Journal of the American Society of Nephrology
Volume 12   February 2001

The discovery of an endocrine link between the heart and the kidneys has its basis in the electron microscopic finding that the striated muscle cells of the cardiac atria in mammals are differentiated both as contractile and as endocrine cells. The demonstration that atria produce polypeptide hormones was established with the discovery of atrial natriuretic factor (ANF). ANF is the founder member of the ANF family of natriuretic peptides that have very important functions in the modulation of volume regulation and cardiovascular growth. 

    The unfolding of this discovery, as many others, has a great deal of human content that often is lost in our technical writings. I hope that students and investigators who are just starting out will find inspiration (and consolation) in the informal account of the ANF discovery that follows.

    When I arrived to the pathology department at Queen's University [Kingston] in 1968, fresh from obtaining a degree in clinical biochemistry from the Faculty of Chemical Sciences in Cordoba, Argentina, my supervisor, Sergio Bencosme, was interested in the functional morphology of the endocrine pancreas. As an aside, Bencosme had taken up the question of secretory-like differentiations found in atrial cardiocytes, a fact known since the early days of electron microscopy and manifesting itself most notably by the presence of storage granules known as "specific atrial granules" whose function was a mystery. He and many other notables, including George Palade then at the Rockefeller Institute in New York, could not advance past their morphologic description. Others considered the atrial granules as an evolutionary remnant. 

    I found myself unable to ignore a secretory phenotype and made it a personal challenge to demonstrate that a combination of morphologic and biochemical techniques would unravel the functional nature of the atrial granules. Perhaps I was influenced by the great endocrine work of Argentinean Nobel Prize laureate B. Houssay, who is an icon of academic excellence for anyone born in Argentina. And so the ANF saga began. It would take 12 yr of investigations (with only 1 month of holidays) before the nature and function of the dual secretory-contractile nature of atrial cardiocytes would become apparent (for a review, see reference ^[1] ).

    I began my studies on the possible secretory function of the heart by trying to isolate the atrial granules armed with the papers produced by Christian De Duve on isolation of subcellular organelles and the paper by Blascho on isolation of adrenal chromaffin granules. There were literature data that hinted that the atrial granules were a storage site for catecholamines, but a careful read of the literature was not very convincing in that sense. At any rate, this was a hypothesis to test and this turned into my project for my master of science project. 

    The isolation of the granules was particularly difficult because they were immersed in the great tangle of myofibrils and connective tissue that represents a homogenate of the heart muscle. Therefore, it took me 2 yr and quite a few 20- to 60-rat ultracentrifugation runs to obtain the purified granules. Bigger animals (cow hearts were suggested many times) were of no use because there is an inverse relationship between the number of atrial granules found in atrial cardiocytes and the size of the animal. Because I had no biochemical marker for the granules, the most tedious job that I found was to look at every fraction by electron microscopy to see where the granules went with the many variations to the isolation technique ^[2] . For this purpose I developed an electron microscopy embedding technique to deal with subcellular fractions. After many trials, I was able to isolate and purify the granules and proved by biochemical means that the granules did not contain catecholamines ^[3] . This was success in one sense, but it also meant that I had no hypothesis left to test.

    I set out to develop techniques to visualize specifically the atrial granules at the light microscopic level. I reasoned that with such a technique, one could correlate the distribution of the granules with histochemical reaction products. It helped me enormously that, by intervention of Divine Providence I am sure, I had managed twice during my undergraduate years to end up working as a research assistant in pathology departments, where I learned many histologic techniques.

    I developed the first method to stain specifically the granules at the microscopic level using lead-hematoxylin following a paper that my wife had found to stain cells in the pituitary gland ^[4]. The stain aldehyde-fuchsin also provided a visualization of the granules. With these techniques at hand, I carried out a whole battery of histochemical investigations ^[5]. A number of cytochemical properties of the atrial granules thus were uncovered. These investigations would later help me to isolate and purify ANF. For example, the poor stainability of the atrial granules following Bouin's fixative (a fixative that contains acetic acid) suggested that the granules' content was soluble in acetic acid. Indeed, ANF and brain natriuretic peptide are highly soluble in acetic acid, which is the basis for extractants of these hormones. 

