovery discussed herein culiminated in a Nobel Prize, many other familiar names are woven into the fabric, and the evolution of ideas from multiple individuals is emphasized.
The Centennial Committee is pleased to sponsor this publication and hopes that the memories of more senior scientists will be relived and that young scientists will find the stories inspiring. We give our thanks to Dr. Rubin for his efforts and for these fine results.
William W. Fleming
On behalf of the ASPET Centennial Committee
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I. Prologue |
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Although pharmacology is a discipline with a rich and enduring heritage, present-day pharmacology is quite a different discipline than the more traditional subject I studied as a graduate student in the early 1960s. The discipline is now deeply rooted in molecular biology and molecular genetics, both of which provide powerful tools for the study of pharmacodynamics. In addition, the development of more sophisticated methods has allowed researchers to make important conceptual advances that may have eluded them for many years. Another aspect that sets present-day science apart from the past is how rapidly it progresses. The number of publications continues to grow to such an extent that many investigators now consider it an inefficient utilization of time to devote their attention to the older literature in their respective fields. However, analogous to the study of history in any format, the recollections of past events are key to understanding the discipline as it exists today and how it may evolve in the future.
There are several other reasons to have perspective on past work. Although scientific progress is viewed by some as being configured by the building of knowledge onto knowledge, I prefer to look upon science as an entity that is constantly permutating, fluctuating, and even vacillating. As a result, basic concepts are constantly reevaluated and modified. In essence, these perturbations make the pursuit of science such an interesting and intriguing endeavor. Furthermore, an historical perspective may enable one to profit from a review of previously missed opportunities to make fundamental advances and thereby avoid the pitfalls experienced by even the most gifted among us. It is also apparent that although molecular biology represents a focus of much of our present day research, the pendulum has recently been swinging back toward integrative and translational research. The genesis of this swing resides in the idea that when experimentation at the subcellular and molecular level is channeled back to the whole animal and ultimately to the patient, the etiology of disease is better understood and the effectiveness of its treatment is enhanced. And finally, it may just be worthwhile to devote time to reflecting upon the development of scientific thought, because it enables one to view his/her own research from a different perspective. This approach may lead to greater insight into present-day problems because "in science, as in life, conceptual progress once achieved sometimes turns out to be the rediscovery of the past" (Hechter, 1978
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In authoring this series of essays, I have omitted the work of some of our most distinguished scientists. This was done to prevent the essays from becoming an overwhelming chore to digest. So I have attempted to limit detailed discussion to selected examples of discoveries that I feel have had important and direct implications for pharmacological research and pharmacotherapy. In addition, each discovery has been selected for inclusion because it had the broadest of implications for humankind. I have also limited the number of references cited in order not to detract from the concepts and/or ideas that I hoped to convey. The personal anecdotes and vignettes embedded in these essays are meant to express a reverence for the gifted scientists with whom I have had the fortune to interact. But my overall objective relates to the fervent hope that the reader will achieve deeper insight into the cultural heritage of present day science, and of pharmacology in particular.
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II. Major Discoveries in Pharmacology |
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Neurotransmitters mediate the transfer of information from one nerve cell to another or from nerve cell to effector by the process of synaptic transmission. The genesis of the concept of chemical synaptic transmission has been attributed to John Newport Langley (Fig. 1), a heralded British figure in the annals of physiology/pharmacology. He determined in 1901 that adrenomedullary extracts (which contained both epinephrine and norepinephrine) elicited responses in different tissues that were similar to those induced by sympathetic nerve stimulation. In the wake of these findings, Langley proposed in 1905 that a "receptive substance" was the site of action of chemical mediators liberated by nerve stimulation. At about the same time, in Germany, Paul Ehrlich developed his own receptor theory of selective binding of toxins and nutritive substances. Drugs were initially excluded because they could be readily extracted from tissues and were therefore not deemed to be firmly bound to the cell. By 1907, Ehrlich revised his concepts to include the binding of drugs to receptors that he called chemoreceptors. The revised concept became the theoretical basis for his subsequent work, culminating in the discovery of the arsenical Salvarsan, the first chemotherapeutic agent used for the treatment of syphilis.
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However, a more rigorous line of research was needed to develop a basic understanding of the fundamental mechanism by which nerves communicate with other nerves or with diverse effectors. A young graduate student named Thomas Renton Elliott was responsible for providing the experimental results and the conceptual advances in our understanding of this most fundamental physiological process. Despite the fact that attributions to Elliott are for the most part buried in the annals of scientific history, the remarkable story of this brilliant young student should be recounted, because scientific lore has unjustifiably assigned Elliott an ambivalent role at best in the early development of scientific thought. He has generally been portrayed as a potentially gifted researcher who failed to follow up on his very promising findings and then summarily abandoned experimental research to pursue a relatively obscure and pedestrian career in clinical medicine. As you will see, my reexamination of his story invites a radically different interpretation.
This story had its origin in 1895 in the UK when George Oliver, a rural medical practitioner, paid a visit to Professor Edward Schaefer at University College London. Oliver's own experiments had yielded a pressor effect of adrenal extracts on various animals, and Oliver wanted to verify his findings. After Schaefer agreed to the collaboration, the two men conducted a series of experiments to examine the effects of adrenal extracts on the systemic circulation. Because of the therapeutic potential of this work, the publication of these findings prompted the search for a purer extract of the active principle. Two years later, John Jacob Abel of Johns Hopkins University, together with A. C. Crawford, isolated and purified the active principle from the adrenal medulla, which Abel later named "epinephrin" (no "e"). Abel, the Father of American pharmacology, would make another important contribution some 30 years later when he crystallized insulin.
Because Abel's extracts did not exhibit strong physiological activity, an industrial chemist named Jokichi Takamine sought to develop and patent a further purification step of the active principle a few years later. Takamine then arranged for Parke, Davis & Company to market the pure crystalline substance as "adrenaline." Takamine's work stimulated much academic and commercial interest, and soon "adrenaline" was recognized as the active principle of the adrenal gland. Because of the availability of this substance (now called epinephrine in the United States), Thomas Elliott, a student in the Department of Physiology at Cambridge, was able to conduct an extensive analysis of the comparative effects of medullary extracts in the form of epinephrine and sympathetic nerve stimulation.
After examining a variety of smooth muscle preparations and glandular tissues in a large number of animal species, Elliott became cognizant of the similarity between the pharmacological actions of epinephrine and the effects of sympathetic nerve stimulation. In May 1904 in a preliminary communication to the British Physiological Society, Elliott introduced the concept of chemical transmission into scientific lore. "But since adrenalin (epinephrine) does not evoke any reaction from muscle that has at no time been innervated by the sympathetic, the point at which the stimulus of the chemical excitant is received, and transformed into what may cause the change of tension in the muscle fiber, is perhaps a mechanism developed out of the muscle cell in response to its union with the synapsing sympathetic fiber, the function of which is to receive and transform the nervous impulse. Adrenalin might then be the chemical stimulant liberated on each occasion when the impulse arrives at the periphery" (Elliott, 1904).
