An Inspired Dream and a Speculative Leap
The background of a man’s achievement is usually the most interesting part of his life story. An advantage of autobiography may be that the author reports more competently than others on his inner experience and its effects; yet, even so, fiction may come into play because a retrospective report may not always truly reflect the past as it happened.
In fact, pharmacology started from physiology, used its methods and has as its main goal the revealing of physiological function from the reaction of living matter to chemical agents. The very conception to use drugs as tools in the study of function oriented me to pharmacology.
By 1914 Henry Dale had established that acetylcholine was the most potent substance known capable of mimicking parasympathetic effects. Moreover, he had also shown that noradrenaline (norepinephrine) was much more potent than adrenaline (epinephrine) in reproducing sympathetic effects. He had the two most important pieces of the puzzle in hand, but he did not risk speculating about what the whole picture might look like and he never hypothesized that these two substances might be secreted by nerves. It is true that it was not known that acetylcholine and norepinephrine were natural substances, but it is also true that Dale, by his own later admission, was especially cautious about speculating and theorizing. In later life he seems to have considered John Langley’s aversion to theorizing a shortcoming, but whether it was because of the example Langley set or because of his own personality, Dale seems to have been similarly inclined. Of course, setting high standards for the evidence necessary to support a theory is not a flaw. Theorizing in science can be either inspired insight or a seductive trap that can cause one to waste years chasing shadows down blind alleys. The problem is that it is usually impossible to know in advance which outcome it will be.
It is also true that Dale’s research was interrupted at a critical point by the outbreak of World War I. From 1914 to 1918 Dale was committed to doing research related to the war effort. After the war ended in 1918, Dale continued his wartime studies of histamine and anaphylactic shock, but there was a fifteen-year interval before he once again did research on acetylcholine. Probably the most important reason Dale did not put together all the evidence he had discovered is that the possibility that nerves secrete chemical substances was not an issue at the time. The evidence might have been right in front of him, but he did not see it because he was not thinking about it. This situation would be changed by Otto Loewi, a man with a very different temperament.
Otto Loewi (1873–1961) was born in Frankfurt into a Jewish family of successful wine merchants. In his youth he spent summers in the Haardt Mountains in southwestern Germany, where his father owned an old manor house surrounded by a large garden and vineyards. Loewi had a classical German gymnasium education with nine years of Latin and six years of Greek. He read original texts in those languages and became well schooled in classical antiquity. Although he performed well in the humanities, by his own admission Loewi did poorly in the sciences, especially in physics and mathematics.
In his youth Loewi developed what would become a lifelong enthusiasm for art. Trips to Belgium had stimulated his interest in the early Flemish painters, and he planned to study art history at the university. His father, however, wanted him to do something more practical and persuaded his son to study medicine. Loewi agreed and started medical school in Strassbourg. He was often bored, however, and would skip classes to hear lectures on the arts. As a result, he barely passed the third-year examination and decided to take a year off in Munich, ostensibly to prepare for the final year of medicine. Munich had a lot to tempt him, and Loewi could not resist spending considerable time at the opera, art galleries, and museums.3
Loewi must have done some work in Munich, because he successfully completed his final year in medicine after returning to Strassburg. He did the required thesis in pharmacology under Oswald Schmiedeberg, a renowned pharmacologist. The thesis involved studying the effects of arsenic, phosphorus, and other substances on the isolated heart of the frog. The topic had attracted him, in part, because of his reading of Walter Gaskell’s 1882 Croonian Lecture on the vagus nerve’s innervation of the frog’s heart. As noted earlier, the isolated heart of a frog had for some time been commonly used by physiologists and pharmacologists to study the effects of drugs on heart rate, and this preparation would be used by Loewi in the experiment that eventually proved that nerves secrete chemical substances.
One of Loewi’s friends in Strassburg was Walther Straub. Later their stays in Marburg overlapped.4 Straub had developed what became known as the “Straub Cannula” for collecting fluid from the isolated heart of a frog. The technique involved removing a frog’s beating heart and tying off all the blood vessels except the aorta. A glass cannula was inserted through the aorta into the ventricle of the heart and then filled with saline. The cardiac nerve in the frog contains both the vagus and the so-called accelerator (or sympathetic) nerve, and this was left attached when the heart was removed from the body. Changes in heart rate were recorded on a kymograph drum before and after the nerve was stimulated or a drug was introduced into the cannula. Loewi used this technique in a number of experiments prior to using it in the critical 1921 experiment, which initiated his investigation of neurohumoral secretions and led to the awarding of the Nobel Prize he would share with Henry Dale.
