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TEACHING WITH CLASSIC PAPERS
Department of Medicine, Bachelor of Health Sciences (Hons) Program, Faculty of Health Sciences, McMaster University, Hamilton, Ontario, Canada
Address for reprint requests and other correspondence: P. K. Rangachari, Dept. of Medicine, Bachelor of Health Sciences (Hons) Program, Faculty of Health Sciences, McMaster Univ., Hamilton, ON, Canada L8N 3Z5 (e-mail: chari{at}mcmaster.ca)
Abstract
Many standard textbooks of physiology have a diagram that shows the transporting elements that lead to the secretion of HCl by the parietal cell. The transporters are neatly aligned, and students see an elegant mechanism that neatly balances the ions to maintain electroneutrality. They little realize the time and effort required to tease out each of those steps bit by bit. This essay uses three papers by Horace Davenport to highlight the experimental evidence for a crucial step in that process: the generation of H+ and HCO3– through the agency of carbonic anhydrase. All three papers form part of the classic papers available through the American Physiological Society Legacy Project.
Key words: classic papers; parietal cell; enzymatic mechanisms; stomach
THOMAS KUHN provoked considerable debate when he presented a paper entitled "The function of dogma in scientific research" at a symposium on the History of Science held at Oxford University in 1961. He considered the anomalous situation that arose in the teaching of science, where there was an excessive reliance on textbook learning. Compared with other creative fields, science education appeared to be conducted largely through textbooks written specifically for students. He noted that except in their introductions, which hardly any students read, these books rarely made attempts to describe the sorts of problems that excited practicing scientists. It was as though the members of a specific scientific discipline had agreed on what students ought to know and focused on the production of a particular mental set. So science, which was in practice exciting, innovative, and open ended, was taught somewhat dogmatically. His assertions did not go unchallenged (12). Nevertheless, there is an element of truth in what he said, and sociologists have reflected on this taken-for-granted aspect of science. In the life sciences, diagrams and cartoons are used widely to frame complex issues. These give students a scaffolding to work with, but rarely is there scope in such texts to deconstruct these figures. Many students rarely appreciate the time and effort that went into establishing the evidence on which those diagrams are based. The exuberance, excitement, and fascination of science are rarely communicated to them.
Consider, for instance, Fig. 1, which is similar to several that can be seen in a chapter on gastric secretion in any standard physiology textbook (e.g., Ref. 18). The sketch shows the parietal cell, where the combination of CO2 and H2O in the presence of carbonic anhydrase generates H2CO3, which subsequently ionizes to H+ transported out of the apical side with HCO3– on the basolateral side. Although different texts may add other elements to the sketch, they all attempt to give an outline of the basic mechanisms of HCl secretion. The transporters are neatly aligned, and the students see an elegant mechanism in place that neatly balances the ions to ensure electroneutrality. They little realize the effort that went into the characterization of each of these steps, the controversies that these engendered, and the final emergence of consensus.
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The papers themselves are fairly easy to read, but a few words could place the situation in context at the time that Davenport began his studies. By that time, the acidity of gastric juices and the presence of HCl had been well demonstrated (7, 15). Between 1823 and 1833, William Beaumont, an Army surgeon, explored the functions of the gastric lining in a young French-Canadian, Alexis St. Martin. St. Martin had been accidentally wounded by a gunshot wound, and, after Beaumont had operated on him, a fistula (an abnormal opening between two compartments) remained through which the inner lining could be seen and the stomach contents sampled. Using this window, the surgeon turned physiologist and explored the production of gastric secretions. This inspired experimental physiologists to produce similar fistulae in experimental animals. It was clear that the parietal cell in mammals or the oxyntic cell in lower vertebrates was the cell responsible for acid production. It had also been long recognized based on studying arteriovenous differences that there was an increase in HCO3– following gastric stimulation. This was referred to as the "alkaline tide."
Anyone proposing a mechanism therefore needed to consider the information available and also provide an explanation for the fact that the stomach appeared to move acid in one direction and base in the other (7).