A total of four publications were authored by Elliott, all dealing with the comparative effects of epinephrine and sympathetic nerve stimulation. In a 68-page treatise published in 1905 (Elliott, 1905), Elliott provided numerous examples of this relationship by demonstrating that the effects of sympathetic innervation and exogenous epinephrine on the bladder exhibited a similar variability among diverse species, which depended upon the density of sympathetic innervation. Armed with this comprehensive evidence, Elliott offered the postulate that the "effector" stimulated by epinephrine was the "myoneural junction" and not the nerve endings or muscle fibers.
Although this study dealt mainly with epinephrine, it was also prophetic in its analysis concerning what is now known about the functions of acetylcholine (ACh) at postganglionic parasympathetic nerves, synapses in autonomic ganglia, and the neuromuscular junction. Lacking convincing experimental evidence, Elliott nonetheless correctly speculated that these other components of the autonomic nervous system possessed a different type of junction. His intuitive recognition of a biochemical link among the three sites of cholinergic transmission would be substantiated by experimental evidence a decade later.
Elliott's last article on this subject reflected a remarkable breadth of knowledge regarding the physiological implications of his findings. However, the basically correct concept of chemical transmission that Elliott delineated in principle and reported in his preliminary note in 1904 was not reaffirmed in his subsequent publications, despite the fact that the scientific establishment failed to offer alternative explanations for his findings. One can only conjecture about the factors that contributed to the growing ambivalence in Elliott's perception of his own original hypothesis. In making no further reference to his original theory in future publications, Elliott never recanted and in fact eventually renounced his proposed theory during his presentation at the Sidney Ringer Memorial Lecture in 1914. In his remarks, he stated that "It is always a pleasure, and therefore a temptation, to accept a theory which harmonizes all the facts into a close-fitting plan. But the evidence at present does not justify us in welcoming this simplification" (Elliott, 1914).
Realizing his singular attributes as an experimentalist, a few associates unsuccessfully tried to dissuade Elliott when he decided to terminate his research activities and resume his clinical training. After fulfilling his medical commitments, Elliott served as a medical officer during World War I, where he eventually rose to the rank of colonel. When the war ended, Elliott returned home to occupy the first of London's full-time Chairs of Clinical Medicine at University College Hospital. During his career in medicine, he continued to publish research articles on clinical topics until 1930. Elliott also won many awards for his service over the years. Most notably, he was elected to the very prestigious Fellowship of the Royal Society of London. When he retired as Chair of Clinical Medicine at University College Hospital in 1939 at the age of 62, his associates paid tribute to his wisdom, high standards, and keen vision. So although Thomas Elliott failed to consolidate his early scientific contributions into a lasting legacy, he was nonetheless remarkably successful in pursuing a distinguished administrative career in clinical medicine.
Some years later, after the evidence became overwhelming that chemical transmission was operative at synaptic sites, the legendary Sir Henry Dale, disregarding his own involvement, attributed the reluctance of Elliott to promote his theory to the perceived lack of interest in his work exhibited by the elite of the scientific establishment. In particular, John Langley, Elliott's mentor and department chair, was known as an individual who disapproved of speculative theories, especially those proposed by relative neophytes working under his direction. So Langley was apparently unwilling to give Elliott's transmitter concept an honest evaluation. In addition, the formulation of the concept of "receptive substance" first proposed in 1905 by John Langley (Langley, 1905) has, at least in part, been attributed to ideas expressed by Elliott about how a muscle cell responds to a chemical stimulus. However, in his publication, Langley failed to give any consideration to Elliott's ideas, which may have further discouraged the young investigator and diverted attention away from what probably was the most important advance in neurobiology up to that time.
Nevertheless, Elliott's contributions to neuroscience, although transient and incomplete, proved to be enduring. His seminal work became a paradigm for later studies, which would ultimately lead to the elucidation of the basic processes involved in nerve function. In 1907, Walter Dixon, a pharmacologist working at Cambridge, attempted to extend Elliott's findings by arguing that parasympathetic nerves similarly liberate a neurotransmitter to activate effector sites (Dixon, 1907). To document his theory, Dixon demonstrated the release of this putative neurotransmitter from the mammalian heart following stimulation of the vagus nerve. After making an extract from canine heart following inhibition of contractility produced by vagal stimulation, Dixon found that the extract produced a depression of contractility of an isolated frog heart. The inhibition produced by the extract, like that caused by vagal stimulation, was blocked by the muscarinic antagonist atropine; however, due to the limitations in methodology and basic knowledge that existed at the time, this study was not continued. As a result, conceptual advances in this field were further delayed.
In his later writings, Sir Henry Dale suggests that the active substance in Dixon's experiment was probably choline, the product of ACh degradation (Dale, 1934). However, at the same meeting of the Physiological Society at which Dixon presented his results, Reid Hunt, an American pharmacologist, reported that the adrenal gland produced a hypotensive substance that was too robust to be attributed to choline. This experiment provided the impetus for Hunt to examine a series of related compounds that were synthesized for him by Rene de M. Taveau. In reporting his findings, Hunt proposed that either a precursor or derivative of choline was the main hypotensive principle. One of the esters investigated was ACh, which was found to be several orders of magnitude more active than choline in producing a drop in blood pressure. However, the transient nature of the hypotensive action exhibited by ACh and other choline analogs argued against any further experimentation to assess their significance as possible therapeutic agents. In a more detailed analysis carried out in 1914, Sir Henry Dale identified the muscarinic and cholinergic actions of ACh (Dale, 1914). While acknowledging the possible physiological significance of the resemblance between the actions of choline esters and the effects of certain elements of the parasympathetic nervous system, Dale felt that any further consideration of the physiological implications of these results should be deferred due to the limited amount of background knowledge on the subject that was available at the time.
Still, the experiments carried out by Dixon, Hunt, and Dale gave credence to the interpretation of Elliott's earlier work and would ultimately vindicate his research. However, Dixon and Hunt did not continue to explore this problem much further; so the attribution of Dixon's and Hunt's role, like Elliott's role, was relegated to brief references in certain historical accounts. One may argue that the scientific community might be forgiven its disinterest in this line of research, since the limitations in methodology made it difficult, if not impossible, to employ a more direct experimental approach to the problem at the time. However, it also seems fitting to conclude that Elliott, Dixon, and Hunt did not possess the burning interest and passion needed to overcome the obstacles presented by this fundamental biological problem (Maehle, 2004).