After completing his medical education in 1896, Loewi acquired additional training in biochemistry during the year following graduation from medical school. He then spent a frustrating clinical year as an assistant doctor in the city hospital in Frankfurt. At the time, there was a high mortality rate among patients with tuberculosis and pneumonia, and Loewi was frustrated by the lack of any effective treatment. He decided not to practice medicine and began to consider a research career. He was familiar with the research of Claude Bernard and others, who had used curare to infer the existence of a specialized neuromuscular site, and he was attracted by the possibility of using drugs to uncover physiological principles.5
Although he had done little research other than what was required for his medical degree, Loewi managed to convince Hans Horst Meyer, an eminent German pharmacologist, to take him on, and he started work in Meyer’s laboratory in Marburg in 1898.6 Loewi spent much of the next six years studying glucose metabolism, nutrition, and diabetes, but he also did some research on kidney and heart function. Loewi’s most notable work during this period involved obtaining evidence that animals could synthesize proteins from amino acids. Prior to that time, it was generally believed that the diet of animals had to contain whole proteins in order for them to maintain a nitrogen equilibrium. He had read a paper published by a colleague who had developed a simple procedure for decomposing proteins into their degradation products.7 This gave Loewi the idea of trying to maintain animals on a diet made up from these degradation products. Although the animals did not like the diet, he found a way to maintain them in good health and thereby proved that animals are able to synthesize the proteins they need to sustain life.8
Loewi wished to widen his knowledge of physiological techniques, and with Meyer’s help it was arranged for him to spend several months in 1902 visiting physiologists in England, mostly in Ernest Starling’s laboratory in University College London. Loewi found Starling’s enthusiasm contagious and was very much attracted by his leadership style and personality, which combined modesty with scientific erudition. This style and personality contrasted favorably with the behavior of some of the more authoritarian German professors Loewi had known. Of this experience, Loewi later wrote in his autobiographical sketch:
When I first saw Starling’s laboratory, I was surprised by the striking contrast between its extremely limited space and primitive equipment and the high level of the work that had come out of it. Like everybody else, I was charmed at first sight by Starling’s appearance, his expressive features, his shining eyes. I soon recognized that he was the most dynamic and contagiously enthusiastic man I had ever met, full of ideas and optimism, as well as very critical of other people’s and his own achievements. He possessed a serenity and simplicity, humility and a kind of naïveté that is characteristic of so many men of genius. He made the atmosphere of his laboratory warm, informal, and stimulating.9
Loewi was equally impressed by William Bayliss, Starling’s brother-in-law and frequent collaborator. It was an exciting time, as Starling and Bayliss had just discovered the hormone secretin and how it is released into the circulation from the duodenum. The work with secretin had led Starling and Bayliss to introduce the term “hormone,” which they defined as a substance secreted into the bloodstream and distributed to other parts of the body, where it often exerted profound effects.
It was during this visit that Loewi first met Dale, who, as was noted in the previous chapter, was working in Starling’s laboratory. Dale found Loewi to be a charming, witty man and an engaging conversationalist, and their lifelong friendship began at this time. Loewi had wide interests, and he made many friends during this brief visit. One of the goals Loewi set for the visit was to improve his English. However, Dale later reported that he was impatient with being corrected, saying: “I have not the time to learn English correctly, I just wish to speak it fast.”10
The following year Loewi visited England again. Humorous reports of his improvised English had spread around the physiology community. When Loewi heard that Sir John Burdon Sanderson, Regius Professor of Medicine at Cambridge, wanted to meet him, he felt honored. Later, when he found out that what Sanderson really had wanted was to experience a sample of his English, he took it with good humor. He enjoyed and was immediately comfortable with the informality of English physiologists and especially their uninhibited style in exchanging ideas with students as well as colleagues. This was quite different from the tradition in Germany, where the “Herr Doktor Professor” was rarely challenged.