The discovery of carbonic anhydrase in erythrocytes sparked considerable interest. This provided a catalytic mechanism for generating HCO3– from CO2 and H2O, and this H2CO3 could readily provide a source for both H+ and HCO3–. In the first paper (2), Davenport described experiments to demonstrate the presence of carbonic anhydrase in the dog gastric mucosa. He had earlier published a detailed analysis of the enzyme in other species: cats, rabbits, and rats (1). But, he needed to confirm that this enzyme was present in the dog stomach, since these were the only available experimental animals on which functional studies could be done.
Davenport began by exsanguinating anesthetized dogs, so that when he took the stomachs out, they would be essentially free of blood. This step was important since erythrocytes were known to contain the enzyme and he needed to minimize contamination. The stomachs were taken out, and cylinders were stamped from the gastric mucosa using a cork borer. These were quickly frozen, and sections were cut until there were three consecutive sections obtained that were deemed satisfactory. The first and third sections were extracted with phosphate buffer, and the extracts were assayed for the enzyme. The intermediate slice was assessed histologically for the presence of parietal cells (2).
Although the paper itself can be read on its own, it may be useful students to read two other papers: an earlier study (1) that Davenport published the previous year in the Journal of Physiology, which was the British equivalent of the American one, as well as another paper (14) in the same journal where Meldrum and Roughton described their technique for measuring the activity of the enzyme. They used a glass boat-shaped trough that had two compartments. In one compartment was placed a solution of NaHCO3 and in the other a buffer containing the material to be assayed in phosphate buffer. The boat was attached by a length of tubing to a U-shaped manometer tube that was open at the opposite end. The boat itself was placed in a water bath. When the boat was shaken vigorously, the two solutions came into contact. The enzyme in the extract reacted with HCO3– to generate CO2, which was measured manometrically.
In the earlier paper (1), Davenport also explained the rationale for the procedure followed in linking enzyme activity to the histological picture. Since there was no histochemical procedure available at the time (in fact, none became available for several decades), his approach was quite reasonable. First, he demonstrated that the number of parietal cells in three consecutive sections did not differ significantly. Therefore, by estimating the enzymatic activity in two sections and counting the number of parietal cells in the middle section, he could relate activity to these cells. He then proceeded to correlate enzymatic activity with the number of parietal cells. Linear regression gave a correlation of 0.95 with the intercept close to 0, suggesting that it was acceptable to link carbonic anhydrase with parietal cells.
Davenport applied the same technique to dogs and obtained nearly identical results (2). He also confirmed that the enzyme was inhibited by heat and by HCN. He concluded that the enzyme was present in high concentrations in parietal cells and in lower concentrations in surface cells and was probably not present in any other cell type. Since he had now demonstrated enzymatic activity in several species, he could legitimately speculate that the enzyme was present in parietal cells of all species and could play a significant role in the mechanism of formation and secretion of hydrochloric acid. To get further evidence, he mentions that when carbonic anhydrase was inhibited, the production of acid was also inhibited. This formed the subject of the next paper in the series (3), where he probed the effects of thiocyanate.
Davenport first demonstrated that increasing concentrations of thiocyanate inhibited the activity of the enzyme in chloroform extracts obtained from human and dog erythrocytes as well as the human and dog gastric mucosa (3). Students will be able to follow his arguments with ease, although they may be puzzled as to why he plots the ratio of inactive to active enzyme rather than total activity. Based on these in vitro studies, Davenport had good reason to believe that thiocyanate given in proper doses should also inhibit the enzyme in vivo and lead to a reduction in acid production.
Although students may have little difficulty with the in vitro experiments, they may need a little more background information to appreciate the in vivo experiments (15). After Beaumont had shown that a fistula could provide a window into the stomach lining, many experimental models were developed to do the same thing, particularly in dogs. In all these cases, the objective was the same: to get access to the inner lining so as to sample the gastric contents. It became quickly evident that just a fistula may not be suitable since gastric secretions were always contaminated by salivary secretions. Ivan Pavlov and his student, Pavel Khizin, in Russia devised a particular variation called the Pavlov pouch for this purpose. This was a pocket of the stomach that had been surgically separated from the body of the stomach by a mucosal septum, allowed to retain vagal innervation and muscular connection, and made to drain to the exterior. This pouch was particularly useful as its secretions mirrored those of the stomach (15). These models were very powerful as one could study the influence of the neural mechanism in conscious animals. Davenport used two such dogs in his study.