    Altogether, these cytochemical studies plus the ones that I carried out later as an independent investigator provided evidence that the atrial granules stored a random-coiled, basic polypeptide that contained cystine and tryptophan. Autoradiographic studies with radiolabeled leucine showed that the content of the granules had a high turnover in a manner similar to secretory cells ^[6]. All of these properties were confirmed later by biochemical means following the isolation of ANF.

    By this time (1973), I had finished my doctoral thesis, my first of our five children had been born, and we had purchased our first home. I was then offered a position to continue at Queen's, moving to the Pathology Laboratory at Hotel Dieu Hospital, a teaching hospital associated with Queen's University, as an assistant professor of pathology. I was to help develop research at this hospital, and it was a leap of faith of the chairman of pathology, Nathan Kaufman, to put me there and for which I am very grateful. Years later, after the discovery of ANF, Nate reminded me that during my thesis defense, I had guessed that the atrial granules, because of their location, might be involved in sensing changes in volume load. I had forgotten that.

    A service-oriented hospital, Hotel Dieu was not the most propitious place for a young scientist. I was given an office, half a lab bench, an old incubator, and a microscope to start. Mine was a windowless office in the basement, across from the autopsy room. The smell of formalin was a constant companion. Looking back, this isolation helped me in continuing with the goal of establishing the endocrine function of the heart.

    My first grant application to the Medical Research Council as an independent researcher was on the status of the cardiac adrenergic innervation in heart failure. This is the reason that I have a publication on a new model for inducing heart failure in the guinea pig. This theme was really a safety net in case the atrial granule business did not work out. As it turned out, the reverse occurred.

    I secured funding from the Heart and Stroke Foundation to continue my graduate studies on atrial granules, but I knew that their patience was wearing thin on this theme. I also collaborated with Jack Kraicer of the physiology department at Queen's on the morphology of the pars intermedia of the pituitary gland. My wife, who had started working with me, and I were able to define a system of canaliculi in this avascular gland using extracellular space markers. In the process, we discovered a new cell type for which my wife developed a silver impregnation technique to demonstrate it at the light microscopic level ^[7].

    Although the nature of the granule's content seemed reasonably well defined by the histochemical studies, there was still no hint as to their function. However, we now had something that was not available previously. Namely, a stain that could demonstrate the granules at the light microscopic level; therefore, we could develop a quantitation procedure to assess changes in the number of granules after different experimental procedures using the light microscope. The difference between a morphometric procedure at the light microscopic level and procedures at the electron microscopic level is that the sample size is made much larger at the light microscopic level. This was particularly important for quantitation of atrial granules because of their irregular distribution in the atria and even within the same cell. 

   The hypothesis thus developed was that the atria produced and stored a polypeptide that helped regulate water and electrolyte balance given the nature of the contents revealed by histochemistry and the changes in the number of granules revealed by the morphometric technique after procedures that were known to alter water and electrolyte balance. I thought that the easiest way for a cardiac hormone to modify water and electrolyte balance was to target the prominent role of the kidneys in maintaining water and electrolyte homeostasis. Besides, the atria were in an ideal spot to sense changes in venous return. Looking for a bioassay for diuretic substances, I found that Harald Sonnenberg of the Department of Physiology at the University of Toronto, whom I did not know, was searching for a natriuretic hormone and had a rat bioassay for that purpose. I phoned him and related to him my quest and hypothesis.