On this second trip to England, Loewi spent time with John Langley and Thomas Elliott in Cambridge. Dale later wrote that the possibility of the existence of neurohumoral secretions may have been planted in Loewi’s mind during this meeting with Elliott. There is no record of their conversation, but years later Dale described a conversation he had had with Loewi at that time:
Loewi was in Langley’s department at Cambridge; and I remember that when Loewi came back to London, before he returned to Marburg, he and I had dinner together one evening and he told me how much he enjoyed the discussion with Elliott and what a high opinion he had formed of his promise. The two had met and interchanged ideas; and the idea of chemical transmission from nerve endings might surely have had some mention between them, though we cannot expect either of them to have any memory now of such talk about it, a half century later. What we do know is that Loewi, later in the same year, threw off the suggestion that muscarine might be liberated to transmit vagus effects.11
Dale was referring to the fact that, in 1904, a year after Loewi and Elliott met in Cambridge, Loewi had remarked to Walter Fletcher, a colleague of Dale, that when the vagus nerve inhibited heart rate a chemical like muscarine might be involved.12 It is impossible to know whether Loewi’s comment had been influenced by Elliott’s work. It could have been an independent thought as Loewi was well aware that Oswald Schmiedeberg, his thesis mentor, had demonstrated more than thirty years earlier that muscarine had the same effect on the heart as vagus nerve stimulation.13 Loewi’s remark, however, was only a casual observation that he did not pursue and apparently completely forgot about until he was reminded of it in 1929.
In 1905, when Hans Meyer accepted a position as professor of pharmacology at the University of Vienna, Loewi followed him. Loewi and Meyer continued to do collaborative research and co-authored several articles on the relative potency of a number of synthetic amines, especially “arterrenol,” which is another name for noradrenaline (norepinephrine). At the time Loewi, like Dale, thought that noradrenaline was just an interesting drug, not a natural substance.
In Vienna, Loewi also collaborated on several studies with Alfred Froelich, who was already well known for the syndrome that bears his name. The Froelich Syndrome is an endocrine disorder characterized by delayed puberty, undersized testes, and obesity. Loewi and Froelich studied the effects of various drugs on autonomic nerves. In one important study they demonstrated that prior treatment with cocaine increases the response to adrenaline, a phenomenon now known as “cross-sensitization.” This cocaine-enhanced response to adrenaline is quite specific and, as will be explained in a later chapter, could be incorporated into a technique for detecting the presence of small amounts of adrenaline. Loewi also worked with the drug eserine (physostigmine), which enhances the response to parasympathetic nerve stimulation.14 These studies would later be useful in proving that nerves secrete chemical substances, but at the time this was not something that Loewi was thinking about.
In 1908 Loewi married Guida Goldschmiedt, the daughter of a professor of chemistry in Prague, and the following year they moved to Graz in Austria, then part of the Austro-Hungarian Empire, where Loewi had been appointed professor and head of pharmacology. Graz is an old and picturesque city built on a hill. Although it was not as cosmopolitan as Vienna, Graz’s university and medical school were highly respected and the city had a good opera company and excellent theater offerings. Loewi was happy there. He had a large laboratory and excellent students and collaborators. He and his wife lived in Graz for thirty years, and three sons and a daughter were born to them there. In 1938, however, as will be described in a later chapter, he had to leave Austria after the Germans annexed that country.
Figure 5.1 The young Otto Loewi, whose passion was art (left); and as an eminent fifty-six-year-old pharmacologist visiting Boston in 1929 (right). Courtesy of the Wellcome Trust.