Other terms that may need some explanation are free and total acidity. Free acidity is what we would now call H+ activity, which was measured by the pH, whereas total acidity included that as well as H+ that were combined with other substances in the gastric juice. These were measured with separate indicators.
Davenport measured gastric acid production in all three dogs. In the two dogs with Pavlov pouches, the gastric juice was taken directly from the pouches, whereas in the third dog, the juice was obtained from intubation. Animals were given the inhibitor in their feed and, 16 h later, were given a subcutaneous injection of histamine to stimulate acid secretion. Chloride and thiocyanate were measured in both the gastric juice and serum. Davenport noted that thiocyanate seemed to inhibit acid production, since it took three times as long to obtain the 6 ml of gastric juice needed for analysis. He tabulated the data and showed that with increasing concentrations of thiocyanate in the gastric juice, there was a decrease in the concentration of free acid, total acid, and chloride. Furthermore, the inhibition was reversible. When toxic symptoms were seen, the administration of thiocyanate was stopped, thiocyanate was slowly eliminated from the blood, and acid secretion returned to near-control values. The data seem clear, but he does not mention anywhere how many experiments were actually done on each dog.
From these observations, Davenport concluded that "the rate of secretion of acid secretion by the gastric mucosa was directly proportional to the rate of formation of carbonic acid by the secretory mechanism."
In the final paper in the series (4), Davenport, along with Fisher, who had been his supervisor at Oxford, explored the mechanism of the secretion of acid by the gastric mucosa. In this paper, they build on the previous observations to propose a clear mechanism for the secretion of acid. Here are their exact words: "Some mechanism in the parietal cells whose rate of action is directly proportional to the rate of formation of carbonic acid in the cells secretes hydrogen ions and uses energy. The principle of electrical neutrality of solutions requires that exactly the same number of anions be present in the secretion as there are hydrogen ions. To satisfy the principle, chloride ions pass from the plasma through the cells and into the secretion, being dragged along by the positive charge on the hydrogen ions. The chloride ions removed from the plasma are replaced by bicarbonate ions formed at the same time the hydrogen ions are formed...Water moves through the cells and into the secretion without osmotic work being done on it." This, of course, provided a plausible mechanism for the alkaline tide that had become generally accepted by then.
Any student today can easily recognize what they have to say and, in many ways, what they proposed is not very different from what appears in current texts, except that the particular transporters on the apical and basolateral sides are not defined.
Their paper (4) attempted to elaborate on the mechanism, focusing on the transfer of chloride ions from plasma to gastric juice. Their objective was to demonstrate clearly that it is the movement of hydrogen ions that is primary with chloride following passively. With another halide, bromide, the authors showed that the concentration of these ions is greater than in plasma and their secretion into the gastric juice closely parallels that of hydrogen ions. Using their defined inhibitor, thiocyanate, they showed that although the halide concentration in gastric juice was lower, the ratio of bromide to chloride remained the same. Therefore, the mechanism responsible for concentrating these halide ions acts because both these ions are negatively charged.
The authors end this paper with the following words: "An incomplete theory of the mechanism of secretion of hydrochloric acid by the gastric mucosa is proposed."
It is interesting to reflect on that particular adjective–"incomplete." Through all these papers, Davenport was careful enough to recognize that the involvement of carbonic anhydrase provided a mechanism to generate hydrogen ions but "another mechanism must be found which is capable of concentrating acid against a diffusion gradient and of providing the necessary energy."
The carbonic anhydrase studies described above were important because they contain the first pieces of evidence regarding the biochemical constitution of parietal cells and, as such, garnered a good deal of attention. The early studies with thiocyanate suggested that inhibition of carbonic anhydrase would lead to a reduction in acid production and, therefore, be a useful mechanism for finding antiulcer drugs. Unfortunately, that promise was not fulfilled with the early inhibitors tested, particularly, sulfanilamides or thiophene-2-sulphonamides. Davenport describes these frustrations in his history of the field (5). He felt obliged to retract his theory and wrote a brief "In memoriam" (5), which was lauded by Hollander (9), who noted that "retraction of an idea after it has received such recognition requires scientific honesty and courage of a high order." But the enzyme did not fade away so easily. Better inhibitors were developed, and one of these, acetazolamide, generated much promise.