B. Sir Henry Dale and Otto Loewi: Chemical Transmission of Nerve Impulses
1. Sir Henry Dale. Although additional pertinent information derived from experiments of the type carried out by Elliott and Dixon would not be forthcoming for another 15 years, further insights into the mechanisms involved in synaptic transmission were fueled by the "applied research" carried out by Sir Henry Dale (Fig. 2) for Wellcome Laboratories from 1904 through 1914. The original firm had been established in 1894 by Henry Wellcome, an American-trained pharmacist, to produce serum antitoxins for clinical applications. Then, in 1895, Wellcome established the Research Laboratories. The second branch of the firm was dedicated to conducting original research and was to be divorced from the commercial subdivision.
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). The appointment of Dale was profoundly significant, not only because it was responsible for guiding biological research into new directions but also because the dual approach inaugurated by Henry Wellcome enabled productive research to develop in concert with a successful business enterprise. In future years, this dual program would be duplicated by other pharmaceutical firms.
However, at the time a schism between academia and industry existed. As an example, when Dale first met John Jacob Abel in 1909, Dale noted that Abel was rather suspicious of him because of his connections with a commercial enterprise. Abel epitomized an academician of the time. Having trained in Germany at the Pharmacology Institute headed by Oswald Schmiedeberg, Abel had returned to the United States to occupy the first Chair of Pharmacology at the University of Michigan in 1891. Then, in 1893, he assumed the Chair of Pharmacology at Johns Hopkins University. He also played a key role in the founding of ASPET in 1908. By such efforts, Abel was responsible for fashioning pharmacology into a discipline primarily concerned with the study of drugs from a systematic and mechanistic perspective, with implications for therapy. His dedication to the discipline of pharmacology also made him wary of anyone whom he believed would sully its reputation by engaging in commercial endeavors. But Abel was eventually persuaded by academic colleagues that Dale did possess strong scientific principles and ultimately accepted him as a colleague (Tansey, 1995).
Wellcome Laboratories had a strong interest in the properties of derivatives of the rye fungus ergot, and Dale was assigned this project. Dale justified this undertaking to himself by reasoning that one component of ergot extracts, ACh, was probably a naturally occurring compound, and therefore its study was of potential physiological significance. In their comprehensive pharmacological analysis published in 1914, Dale and Laidlaw found that the actions of ACh on cat blood pressure and exocrine glands, as well as rat smooth muscle, resembled those of the alkaloid muscarine. They also observed that the pharmacological effects of exogenous ACh exhibited a striking similarity to the effects of parasympathetic nerve stimulation, which was also comprised of muscarinic (blocked by atropine) and nicotinic actions (mimicked by nicotine).
In reporting the transient nature of the action of ACh, Dale suggested that an esterase in tissues or blood was probably responsible for its rapid metabolism. In this article, Dale alluded to the possible presence of ACh in humans and its potential biological significance. Although the key physiological implications of his work seemed to elude Dale at the time, this study did provide the theoretical basis for defining the pharmacology of autonomic drugs. The physiological relevance of ACh would be established by the classic experiments performed by Otto Loewi a few years later.
In 1910, Dale also published a detailed account of the sympathomimetic actions of a number of biogenic amines synthesized by George Barger. By demonstrating that several structurally diverse amines reproduced the effects of sympathetic nerve stimulation, Dale provided support for the hypothesis elaborated several years earlier by Thomas Elliott that epinephrine, or some other catecholamine, transmitted the response elicited by sympathetic nerve stimulation to the postsynaptic effector site (Barger and Dale, 1910). The luxury of hindsight enables us to conclude that by unwittingly excluding from their investigations the epinephrine (adrenaline) series of sympathomimetics, Dale and Barger overlooked the most physiologically relevant derivative, norepinephrine (noradrenaline). The fact that at the time norepinephrine was available commercially and did not require its synthesis by Barger made Dale's oversight even more vexing. As a result, the correct identification of the putative neurotransmitter of postganglionic sympathetic nerves would be delayed for many more years.
In reflecting on the reasons why he did not initially champion the concept of chemical neurotransmission as elaborated by Thomas Elliott, Dale noted that exogenous administration of epinephrine produced several inhibitory actions on sympathetically innervated end organs that were not duplicated by sympathetic nerve stimulation. This inconsistency suggested to him that some alternative process was operative. Years later, Dale tried to rationalize his missed opportunity by noting that even if he had suggested that norepinephrine was the putative neurotransmitter, because of the limited technologies available at the time (c. 1915), it would have been very difficult to identify each of the various catecholamines that might be present. So, until 1921, the physiological mechanisms involved in the transmission of signals across synapses were a subject of intense debate. In fact, certain distinguished scientists of the time gave credence to the hypothesis that synaptic transmission was an electrical event, brought about by transmission of the activation wave from the nerve ending to the effector. All of that began to change at the beginning of the 1920s, when the classic demonstration of chemical transmission was finally achieved by a simple, yet ingenious experiment carried out by Otto Loewi.
2. Otto Loewi.
Otto Loewi (Fig. 3) had been trained as a pharmacologist at the University of Marburg in Germany at the beginning of the 20th century. Fortunately for Loewi, the conditions that prevailed during the early 1900s in Germany were most favorable for the development of scientific thought, with no government intervention (Loewi, 1961). Loewi took advantage of these positive conditions to learn to view scientific theory through a wide lens. As a result, his ideas were not constrained by existing dogma. After he was invited to accompany his superior Hans Meyer to Vienna, Loewi accepted the Chair of Pharmacology at the University of Graz (Austria) in 1909, where he conducted his classic experiments.
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After Loewi placed two frog hearts into a single bath, the vagus nerve of one heart was stimulated, thereby slowing it, while causing the rate of the second heart to also diminish. From this experiment, Loewi reached the obvious conclusion that a substance liberated from the first heart was responsible for causing inhibition of the second heart. He termed the unknown substance vagus-stoff, which was later identified as ACh. Subsequent articles by Loewi provided additional evidence favoring the similarity of this substance to ACh, including its characteristic sensitivity to destruction by an esterase that Loewi had extracted from heart muscle.
Loewi also used the frog heart preparation to demonstrate that sympathetic nerve stimulation caused the liberation of a substance, which he called acceleransstoff. He showed that it shared many of the properties of epinephrine in that it could be destroyed by alkali, fluorescence, and UV light. In addition, its activity at adrenergic effector sites was blocked by ergotamine and augmented by cocaine. Loewi also observed that the effects of sympathetic nerve stimulation and epinephrine on the heart declined very slowly, in contrast to the transient effects of ACh. These findings that suggested different modes of inactivation were operative for the two putative neurotransmitters would be substantiated by the work of Julius Axelrod and his colleagues some 40 years later. On the basis of these experiments, Loewi proposed that parasympathomimetic effects were mediated by ACh and sympathomimetic effects were transmitted by epinephrine.