    Because the existence of atrial granules was not widely known, even by morphologists, it was specially generous of Harald to accept my invitation "to take a shot in the dark." He invited me to give a seminar in Toronto, and we agreed that I would send him atrial extracts. The first extracts were, in fact, atrial granule extracts that contained a high concentration of potassium chloride because of the composition of the solutions used for isolation. This promptly killed the bioassay rats upon injection. I then more or less supplicated Harald to be patient and please to try just crude extracts of atria, and of ventricles as a control, prepared in simple phosphate-buffered saline.

    Some weeks went by, and then, to my unbelieving ears, Harald phoned me to say that the injection of atrial extracts produced a diuresis and natriuresis that was immediate and incredibly strong, just like furosemide. Always a worrier, I started to wonder about what contamination would produce such effects. We repeated the experiments many times in my lab, and the results were equally impressive. Also, proteinase destroyed the activity, which went right along with the hypothesis that the atrial granules contained a polypeptide hormone.

    The potential importance of the finding prompted us to send our findings to the prestigious Journal of Clinical Investigation. It was tersely rejected in a letter dated May 28, 1980, because the finding "was not thought to be suitable for publication." Because I had disclosed the findings previously at a meeting of the Canadian Society for Clinical Investigation, we decided to publish the findings as quickly as possible. For this reason, it was sent to Life Sciences, where it was quickly accepted and published in 1981 ^[10]. By 1983, the first publications on ANF from other centers started to appear. Not a single laboratory failed to confirm our findings, given that the natriuretic and diuretic activities of atrial extracts were so powerful that nothing short of a dead bioassay rat could stop such action.

    The article in Life Sciences [1981] spurred a flurry of activity and went on to become a Citation Classic as qualified by the Institute of Scientific Information. Needless to say, the researchers in the hypertension field were ready to exploit the finding of a hormone that was diuretic, natriuretic, and hypotensive. It is of interest to note the different reactions by different groups of investigators. 

    Our laboratory also was the first to isolate, purify, and sequence ANF ^{[11] [12] [13]}. The way that this was accomplished was not less heroic than the 12 yr of work that preceded the ANF discovery. It was very opportune for me to find in the United States a company that provided us with rat atria. In total, approximately 200,000 rat atria were used. It was also fortunate that the techniques for isolation of peptides by HPLC were coming into use. The only problem was that I did not have an HPLC. The clinical laboratory in our hospital, however, had just purchased one to measure theophylline in serum. Luckily, I was put in charge of that technique, so it was not very noticeable that I came during the night to reconfigure the machine and fitted it with a chromatographic column to purify peptides.

    Three people essentially did the isolation and purification of ANF in my laboratory: my wife would extract the atria, I would purify the extracts, and a technician would test the different fractions obtained during purification in the bioassay rat. No other resource or person was involved in this effort.

    Once the peptide was purified to chemical homogeneity, my next problem was to sequence it. The only person at Queen's involved in amino acid analysis and protein sequencing was Geoff Flynn, to whom I offered collaboration. We had various false starts because of antiquated equipment, both in the amino acid analysis and in the sequence results. The problems were resolved when we obtained funding from the government of Ontario to purchase a gas phase sequencer; thus, we were the first laboratory to produce a sequence in 1983 ^[13]. The Japanese workers produced the human sequence the following year.

    Students often ask for advice to succeed in research, and my standard answer is, "Have a dream, don't think small, work hard, and believe in yourself." I finish this in my mind with, "...and pray that you are right."

References

1. de Bold AJ, Bruneau BG: Natriuretic peptides. In: Handbook of Physiology, Section 7: The Endocrine System, Volume III: Endocrine Regulation of Water and Electrolyte Balance, edited by Fray JCS, Goodman MH, New York, American Physiological Society by Oxford University Press, 2000, pp 377-409  

2. de Bold AJ, Bencosme SA: Studies on the relationship between the catecholamine distribution in the atrium and the specific granules present in atrial muscle cells; 1. Isolation of a purified specific granule subfraction. Cardiovasc Res 7; 351-363, 1973  