Once again, it was seen to be but the first step, and Hollander, who lauded Davenport's courage, wrote the following statement in 1955 in the first issue of Gastroenterology, which became the official organ of the American Gastroenterology Association (10): "We have serious hope that, within the next few years, there will be available to clinicians some chemical substance which blocks an enzyme which is more or less specific for the parietal cell–perhaps an enzyme which operates within the wall of the intracellular canaliculus, where we believe the hydrogen ion is separated to form hydrochloric acid. When this happy day comes, I think that duodenal ulcer will, in great measure, be removed from the category of surgical disease and became one which responds very early, very simply to medical therapy."
That happy day was postponed for several decades as more powerful biochemical techniques were needed to open the "black box" and reveal the molecular mechanisms involved. In his 1940 papers, Davenport noted that he defined the enzyme in dogs, since these were the best experimental models for exploring acid secretion at that time. Ironically, in that same year and in the pages of the same journal, Gray et al. (8) described their studies with the isolated frog gastric mucosa, which was to prove a more amenable model for exploring the biophysical and biochemical aspects of acid secretion. These authors followed up initial studies that had been published in French by Georges Delrue, who has rarely been cited subsequently (17).
Part of the interest in defining a molecular mechanism for acid production was to design better treatments for peptic ulcer. In that regard, carbonic anhydrase was the first but by no means the last. Although inhibitors developed for that enzyme were to prove far more effective in the treatment of other conditions, the enzyme still forms part of our current model of acid production. Treatment of ulcers now focuses on the use of proton pump inhibitors and antibiotics as part of a strategy to eradicate Helicobacter pylori. Ironically, more recent studies have shown that a periplasmic form of carbonic anhydrase may be present in H. pylori, providing it with molecular machinery to acid adapt. Reappraisals of some earlier studies with acetazolamide suggest that part of its success may have been due to eradication of H. pylori in these patients (13).
In sketching out these studies, I have focused narrowly on one small aspect of Davenport's contributions to physiology. He had an illustrious career that started at Caltech, moved onto Oxford as a Rhodes Scholar with stints at Pennsylvania and Utah, and then became the Chairman of Physiology at the University of Michigan. He was the doyen of American physiologists who had been a member of the National Academy of Sciences and former President of APS and, when he died in 2005, had been appropriately enough the William Beaumont Professor Emeritus of Physiology at the University of Michigan at an institution with which he had been associated for nearly half a century. In a brief autobiographical sketch published 20 years ago (6), he called himself a "second-class man," since that is where his examiners in Oxford placed him after the Final Honor School of Animal Physiology. In that sketch, he notes that he "never intended to be anything but an academic. An academic's obligations are teaching, service and research...," an interesting order of responsibilities. He fulfilled them all. He stood tall in more ways than one. I met him just once, over 30 years ago, when I was a postdoc in Richard Durbin's laboratory in San Francisco, CA. His talk on his studies exploring the gastric mucosal barrier was a model of lucidity. Through his many texts, he communicated complex ideas with that same facility. As an administrator, he left his mark on the Department of Physiology at the University of Michigan, where, for 22 years, he provided the right atmosphere to ensure that good work got done. He was acutely conscious of history, and, in his monumental book, he carefully traced the history of his own chosen field from the 18th century to 1975 (7).
Teaching Points
How can teachers use the papers described above in the classroom setting and what could their students hope to gain from it? That would depend to a certain extent on the objectives of the course and the level of the students. I give several options below, and individual teachers can adapt them to their particular needs.
Option 1.
Begin with a figure from a standard text showing the asymmetric arrangement of transporters across the parietal cell–but leave out the carbonic anhydrase step. Ask the students whether the information provided is adequate to explain the production of HCl. Depending on the answer given, prompt them to think about the source of the H+ and HCO3– and then introduce them to the earliest explorations of the molecular mechanisms involved, namely, the Davenport papers discussed here. Another variation would be to simply have the apical transporters shown and prompt students to think of the consequences of such a mechanism. A probing question as to what would happen to the interior of the cell if there was an excess of base left behind would provoke some discussion.
Option 2.