Despite the potential far-reaching implications of this work, Loewi faced some formidable challenges from colleagues concerning the validity of his conclusions. Their skepticism was based primarily on the technical limitations of Loewi's experiments, which were deemed responsible for contradictory results obtained by other investigators. Most importantly, the frog heart preparation was widely considered to be an unpredictable experimental model, with regard to the reproducibility of responses that various stimuli were able to elicit. In addition, because the preparation used by Loewi functioned as a hypodynamic heart, it was viewed by some as nonphysiological in terms of its functionality. Unfortunately for Loewi, the hypodynamic preparation yielded the most favorable results in support of his theory.
Thus, progress in this field was shackled by the controversy that Loewi's experiments and conclusions engendered among his colleagues. However, decisive evidence in favor of Loewi's hypothesis was eventually produced when the liberation of vagus-stoff was observed in a nonhypodynamic heart. Moreover, much of the conflicting data obtained by various laboratories was discounted because of the known instability of vagus-stoff, which Loewi had identified as ACh. Dale had already proposed in 1914 that the rapid breakdown of ACh was due to the presence of esterases in blood and tissues. This idea was confirmed in 1926 by Loewi and Navratil, who reported that extracts of frog heart tissue rapidly degraded ACh, presumably by a form of acetylcholinesterase (Loewi and Navratil, 1926). They also found that eserine could not only inhibit the enzyme but could also markedly enhance the inhibitory effects of ACh and vagus-stoff on the frog heart. So vagus-stoff could now be defined pharmacologically as a substance whose action was inhibited by atropine and enhanced by eserine. Because the properties of vagus-stoff were identical to those exhibited by the muscarinic actions of ACh, this work left little doubt that the neuronal stimulus was transmitted to the postsynaptic effector by chemical means rather than by electrical transmission.
Although one might argue that Loewi's original experiments were not very convincing, he did possess the tenacity of purpose to doggedly pursue his theory, until it was ultimately confirmed in its most basic form. In 1926, after Loewi reproduced his basic experiment 18 times on the same frog heart preparation at the famed Karolinska Institute in Sweden, his colleagues began to comprehend what he had accomplished. His work and conclusions were finally vindicated in 1933, when the introduction of the leech muscle preparation for bioassay enabled Wilhelm Feldberg and Otto Krayer to demonstrate definitively that the stimulation of the vagus nerve liberated ACh into the coronary vasculature of mammals. A major advantage of the leech muscle for the bioassay of ACh was that it was extremely sensitive to very low levels of endogenous ACh but was not responsive to catecholamines. So, by the early 1930s, it was generally accepted that the autonomic nervous system was regulated by two substances with antagonistic actions: an ACh-like agent liberated by parasympathetic fibers and an epinephrine-like substance released by nerve fibers of the sympathetic system.
At this point, evidence was needed to assess whether the substance released from parasympathetic fibers might be a choline ester with pharmacological properties similar to ACh. Dale and Dudley made progress on this issue in 1929, when they reported the extraction and identification of ACh as a natural product of oxen and equine spleen (Dale and Dudley, 1926). By this time, Dale, now working as Chief Pharmacological and Biochemical Officer at the National Institute for Medical Research in London, was a strong proponent of the chemical theory, and he coined the terms adrenergic and cholinergic to describe the actions of autonomic and motor nerve fibers.
The hypothesis that described an ACh-like transmitter was later extended by Dale and his colleagues to synaptic transmission at autonomic ganglia. He and his distinguished associates, including Wilhelm Feldberg, Sir John Henry Gaddum, and Marthe Vogt, demonstrated by bioassay the presence of ACh in isolated perfused cat sympathetic ganglia following nerve stimulation. Not surprisingly, the detection of ACh in the venous effluent of perfused ganglia was predicated upon the presence of eserine in the perfusion solution. These findings and those previously made by Loewi suggested that a fundamentally similar process was operative in synaptic transmission of excitatory effects at all autonomic ganglia and postganglionic parasympathetic effector sites.
Despite the mounting evidence in support of the theory of chemical transmission, debates still raged during the 1930s concerning the general applicability of this theory. Dale and his colleagues maintained their important role in endorsing this concept, despite being challenged by colleagues who continued to argue in favor of electrical transmission. The most renowned proponent of this latter view was the Nobel Laureate Sir John Eccles, who continued to perpetuate this outdated theory. It was reported that rather harsh words were sometimes exchanged between Dale and Eccles on this issue. But by the 1950s, when overwhelming evidence finally resolved the argument, the debates finally ended in mutual respect between the two Nobel Laureates, exemplified by their frequent correspondence of more than 20 years.
Decisive experiments were also conducted by Dale and his colleagues on the neuromuscular junction in the 1930s, which established that the action of ACh was not confined to the autonomic (involuntary) nervous system. Together with Wilhelm Feldberg and Marthe Vogt, Dale published two articles that not only provided a clear demonstration that ACh was released from motor nerve endings following nerve stimulation but also embellished their results by showing that when injected close to the muscle, ACh produced a depolarizing effect similar to that of nerve stimulation (Dale et al., 1936). The fact that ACh release was detectable even when the responsiveness of the motor end-plate was impaired by curare represented an analogous result to that obtained by Loewi on postganglionic parasympathetic effector sites. Loewi had already shown that atropine blocked the postsynaptic action of ACh on cardiac muscle but did not modify its release elicited by vagal nerve stimulation. Thus, incontrovertible evidence eventually persuaded Dale to lend his unequivocal and influential support for the theory of chemical transmission. One cannot overemphasize the importance of Dale's endorsement, because many of the cognoscenti at the time still firmly believed that the data were not sufficiently strong or convincing to incontrovertibly validate the new concept that would ultimately alter the scientific world's view of neuronal function.
However, final validation of the concept of chemical transmission had to await studies that would conclusively identify the agent involved in synaptic transmission at postganglionic sympathetic nerves. Although Elliott had shown that the effects of epinephrine were similar to those of sympathetic nerve stimulation, innumerable experiments, including those of Dale, showed that the inhibitory effects elicited by injected epinephrine were not prominent following nerve stimulation. A few years later, the renowned Harvard physiologist Walter Cannon observed quantitatively different effects on the chronically denervated and supersensitized pupil of the cat, when he compared the effects of exogenous epinephrine with those induced by hepatic or cardiac nerve stimulation. To address such disparities, Cannon and Rosenblueth proposed in 1933 that sympathin, a hypothetical mediator elaborated by sympathetic nerves, combined with either excitatory or inhibitory substances at the postsynaptic site, forming either sympathin E (excitatory) or sympathin I (inhibitory). These two substances were then released into the blood stream, leading to either a stimulatory or inhibitory response (Cannon and Rosenblueth, 1933).