Loewi was a lively lecturer, popular with students and highly respected by the medical faculty, which elected him dean in 1912. He continued to work on carbohydrate metabolism and experimental diabetes, but he also did research on the action of digitalis on the heart and published several papers on how calcium and other drugs affect the vagus nerve’s capacity to slow heart rate. This work further prepared Loewi to pursue his seminal experiment, which eventually proved that the vagus nerve secretes acetylcholine. The idea for this experiment occurred to him in a dream at a time when he was not consciously thinking about neurohumoral secretions. Later in his life Loewi remarked that he had occasionally thought about the possibility of neural secretions, but because he saw no way of attacking the problem: “it entirely slipped my conscious memory until it emerged again in 1920.” Loewi described the experience of his dream on many different occasions, always in a similar manner:
The night before Easter Sunday of that year I awoke, turned on the light, and jotted down a few notes on a tiny slip of thin paper. Then I fell asleep again. It occurred to me at six o’clock in the morning that during the night I had written down something most important, but I was unable to decipher the scrawl. The next night, at three o’clock, the idea returned. It was the design of an experiment to determine whether or not the hypothesis of chemical transmission that I had uttered seventeen years ago was correct. I got up immediately, went to the laboratory, and performed a simple experiment on a frog heart according to the nocturnal design.15
Loewi’s version of these events seems to be somewhat more dramatic than what actually occurred. Dale, who remarked later that he was one of the first to hear Loewi’s account of “the remarkable story of the dream,” recalled that Loewi had originally told him that when he awoke on the second night at 3:00 A.M. he made careful notes so that he would have no trouble deciphering his thoughts the next morning.16 Loewi’s more dramatic later version is that he went directly to the laboratory at 3:00 o’clock in the morning and that by “five o’clock the chemical transmission of the nervous impulse was conclusively proved.”17
Moreover, Loewi’s recollection that the dream occurred “on the night before Easter Sunday” does not seem to be accurate. He performed the experiment in late February, perhaps extending into early March, and he sent the manuscript describing the experiment to the journal later in March. The article is marked as having been received in the editorial office of Pflügers Archiv on March 20, 1921. However, in 1921 Easter Sunday was on March 27, a week after the manuscript was received by the editor.18
While these discrepancies do not subtract from the importance of the experiment, they would seem to reflect the artistic bent of Loewi’s temperament, which might have made it difficult for him to resist making the description of what transpired even more dramatic than it actually was. Dale, in contrast, would probably never have strayed from the facts, no matter how inconsequential. It may also be true that Loewi’s different temperament played a role in allowing him to risk drawing a conclusion from quite preliminary and inconclusive experimental results.
The experiment inspired by the dream was also described by Loewi on many occasions:
The hearts of two frogs were isolated, the first with its nerves, the second without. Both hearts were attached to Straub cannulas filled with a little Ringer solution. The vagus nerve of the first heart was stimulated for a few minutes. Then the Ringer solution that had been in the first heart during the stimulation of the vagus was transferred to the second heart. It slowed and its beats diminished just as if its vagus had been stimulated. Similarly, when the accelerator nerve was stimulated and the Ringer from this period transferred, the second heart speeded up and its beats increased.19
Loewi reported that he used the hearts from fourteen frogs of two different species (Rana esculenta and R. temporaria) and from four toads.20 As already noted, in these amphibians the heart is innervated by a mixed nerve; the vagal-sympathetic trunk contains the sympathetic nerve as well as the vagus, a parasympathetic nerve. For that reason, electrical stimulation of the nerve trunk sometimes produced an acceleration and sometimes a slowing of heart rate, and it was not possible to predict which would occur.21 Loewi initially gave the name “Vagusstoff” to the humoral substance that slowed heart rate, and he called the substance responsible for accelerating heart rate “Acceleransstoff.”
It was certainly an exaggeration to state, as Loewi did many times, that the initial experiment had “immediately and conclusively” proven “that the nerves do not influence the heart directly, but liberate from their terminals specific chemical substances.” In fact there was a protracted and sometimes acrimonious dispute over Loewi’s data and what, if anything, his experiment had proved. It took almost a decade before Loewi’s conclusions were widely accepted.