Start the class off with a "thought experiment." Ask them to imagine a gigantic sentient cell capable of solving problems. The cell is dropped into a beaker containing physiological salt solution. The cell has an intracellular pH of 7 and is given the task of acidifying the lumen on just one side. The students are asked to consider possible ways by which this could be done. Essentially, they are asked to "design" a parietal cell. With proper guidance, the ensuing discussions can be tailored to a consideration of the basic underlying mechanisms and the role of carbonic anhydrase. The Davenport papers can then be introduced for discussion.
I used this particular approach a short while ago with a freshman class in biology for students in a Health Sciences program. In my talk, I began with a basic overview of gastric anatomy and the cellular organization of a gastric gland. I then interrupted the talk to get the students actively engaged by introducing my thought experiment. This was based on an approach I had used to teach the functioning of the intestinal lining (16), where I had students design an "enterocyte." Here, I applied the same approach to the parietal cell.
There were close to 200 students in the class and obviously not all participated. Nevertheless, a very lively discussion ensued. One student did not think there should be any difficulty. All one needed was a channel selective for protons. This provoked a counterargument; a channel alone was insufficient, but a source of metabolic energy was needed. The source of the protons was identified as an issue, and the consensus was that it could only be from water. This raised another issue as to what would be the state of the cell if the protons were removed and the interior of the cell became alkaline. Then, the issue of back diffusion came up. To maintain an acid side, there must be some way of preventing the protons from moving around the cell. Unfortunately, I had limited time and so had to cut short the lively discussion, and I presented to them what has emerged as a consensus view, a textbook diagram. I used the opportunity to sensitize them to historical issues and pointed out to them that each one of the steps that appears so simple today required enormous effort to define. With more time, I could have gotten them to discuss the Davenport papers.
Option 3.
Divide the class into groups. Give each group figures showing the secretion of HCl from different editions of the same text (e.g., Vander). Ask students to compare the mechanisms depicted. They would quickly recognize that the role of carbonic anhydrase in generating H+ and HCO3– has remained constant, whereas other elements have been added sequentially. This would lend itself to a discussion on the evolution of our concepts highlighting the Davenport studies as being the earliest to propose a biochemical mechanism.
Once teachers have introduced their students to these papers, they can be used to illustrate several key elements about the practice of modern science. These are that science is a cumulative process, where information obtained in one context can be transferred to another. In this case, knowledge of an enzyme found in erythrocytes was found to be crucial to understanding the secretion of gastric acid. In this instance, even the same techniques were used. Also, the availability of a specific technique limits the questions that can be asked and the answers that can be obtained. Despite this, investigators can glean much by designing experiments carefully and well. Other interesting points can be discussed. When Davenport used histamine to stimulate gastric secretion, he was following what had become a standard procedure by then. Although most students may be aware of histamine's role as an inflammatory mediator, they may not be aware that its role in gastric function has an equally illustrious history. Although the precise role played by endogenous histamine in gastric function was debated, exogenous histamine had been used as gastric stimulant both in experimental studies as well as in assessing gastric function in humans.
The Davenport papers can also be used to emphasize another crucial element: the importance of clarity in communicating scientific information. This was a matter of great importance to early scientists such as Robert Boyle, who advocated the use of plain, simple language to communicate clearly and effectively. Passages from the Davenport papers could be selected as excellent examples of those ideals. This plain manner of speaking contrasts with the denser, sometimes unreadable, prose of more contemporary papers. Tracing the history further will get students to recognize that high expectations often founder on the bedrock of reality, but new information can cast a different light as long as the original information has been soundly gathered. Finally, students will realize that what ends up as a line or an item in a textbook took years to unravel.
In a sense, that itself may be the ultimate triumph; that such effort can be so easily obscured. This point was made by an outstanding biophysicist, Aharon Katzir-Katchalsky, whose life was tragically cut short by a terrorist attack at Lod airport near Tel Aviv, Israel, in 1972 (11). "Whether we like it or not," he wrote, "the ultimate goal of every science is to become trivial, to become a well-controlled apparatus for the solution of school book exercises or for practical application in the construction of engines."
Acknowledgments
The author thank Stash Nastos for help in drawing the figure and Liz Penny for cleaning up erratic typing.
Received for publication August 17, 2006. Accepted for publication April 16, 2007.
REFERENCES
-carbonic anhydrase activity of Helicobacter pylori is essential for acid acclimation. J Bacteriol 187: 729–738, 2005.
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