The apparent conundrum resulting from the analysis of the comparative effects of epinephrine and sympathetic nerve stimulation was eventually resolved by a less complex and more physiologically relevant explanation. In 1948, Raymond Ahlquist (Fig. 4) at the Medical College of Georgia reasoned that if the rank order of potency of a series of catecholamines was the same in all tissues, then the variation in their relative activities must be due to differences in their chemical structure. However, if the rank order of potency varied from tissue to tissue, the observed variations must be due, at least in part, to inherent differences in the receptors. To test this postulate, Ahlquist compared the relative potencies of several sympathomimetic amines (including epinephrine) with sympathetic innervation on several isolated mammalian preparations. Only two orders of relative potency were observed with regard to inhibitory actions such as vasodilation and brochodilation (isoproterenol > epinephrine > norepinephrine). For the excitatory actions such as vasoconstriction and pupillary dilation, the rank order of potency observed was epinephrine = norepinephrine > isoproterenol. The differential sensitivity of the various tissues to the agonists could not be readily explained by the theory of Cannon and Rosenblueth, which centered on two types of transmitters. Rather, the different patterns of relative efficacy more likely represented a preferential affinity of each agonist for one of two types of adrenoceptors.
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Capitalizing on the additional discovery in 1946 by Ulf von Euler that norepinephrine was the adrenergic neurotransmitter, Ahlquist postulated in 1948 that the action of norepinephrine on postsynaptic sites was mediated by two types of adrenergic receptors, which he called 
and β. It is of interest to note that the original manuscript submitted by Ahlquist was rejected by the Journal of Pharmacology and Experimental Therapeutics, despite the fact that it contained strong evidence to support Ahlquist's concept. Eventually, with the help of a friendly colleague, Ahlquist's manuscript was published in the American Journal of Physiology. But the scientific community was reluctant to accept this concept because of its novel approach to pharmacology and the mathematical modeling that Ahlquist used to explain his theory.
All of that began to change, however, when in 1954 Ahlquist was invited by Victor Drill to write the chapter on adrenergic pharmacology in Drill's Pharmacology in Medicine. As author of this chapter, Ahlquist took advantage of the opportunity to promote his theory, which ultimately enabled it to gain general acceptance. The concept not only prompted fresh thinking about adrenergic receptor pharmacology, it also vaulted scientific research into new directions that would guide future drug development. In particular, Ahlquist's ideas presented in Drill's textbook were adopted by Sir James Black in his quest to develop an agent that would reduce the demand for oxygen by the heart. In fact, Black maintains that Ahlquist's concept provided the conceptual framework for the development of β-receptor blockers, which was to earn Black the Nobel Prize (see section II.F.1.). Moreover, this fundamental concept led to the identification of adrenergic receptor subtypes, which subsequently spawned the development of more selective and useful therapeutic agents such as the β-1 receptor-blocking agent atenolol, the β-2 agonist terbutaline, and the -1 antagonist prazosin.
Despite the irrefutable and overwhelming evidence favoring chemical transmission at synapses of the autonomic nerve system, during the 1950s, a few members of the scientific establishment maintained an obdurate refusal to relinquish their outdated theory. Sir John Eccles resolutely remained a dissenting voice, arguing that transmission at the neuromuscular junction was too rapid to be mediated by a chemical event. To quash these dissenters once and for all, Sir Bernard Katz and his skilled associates took it upon themselves to develop precise and sophisticated intracellular recording techniques to conduct comparative studies on the effects of exogenous ACh and nerve stimulation at the motor end-plate. As a result of their work, the proposition that synaptic transmission at the neuromuscular junction involved a chemical process was finally rendered indisputable once and for all. The final resolution of this elusive question was obviously important from many perspectives. However, it should be emphasized that by establishing the concept of chemical transmission in peripheral nerves, Dale, Loewi, and Katz provided the foundation for further experimentation by others to probe the mechanisms of synaptic transmission in the central nervous system. As a result, major progress in our understanding of cell signaling mechanisms in the nervous system has led to better treatment and management of neurological and psychiatric disorders, which plague a major segment of our population.
In recognition of their extraordinary achievements, Dale and Loewi shared the Nobel Prize in 1936 for their work on chemical transmission of nerve impulses. Dale's contributions to pharmacology were legion, but it is important to single out his unique ability to distinguish, characterize, and classify drugs by virtue of their selective actions. In this way, Dale made fundamental and lasting contributions to the development of pharmacology as a discipline. The legacy of Loewi also endures, although his later career was tainted by intrigue and politics. Two years after becoming a Nobel Laureate, Loewi was jailed by the Nazis for his religious beliefs. However, eventually Loewi was granted safe harbor to leave Austria, but only after he transferred his prize money to a Nazi-controlled bank and was deprived of all properties and belongings.
For the next few years, Loewi experienced a rather nomadic existence. He first sought haven at the Universite Libre in Brussels, followed by another move to the UK in 1939. Once in the UK, he then traveled overseas in an attempt to establish himself at Harvard under the auspices of Walter Cannon. However, because of the massive influx of European scientists at that time, Harvard was reluctant to offer even a Nobel Laureate a faculty position. So, in 1940, Cannon contacted Homer Smith, his former research fellow and now a famed renal pharmacologist/physiologist, to offer Loewi a professorship in pharmacology at New York University (NYU) School of Medicine. Like so many other expatriated scientists of that era, Loewi expressed his gratitude by embracing his new country. For the rest of his life, Loewi worked at NYU during the winter and at the Marine Biological Laboratory in Woods Hole in the summer, where he continued to conduct research until his death in 1961. The life of Otto Loewi clearly illustrates how one unique individual envisioned and proved a scientific truth that had been ignored and even ridiculed. The full importance of Loewi's contributions can be better understood from the perspective that his breakthrough discovery led to the recasting of ideas about how nerves function.
C. Daniel Bovet: Synthetic Compounds That Inhibit the Action of Certain Body Substances, and Especially Their Action on the Vascular System and Skeletal Muscle
By identifying the physiological roles played by biogenic amines in cell function during the 1920s and 30s, Otto Loewi, Sir Henry Dale, and their colleagues set the stage for pharmacologists and chemists to pursue drug development in a structured and systematic manner. Toward this end, Daniel Bovet (Fig. 5), noting that drugs such as arsphenamine and sulfonamides had been introduced into therapeutics empirically, decided to take a more rational approach toward synthesizing and testing new pharmacological agents. Bovet based his strategy on the principles inherent in the antimetabolite theory of Woods and Fildes (Fildes, 1940; Woods, 1940). This theory explained the bacteriostatic action of sulfa drugs by virtue of their ability to competitively antagonize the normal cellular utilization of p-aminobenzoic acid, a metabolite with a chemical structure very similar to that of the sulfonamides.
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Adopting this general approach for studying pharmacological activity, Bovet examined a series of chemical derivatives through several steps to determine which chemical groups were responsible for the antagonist or agonist activity in question. Bovet defined the steps as 1) an analysis of the structure of parent compounds possessing a specific pharmacological activity followed by 2) the synthesis and testing of various chemical derivatives or analogs for agonist or antagonist activity. In this way, Bovet applied the concept of competitive interactions to pharmacodynamics in the hope of providing clues as to the nature of the receptor site and possible mechanism of drug action. These advances, in turn, might then lead to the development of more useful and effective therapeutic agents.