Many physiologists were simply unwilling to believe that nerves produce their effects by secreting chemical substances, let alone that different nerves secrete different substances. Others reported that they were unable to replicate Loewi’s results.22 There are many reasons why the initial attempts to replicate Loewi’s results may have failed. First, as it was later learned, the “Vagusstoff” turned out to be acetylcholine, a neurotransmitter that is, as Dale hypothesized in 1914, rapidly degraded by an enzyme now known to be cholinesterase. Loewi was most fortunate in using frogs and toads in his experiment and particularly in working with these cold-blooded animals during February and early March. In the colder months, acetylcholine is said to be more stable because there is less cholinesterase available. Had Loewi’s initial experiments been done during the warmer months, it is unlikely that the experiment would have demonstrated any effect at all. This factor may have been partially responsible for the failure of others to replicate his results. It is also now known that frog species differ in the amount of cholinesterase available, and this may have contributed to the failure to confirm Loewi’s initial report. Loewi also pointed out in 1924 that successful results required the selection of hearts that are maximally responsive.23
Moreover, Loewi pipetted the fluid from the stimulated heart and then often delivered it back to the same heart, although he did use a second heart in some tests. Thus, in much of his initial work, the same heart served as both donor and recipient.24 This meant that Loewi had to withdraw some fluid from the ventricle of a heart that had been stimulated and wait several minutes until the heart rate returned to normal base levels. There are good reasons for questioning whether the little acetylcholine released could have remained active over this period of time. Even Loewi admitted later that it was remarkable that the initial experiment worked:
If I had carefully considered in the daytime I would undoubtedly have rejected the kind of experiment I performed. It would have seemed likely that any transmitting agent released by the nervous impulse would be in amount just sufficient to influence the effector organ. It would seem improbable that an excess that could be detected would escape into the fluid which filled the heart.25
Another problem that may have been responsible for a failure to replicate Loewi’s results was the physical disturbance produced when fluid is pipetted back into a heart. It was found that even the small changes in hydrostatic pressure introduced by dripping fluid into the cannula could produce changes in heart rate, even if there is no active substance in the solution. The recipient heart sometimes slowed down when the perfusate came from a heart beating at a normal rate or when the vagus nerve had not been stimulated.26
Considering all that could have gone wrong, Loewi was clearly lucky that his initial experimental procedure produced any interpretable result. Actually, the data presented in the first publication was not at all convincing, as it consisted solely of a few not very persuasive kymographic tracings of changes in heart rate. Moreover, different results were obtained with the two frog species and the results with the toads were also different.27 A more cautious scientist would probably have collected much more additional evidence, and ruled out alternative explanations, before submitting a paper proposing such a bold and controversial hypothesis.
Loewi did include the phrase “First Communication” in the title of the article, indicating that more information was being collected. Indeed, Loewi soon published additional papers, each with more evidence on the same effect. By 1926 Loewi had published eleven communications in a numbered series all bearing the same introductory title: “Concerning a humoral transfer of a heart nerve effect” (in German).28 He also wrote another six articles related to different aspects of the same effect during this period. It was as though the criticism he received as well as the recognition of the potential importance of the work had provided an energy and focus to Loewi’s work that exceeded anything evident before this.
There were so many criticisms of Loewi’s claims that in 1926 he was asked to respond to his critics by demonstrating the experiment at the twelfth International Physiology Congress held in Stockholm. By this time Loewi had apparently removed many of the sources of difficulty in replicating his results, but he was nevertheless somewhat hesitant about accepting the invitations, remarking that, “Like most experimenters, I had experienced time and again that experiments before a large audience often fail although they never did in the rehearsals.”29
Nevertheless, Loewi was successful in demonstrating his results eighteen times during the course of the Stockholm meeting. As Loewi later told William Van der Kloot, his colleague in pharmacology at New York University, he “had been obliged to stand at one end of the room and simply give instructions, so that the possibility of his secreting some chemical under his fingernails and dropping it on the preparation could be eliminated.”30 One of the changes in his procedure that made it easier to replicate his initial results was the addition of physostigmine (eserine) to the solution to prevent inactivation of the “Vagusstoff” (acetylcholine).31 Ulf von Euler, who was at the congress in Stockholm, later wrote that Loewi’s demonstration was what first aroused his interest in neurohumoral transmission.32 Later, von Euler, who spent part of 1930 and 1931 in Dale’s laboratory, proved that the sympathetic neurotransmitter is noradrenaline (norepinephrine), not adrenaline (epinephrine).33
Although R. H. Kahn, a physiologist in Prague, reported in 1926 that he had confirmed Loewi’s results using an improved technique, there continued to be a number of reports of failure to replicate the effect.34 Loewi’s successful demonstrations in Stockholm had by no means convinced all his critics. In 1927, for example, Louis Lapicque, a renowned Parisian neurophysiologist, declared that the idea of humoral transmission of nerve impulses was “unthinkable.”