3. de Bold AJ, Bencosme SA: Studies on the relationship between the catecholamine distribution in the atrium and the specific granules present in atrial muscle cells: 2. Studies on the sedimentation pattern of atrial noradrenaline and adrenaline. Cardiovasc Res 7: 364-369, 1973  

4. de Bold AJ, Bencosme SA: Selective light microscopic demonstration of the specific granulation of the rat atrial myocardium by lead-hematoxylin-tartrazine. Stain Technol 50: 203-205, 1975  

5. de Bold AJ, Raymond JJ, Bencosme SA; Atrial specific granules of the rat heart: Light microscopic staining and histochemical reactions. J Histochem Cytochem 26: 1094-1102, 1978  

6. de Bold AJ, Bencosme SA: Autoradiographic analysis of label distribution in mammalian atrial and ventricular cardiocytes after exposure to tritiated leucine. In: Recent Advances in Studies on Cardiac Structure and Metabolism. The Cardiac Sarcoplasm, edited by Roy PE, Harris P, Baltimore, University Park Press, 1975, pp 129-138  

7. de Bold ML, de Bold AJ, Kraicer J: Demonstration of stellate cells of the pars intermedia of the pituitary gland using a new silver impregnation technique. Stain Technol 59: 49-52, 1984  

8. de Bold AJ: Morphometric assessment of granulation in rat atrial cardiocytes: Effect of age. J Mol Cell Cardiol 10: 717-724, 1978  

9. de Bold AJ: Heart atria granularity effects of changes in water-electrolyte balance, Proc Soc Exp Biol Med 161: 508-511, 1979  

10. de Bold AJ, Borenstein HB, Veress AT, Sonnenberg H: A rapid and potent natriuretic response to intravenous injection of atrial myocardial extracts in rats. Life Sci 28: 89-94, 1981  

11. de Bold AJ, Flynn TG: Cardionatrin I--A novel heart peptide with potent diuretic and natriuretic properties. Life Sci 33: 297-302, 1983  

12. Flynn TG, Davies PL, Kennedy BP, de Bold ML, de Bold AJ: Alignment of rat cardionatrin sequences with the preprocardionatrin sequences from complementary DNA. Science 228: 323-325, 1985  

13. Flynn TG, de Bold ML, de Bold AJ: The amino acid sequence of an atrial peptide with potent diuretic and natriuretic properties. Biochem Biophys Res Commun 117: 859-865, 1983

Figure. Reproduction of a typical chromatographic run showing the final purification step of what later sequencing demonstrated to be ANF99-126.     This particular run was completed on July 7, 1982, and represents the peptide obtained from a pool of several hundred rat atria extracts. The absorbance unit at full scale is 0.02 at 280 nm. A small amount of peptide was recovered because our isolation procedure prevented peptide breakdown; thus, most of the tissue ANF was present as proANF, which eluted in another, higher molecular weight fraction. The technician has written that the test to confirm natriuretic activity in the bioassay rat was carried out on July 8, 1982. He wrote "with Strong R," meaning strong response after injection of 0.2 ml of sample eluate mixed with 0.3 ml of phosphate- buffered saline.      The peak shows good symmetry, suggesting that purification had been taken to homogeneity. Samples such as this then were sent for amino acid analysis and sequencing.

 

In 1986 a Gairdner Foundation International award was given "for the discovery and characterization of atrial natriuretic factor" to Adolfo J. de Bold, T. Geoffrey Flynn and Harald Sonnenberg. The Toronto-based "Gairdner Foundation is a non-profit corporation devoted to the recognition of outstanding achievement in biomedical research worldwide.... The purpose of these awards is the recognition of individuals whose work or contribution constitutes tangible achievement in the field of medical science." "An award of $30,000 CAD (each) payable in Canadian funds to individual winners from a diversity of fields for outstanding discoveries or contributions to medical science. A joint award for the same discovery or contribution may be given, usually to no more than two individuals." In 1994 a Ciba Foundation award was given to Adolfo J. De Bold "for his discovery of atrial natriuretic factor."