Bovet favored the view that substances of natural origin such as ergotoxin, curare, and atropine could serve as useful models for the development of more selective receptor antagonists. Thus, he initially directed his attention to the ergot alkaloids, a fecund resource of pharmacological activity that was investigated by Sir Henry Dale many years before. In carrying out his work, first at the Pasteur Institute in Paris and then after 1947 at the Instituto Superiore di Sanità in Rome, Bovet initially sought the identity of the active principle of the ergotamine moiety. Toward this end, Bovet and his colleagues synthesized and characterized a voluminous number of compounds whose actions would mimic or inhibit those of the naturally occurring ergot. However, the search for the active principle proved formidable, since the structure of ergot was quite different from that of epinephrine and other derivatives of phenylethylamine. Nevertheless, by using the autonomic nervous system as the test model, Bovet observed a gradual reduction in sympathomimetic activity and the emergence of antagonistic activities of compounds with structures of increasing complexity. However, when Bovet observed a lack of correspondence between the results obtained in animals and those found in humans, he discontinued the work with adrenergic blocking agents and redirected his attention to agents that acted at cholinergic sites.
Bovet realized that the study of anticholinergic agents would also prove to be a daunting task because of the multiple systems on which ACh acted, including postganglionic parasympathetic effector sites, autonomic ganglia, and the neuromuscular junction. Noting that ACh antagonists differed structurally, depending upon their site of action, Bovet decided to confine his investigations to an examination of the neuromuscular junction. Identifying the synapse as the primary locus of action of curare had been accomplished during the 1850s by the classic experiments of Claude Bernard. The specific mechanism of action of d-tubocurarine and other competitive neuromuscular blocking agents was elucidated more than a century later by Sir Bernard Katz and his colleagues using intracellular recording and microiontophoretic techniques.
Curare was a generic term for various South American arrow poisons. The crude preparations that were initially available consisted of a thick, black, gelatinous-like substance obtained from various remote sources in South America. These crude preparations made any analysis of the pharmacological properties of curare very difficult. In addition, to become effective therapeutically, a stable preparation had to be developed that possessed pharmacological actions that were free from undesirable side effects. So the study and therapeutic use of curare was thwarted for almost a century until the pure alkaloid, d-tubocurarine, finally became available in the 1940s. The alkaloid then was used as an adjuvant during general anesthesia (West, 1984).
With the advent of the pure alkaloid, Bovet and his colleagues could now use diverse methods of biological testing to compare the pharmacological properties of d-tubocurarine with analogs that were less complex structurally. Bovet's overall strategy was based upon the principle that manufactured pharmacological agents would prove more useful than naturally occurring substances, because they were more selective in their sites of action, relatively free from side effects, and had a shorter duration of action. Concentrating on phenolic esters containing quaternary ammonium activity, Bovet's team synthesized gallamine (Flaxedil). This drug had a more rapid onset of action than tubocurarine, as well as a more rapid recovery time. Although the clinical use of gallamine was limited by its positive chronotropic effect and its proscription in patients with kidney disease, the rigorous analysis of structure-activity relationships identified several anticholinergic compounds that were less complex structurally and more useful than their naturally occurring counterparts in terms of specificity and absence of undesirable side effects.
At about the same time, Bovet initiated a study of tubocurarine analogs by varying the distance between the two quaternary ammonium moieties. He found that there were two key characteristics that defined the activity of such bis-quaternary derivatives, the distance between the quaternary groups and the size of the substituents added to the molecule. When the chain between the quaternary groups contained 10 carbon atoms (decamethonium), maximum pharmacological activity was observed. Coincidentally, William Paton and Eleanor Zaimis made the identical discovery of decamethonium in the UK at about the same time. They demonstrated that this agent acted by depolarizing the end-plate and thereby prevented it from responding to ACh. However, because it resembled curare in not being degraded by cholinesterase, decamethonium produced a muscle paralysis that was excessively prolonged and not reversible by an esterase inhibitor (Paton and Zaimis, 1949).
Among the drugs tested for their paralytic effects at the neuromuscular junction was succinylcholine, which was composed of two molecules of ACh attached end-on-end. Bovet's meticulous analysis of the properties of this agent was perhaps his most celebrated discovery. Its development provided a major contribution to pharmacotherapy, when it was shown that the drug was rapidly hydrolyzed by pseudocholinesterase. As a result, succinylcholine possessed a short duration of action as a muscle relaxant compared with d-tubocurarine and decamethonium. It therefore could be employed as an adjuvant in the form of a drip to more precisly titrate the level of muscle relaxation during general anesthesia. In this way, the potential hazards of surgical anesthesia were reduced.
It should be noted that many years earlier, Reid Hunt and Rene de M. Taveau reported on the pharmacological actions of a number of choline derivatives they had synthesized, including succinylcholine (Hunt and Taveau, 1906). However, these investigators failed to identify the neuromuscular-blocking properties of the agent, because they employed curare-pretreated animals as their model for drug testing. As a result, the introduction of succinylcholine into clinical use was delayed until 1942. So, just as Dale had shown that atropine blocked the action of ACh at muscarinic sites and ergot alkaloids annulled the effects of epinephrine and norepinephrine at postganglionic sympathetic sites, Bovet and his associates were responsible for providing the conceptual framework for analyzing the pharmacology of cholinergic antagonists at the neuromuscular junction.
Because of perceived similarities thought to exist among epinephrine, ACh, and histamine with respect to their pharmacological properties, Bovet extended the scope of his work in 1937 to include histamine antagonists. Pharmacologists had long understood that the development of drugs that were capable of blocking the actions of histamine would not only help to provide deeper insights into various aspects of physiological and pharmacological mechanisms but would also be of inestimable value in the treatment of allergic disorders. However, 25 years elapsed between the articles by Sir Henry Dale and Patrick Laidlaw (1910, 1911) on the pharmacological actions of histamine and the genesis of Bovet's work to develop drugs with histamine-blocking activities. But when histamine was identified as a constituent of the body, intense interest in this autacoid was created. Daniel Bovet was among the researchers who initiated efforts to produce antagonists of histamine. He first examined the adrenoceptor-blocking agent piperoxan, which was known to possess inhibitory activity against histamine in isolated intestine. Although Bovet found that several related compounds afforded some protection against the effects of histamine, they proved too toxic for clinical use.
Bovet and Anne-Marie Staub were able to obtain a better grasp on histamine antagonists when they investigated thymoxyethyldiethylamine (Bovet and Staub, 1937). This agent protected guinea pigs against lethal doses of histamine, antagonized histamine-induced spasm of smooth muscle, and diminished the symptoms of anaphylactic shock. Although this compound, like several others initially developed, was found to be relatively ineffective and too toxic for clinical use, Bovet and his team persevered and in 1944 produced Neo-Antergan (pyrilamine) (Bovet et al., 1944). This drug is still used today as a selective H1 antagonist in treating symptoms associated with acute allergies such as urticaria, rhinitis, and conjunctivitis. In describing the selective antagonism of responses to histamine, Bovet and Staub made it possible to construct certain general criteria for developing histamine antagonists, which are now mainstays in the treatment of a variety of allergic disorders. In addition, the contributions made by Bovet and Staub formed the basis for future structure-activity studies on histamine antagonists employed by other investigators, most notably Sir James Black.