In 1932 W. A. Bain in Edinburgh modified the apparatus used by Kahn and was able to obtain even more robust evidence supporting Loewi’s conclusion about neurohumoral secretions. In the introduction to his paper, Bain described the shortcoming in Loewi’s method that might have accounted for the difficulty in replicating his results:
Many who have attempted to repeat Loewi’s fundamental work on the frog-heart have found it difficult to get confirmative results, and some have failed to get any results at all…. Others again, while obtaining a percentage of positive results, are unable for various reasons, to accept Loewi’s conclusions…. Loewi, in his experiment, collected the fluid from a vagus-stimulated heart and applied this to the same or to another heart. He considered that it was important in such experiments to apply the smallest possible quantity of fluid in the nerve-stimulated heart, his idea being that the smaller the quantity of fluid in contact with the heart the more concentrated would be the resulting neuromimetic fluid, and thus the more definite the effects on the heart to which it is applied.
Nevertheless it would appear that in Loewi’s method a considerable amount of the vagus substance formed during stimulation must be destroyed before it can be applied to another heart…. From this consideration it appeared possible that better results might be obtained by a method in which the irrigating fluid was passed somewhat rapidly through the heart in the hope that the “vagus substance,” almost as soon as it is formed, would pass into the irrigating fluid and thus away from at least one of the factors operating for its destruction.35
Loewi never published a diagram of the technique he used, and in most later accounts of his work the improved technique introduced by Bain (see fig. 5-3) is reproduced, with the implication that it is the apparatus Loewi had originally used.
Figure 5.2 Otto Loewi demonstrating neurohumoral transmission in 1926 at the International Physiology Congress in Stockholm. Picture reproduced (without credits) in B. Holmstedt and G. Liljestrand, Readings in Pharmacology (New York: Macmillan, 1963), p. 194.
Figure 5.3 Apparatus used by R. H. Kahn. This was an improvement over the technique used by Otto Loewi and produced much more reliable evidence of neurohumoral transmission.
The same year that Bain replicated Loewi findings with his improved technique, Leon Asher of Basel wrote that one had to be delusional to believe that Loewi’s experiment proved anything:
Only the failure of the necessary experimental critique and a little “autistic” thinking by definition of Bleuler could allow one to consider experiments which in the present situation do not prove anything, much less than absolutely proving the existence of a vagus hormone.36
Later that year Asher was finally convinced that nerves do indeed secrete inhibitory and excitatory substances. Although he reluctantly agreed that Loewi was right, Asher felt compelled to add that “it was just a piece of luck,” as Loewi’s experiment had not proven anything. Much earlier, in 1917, Asher had tried an experiment somewhat similar to that of Loewi’s, but it failed to prove anything and he may have been reluctant to acknowledge Loewi’s success where he had failed.
Early on Loewi had recognized that there were two possible origins of the humoral substances responsible for slowing and speeding up heart rate:
On the one hand, they may originate directly from the effect of nerve stimulation independent of the type of cardiac activity…. From a different viewpoint, there is also the possibility that these substances are only products of the specific type of cardiac activity which is released by the nerve impulse; under such circumstances, therefore, the identification of their action with the nerve stimulus would be only accidental, so to speak.37
Loewi was referring to the persistent problem of trying to determine whether the humoral substance detected originated in the innervated organ (in this instance the heart muscle) or from the nerve.
In his second publication in the series on neurohumoral substances, also published in 1921, Loewi attempted to rule out the innervated heart as the source of the chemical substance. When he paralyzed the donor heart with a high dose of nicotine, for example, he found that it was still possible to transfer the “Vagusstoff” after the vagus nerve was stimulated. Although the evidence did not completely rule out the possibility that the “Vagusstoff” had come from the heart, Loewi was apparently satisfied that he had shown that it was, at least, not dependent on any change in heart rate.
In the same second publication in the series, Loewi began the process of identifying the chemical nature of “Vagusstoff.”38 He was well aware that Henry Dale’s 1914 publications on acetylcholine had provided a good basis for suspecting that it was the most likely candidate for being “Vagusstoff,” and he began to accumulate evidence that this was the case. He showed, for example, that of all the known substances that could mimic the action of the vagus nerve—muscarine, pilocarpine, choline, and acetylcholine—only acetylcholine satisfied all the pharmacological tests.39 Choline had been a possibility until he demonstrated that not enough was present in the “Vagusstoff” to produce the effect observed. Moreover, he noted that choline, even in high concentrations, has only a relatively weak action on the heart and hardly ever arrests it in diastole.
In regard to the site of action of “Vagusstoff” and “Acceleranstoff,” Loewi raised the long-standing question of whether the action of acetylcholine is on the heart muscle or back on the nerve that secreted it. He answered this question, much as Langley had done many years earlier in another context, by demonstrating that “Vagusstoff” had the same effect even after he had severed the vagus nerve and it had degenerated. Loewi concluded that neither vagal nor sympathetic substances act upon the nerve, but directly on the effector organ, in other words, the heart.
As already noted, Loewi had, with his colleague Emil Navratil, demonstrated that physostigmine (eserine) increases the response to “Vagusstoff.”40 It had previously been shown that this drug potentiates the slowing of the heart caused by vagal stimulation. Since it was known that eserine (physostigmine) inhibits the esterase enzymes that inactivate acetylcholine, Loewi and Navratil postulated, as had Dale much earlier, that the heart probably contains an endogenous esterase, which they called “cholinesterase.”41 In the eleventh paper in their series, Loewi and Navratil wrote:
We proposed in our tenth paper that the administration of vagal substance or acetylcholine produces only a very short-lasting effect on the heart since both were speedily metabolized. We therefore investigated whether the long duration of action of vagal substance and acetylcholine, which is seen when physostigmine or egotamine are given beforehand, was due to an inhibition of their metabolism. These experiments showed that the metabolism of vagal substance and acetylcholine by heart extracts, was indeed inhibited by physostigmine and ergotamine. This is further evidence for the analogous behavior of the two substances [i.e., “Vagusstoff” and acetylcholine].42
It was presumed that some enzyme like cholinesterase must be responsible for metabolizing acetylcholine, and in 1930 Loewi reported finding traces of cholinesterase in the heart.
While eserine (physostigmine) enhanced the slowing effect following stimulation of the vagus nerve, atropine blocked this response. The atropine and eserine were specific in their effects, as they had no influence on the acceleration of heart rate produced by stimulating sympathetic nerve fibers. Loewi and his colleagues found, for example, that eserine did not enhance the heart-rate response to muscarine or any of the other candidates being considered, except for choline, which had already been ruled on other grounds. All of these tests had provided strong support for the conclusion that acetylcholine was mediating the slowing of heart rate produced by vagal stimulation.
Loewi also demonstrated that the drug ergotoxine, an alkaloid substance that Dale had earlier shown would block and even reverse the effect of adrenaline, prevented the acceleration of heart produced by sympathetic stimulation.43 Even though the results with ergotoxine suggested that the Acceleransstuff is adrenaline, Loewi hesitated to commit himself. He did conclude that some amine substance closely related to adrenaline, if not adrenaline itself, was probably involved.44
Loewi had taken a risk in initially claiming that he had demonstrated the existence of neurohumoral secretions based on what was initially a rather weak experiment, which lacked many controls that were needed to make it convincing. However, after being challenged, he focused all work in his laboratory on collecting supporting evidence and eventually proved that he was right, but it took about a decade before he had convinced the hardened skeptics.
Although neurophysiologists eventually conceded that Loewi was correct in his conclusion that neurohumoral secretions regulated heart rate, many regarded this as a special case that did not apply to other peripheral nerves, let alone to the central nervous system. Through 1933, even Loewi was reluctant to extend the idea of neurohumoral transmission, probably regarding it, as most neurophysiologists did, as too slow a process for transmission at most synapses. Loewi was particularly skeptical of the possibility of neurohumoral transmission at either the autonomic ganglion or at the synapse between spinal motor nerves and skeletal muscles. He expressed this reservation in his Harvey Lecture in 1933, when he stated that: “I personally do not believe in a humoral mechanism in the case of striated muscle.”45
This was at the very time Dale and his colleagues had started to collect evidence that acetylcholine might be involved in innervating skeletal muscles. Dale once described Loewi’s reluctance to accept the possibility that acetylcholine is secreted by spinal motor nerves as “an attitude of almost obstinate skepticism.” Dale wrote that Loewi seemed almost frightened by the idea:
Loewi seems to have taken alarm, for the time being, at the thought of such a possible extension of his discovery, and to have gone to the length of establishing his own alibi by a public disclaimer of belief in chemical transmission at the motor nerve endings.46
In 1935, however, Loewi made it clear in his Ferrier Lecture to the Royal Society that he was now convinced that acetylcholine was secreted at these additional sites.47 This evidence, collected in Dale’s laboratory, led to Dale sharing the Nobel Prize with Loewi, and its discovery is described in the next chapter.