In addition to publishing more than 300 articles that documented his work on drugs affecting the autonomic nervous system, the neuromuscular junction, and the actions of histamine, Bovet also probed various aspects of the pharmacology of the central nervous system. His observations with lysergic acid and its derivatives exerted a marked influence in the field of psychopharmacology, and in particular psychedelic drugs. By demonstrating that relatively simple molecules can modify changes in perception and mood, Bovet's work helped to shape scientific thought regarding psychoactive drugs that are used in therapy today.
Perhaps Bovet's greatest legacy, however, is that he advanced the evolution of pharmacology as an established discipline and helped to promote the era of pharmacodynamics and a more mechanistic approach to pharmacology. In applying his expertise as an organic chemist to therapeutics, Bovet also helped to advance the discipline of pharmaceutical chemistry by conveying how chemical structure relates to pharmacological activity. Yet Bovet possessed the depth of understanding to realize that, because of the diversity of the chemical classes to which pharmacologically active agents belong, experimental findings cannot always be explained in terms of predictable scientific paradigms. For him, such atypical results could only be characterized as empirical findings. In honor of his groundbreaking contributions leading to the discovery of synthetic compounds that selectively inhibit the action of endogenous substances, Daniel Bovet was awarded the Nobel Prize in 1957.
D. Ulf von Euler, Julius Axelrod, and Sir Bernard Katz: Humoral Transmitters in the Nerve Terminals and the Mechanism for Their Storage, Release, and Inactivation
As previously chronicled, chemical transmission as a concept was attributed to a British student named Thomas Elliott, who in 1904 reported that there was a striking similarity between the action of epinephrine (which he called adrenaline) and sympathetic nerve stimulation. In 1910, Barger and Dale coined the term sympathomimetic amine to characterize the actions of a large series of amines that elicited physiological responses similar to those exerted by sympathetic nerve stimulation. Many years later in a volume composed of a compilation of several of his many articles, and having the advantage of hindsight, Dale laments the "opportunities missed" in the second decade of the 20th century to examine the analogs of epinephrine (adrenaline) (Dale, 1965). This oversight permitted a major discovery (chemical transmission) to elude him. It was only later that Dale realized that Elliott had been correct in principle and erroneous only with regard to the actual identification of the mediator. To profit from Dale's experience of a "missed opportunity," the reader may find it worthwhile to dust off his book and read his justification for not reaching what we now know is an obvious conclusion. Dale's comments seem generic for all who are engaged in scientific research.
1. Ulf von Euler. During the 1930s, certain investigators alluded to the possibility that norepinephrine might be the neurotransmitter liberated at adrenergic nerve endings. However, it was not until the mid-1940s that Ulf von Euler (Fig. 6) used various pharmacological and chemical assays to correctly identify the major catecholamine as norepinephrine (noradrenaline) in extracts of adrenergic nerves from different species. When it became necessary to differentiate epinephrine from norepinephrine, von Euler used two bioassays with different sensitivities to the two amines, such as the cat blood pressure and hen rectal caecum. A fluorometric technique for independently measuring epinephrine and norepinephrine, which was developed in von Euler's laboratory, also helped to raise the bar of research in this field.
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The discovery of norepinephrine as the neurotransmitter at postganglionic sympathetic nerve endings positioned von Euler at the frontier of research in biogenic amines. In addition to demonstrating the presence of norepinephrine in almost all sympathetically innervated tissues of mammals, von Euler and his colleagues built upon these findings by later showing that adrenal glands of various mammalian species not only contained varying amounts of epinephrine and norepinephrine but also released them differentially, depending upon the mode and duration of stimulation. Ulf von Euler and Nils-Ake Hillarp also showed that a particulate fraction isolated from a homogenate of adrenergic nerve tissue sequestered a disproportionately large amount of norepinephrine (von Euler and Hillarp, 1956). Electron microscopy revealed that this particulate fraction was composed of granular structures that were later found to sequester biogenic amines. These studies placed a great deal of emphasis on events taking place at adrenergic nerve endings during synaptic transmission and complemented the key investigations relating to the synthesis and metabolic fate of the adrenergic neurotransmitter subsequently conducted by Julius Axelrod.
2. Julius Axelrod. Julius Axelrod (Fig. 7) was arguably one of the most beloved Nobel Laureates, who possessed all of the qualities that represent the best in our profession. His professional and personal attributes were characterized by systematic thinking, diligent effort, and humanity. Being one of the pioneers of neurotransmission and drug metabolism, Axelrod was responsible for developing treatments for the relief of pain and depression. But just as importantly, his determination in the face of the many obstacles that he encountered represents a shining example to anyone who is committed to fulfilling his/her life's ambitions.
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Axelrod was born in 1912 on the east side of Manhattan and during his early education expressed a keen interest in attending medical school. In 1933, after graduating from City College of New York with a bachelor's degree, Axelrod's application to medical school was rejected. He lamented later that in those days religious bias may have played a role in the negative decision rendered to him. Because of the Great Depression, Axelrod found it difficult to obtain a suitable position in a scientific field, although for a brief time he worked as a laboratory technician at NYU at a starting salary of $25 per month. He then went on to work for 10 years at the New York City Department of Health, where he was tasked with modifying methods that were used for evaluating the amounts of vitamin supplements added to foods. Although his work was tedious and uninspiring, the experience he gained in modifying methods for assaying vitamins proved invaluable in his later research. It was at NYU that Axlerod lost sight in one eye when a bottle of ammonia exploded in his face. This disability made Axelrod unfit for military duty, which enabled him to obtain his master's degree at NYU in 1941.
In 1946, while working at the Laboratory of Industrial Hygiene, Axelrod reached a crossroads in his scientific career when he became involved in studies concerned with the toxicity of the analgesic acetanilide. To prepare for this new project, Axelrod's supervisor suggested that he consult with Bernard Brodie (Fig. 8), Professor of Pharmacology at NYU. Brodie was also carrying out research at Goldwater Memorial Hospital, which had been established during World War II to test the clinical utility of various antimalarial drugs. Axelrod's meeting with Brodie spawned an 8-year association, first at Goldwater Memorial Hospital and then at the National Heart Institute (a branch of the NIH). Brodie, a man of extraordinary energy and an infinite source of ideas, would be responsible for directing Axelrod's early scientific career and for fostering Axelrod's long-term commitment to pharmacology.
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Relying on his previous laboratory experiences, Axelrod developed methods for analyzing acetanilide. He soon discovered that it was a prodrug and exerted its actions by being metabolized to N-acetyl-p-aminophenol (acetaminophen) (Axelrod, 1948). In the early 1970s, Johnson & Johnson marketed the drug as Tylenol, which proved to be an effective and profitable alternative to aspirin. As a result of this work, Brodie invited Axelrod to remain at Goldwater Memorial Hospital to study the metabolic fate of other analgesics. As time passed, Brodie became ever more aware of Axelrod's prodigious talents. So when Brodie was recruited to the NIH as the Chief of the Laboratory of Chemical Pharmacology in the early 1950s, he invited Axelrod to accompany him.
Using in part the knowledge gained from his studies on acetanilide, Axelrod's first project at the NIH involved determining the metabolic processes by which ephedrine and amphetamine were metabolized (Axelrod, 1954). After only 1 year, Axelrod identified an enzyme localized in rat liver microsomes that deaminated amphetamine in the presence of NADPH and oxygen (Axelrod, 1955). At about the same time, Axelrod also found that ephedrine was demethylated to norephedrine by microsomal enzymes (Axelrod 1953). These studies disclosed the extraordinary talent that Axelrod possessed as a research scientist, even though he had no doctoral degree.
During this period, several colleagues attempted to persuade Axelrod to leave Brodie's laboratory and continue his education. However, Axelrod always contrived reasons for not breaking the close ties with his overbearing mentor. In addition to not having the independence that he desired, Axelrod, after publishing 25 articles, many of them independently, was denied a promotion at the National Heart Institute because he did not have a doctoral degree. By the mid-1950s, the situation reached a climax when Brodie usurped major credit for the discovery of the microsomal enzyme system that is responsible for metabolizing drugs and other foreign substances. As time passed, Axelrod became more and more resolute in his feeling that Brodie had denied him primary credit for his discovery of the microsomal enzyme system. Although there was some difference of opinion regarding the validity of Axelrod's feeling of betrayal, the ill will that it generated caused a lasting schism between him and Brodie. Although Axelrod was ultimately afforded recognition for helping to lay the foundation of modern drug metabolism, he had become sufficiently disillusioned that he decided to take a leave of absence from the NIH and enrolled in the Department of Pharmacology at George Washington University. His tenure as a predoctoral student was unusually brief, because he had taken a number of requisite courses while pursuing his master's degree, and the doctoral thesis represented a virtual compilation of reprints of his previously published articles.
So, by 1955, at the rather advanced age of 42, Julius Axelrod had earned a doctoral degree and was now in a position to set up an independent research program (Axelrod, 2003). Because of his extensive experience working at Goldwater Memorial Hospital and then at the Laboratory of Chemical Pharmacology at the NIH, Axelrod soon was appointed Chief of the Section of Pharmacology at the National Institute of Mental Health. Fortunately for Axelrod, Seymour Kety was appointed the first Director of the Intramural Program at the National Institute of Mental Health and established a world-class program in which Axelrod would thrive.
Axelrod intended to launch an extensive study relating to the metabolism of biogenic amines. In searching for a specific project to pursue, Axelrod contemplated a biochemical analysis of the central nervous system and psychoactive drugs. In this context, Axelrod was apprised of an article that reported that epinephrine would turn pink when exposed to the air for several hours. This pink material, called adrenochrome, elicited great interest from the new independent investigator because it produced hallucinations when injected into animals. This action of adrenochrome led Axelrod to speculate that abnormal metabolism of catecholamines might provide an important clue to explaining the biochemical basis of schizophrenia. However, very little was known at the time about how norepinephrine was normally released and metabolized so that its physiological action could be rapidly terminated.
Fortunately, Axelrod had the expertise to address this question, since he had previously investigated the disposition of sympathomimetics and other drugs with similar chemical structures. However, Axelrod understood that it was essential to first define the normal characteristics of catecholamine metabolism if he was to determine whether anomalous metabolism was responsible for symptoms of schizophrenia. Although initially unsuccessful in finding an enzyme responsible for converting epinephrine to adrenochrome, in 1957 Axelrod came across a brief abstract by Armstrong et al. (1957). This abstract reported the excretion of large quantities of an O-methylated product, 3-methoxy-4-hydroxy-mandelic acid, by patients with pheochromocytoma, a tumor of adrenomedullary chromaffin cells. To verify that this compound was a metabolite of norepinephrine, Axelrod launched a study to identify an enzyme that would O-methylate catecholamines. He found that when an extract of rat liver was incubated with S-adenosylmethionine, catecholamine was metabolized to metanephrine, the m-O-methylated product of epinephrine.
On the basis of this work, Axelrod postulated the existence of a pathway by which norepinephrine is converted to a methylated metabolite, with S-adenosylmethionine serving as an obligatory cofactor. Axelrod went on to find that other catechols, such as epinephrine, dopamine, and isoproterenol could also be converted to O-methylated products. Despite initial trepidation about introducing enzymology into his research program, Axelrod proved successful in isolating and purifying the enzyme, which he named catechol-O-methyl transferase (Axelrod, 1959). Fortunately, a colleague, Bernard Witkop, helped to advance the project even further by synthesizing crystals of the enzyme. So only 2 years after gaining independence as a scientific investigator, Axelrod made a fundamental discovery that is now included in textbooks of pharmacology, biochemistry, and physiology. The success that he achieved in carrying out these studies provided him with fresh thinking for pursuing research on the mechanisms involved in adrenergic neurotransmission.
For many years, it was believed that the actions of neurotransmitters were terminated by enzymatic transformation, with ACh being cited as the classic example. While realizing that norepinephrine must somehow be inactivated for the nerve to successfully transmit a subsequent stimulus, Axelrod was also cognizant of the fact that the mechanism involved in the termination of adrenergic transmission was a much slower process than that of ACh, which was very rapidly degraded by cholinesterase. These facts suggested to him that alternate mechanisms for inactivating catecholamines might exist.
Confirming that neurochemical transmission in the sympathetic nervous system was still extant when enzymatic metabolism (by oxidative deamination and O-methylation) was blocked, Axelrod was now confronted with the basic question of how the body dealt with the nonenzymatic disposition of norepinephrine. Because of the low endogenous levels of catecholamine in urine, Axelrod was aware that he would have to employ radiolabeled catecholamine for this study. Coincidentally, at about the same time Seymour Kety had ordered from New England Nuclear a batch of [3H]norepinephrine of relatively high specific activity. His intent was to investigate possible alterations in the metabolism of biogenic amines among schizophrenics. To add to the high cost of this endeavor, Kety had also purchased an early version of a liquid scintillation counter to quantitate radioactivity.
When Axelrod made the case for using a small aliquot of the expensive radioactive material for his experiments, Kety exhibited a lack of enthusiasm about donating the valuable isotope tracer for Axelrod's experiments in which he had
