It's all about bile
Authors: James L. Boyer
Journal: Hepatology (2009)

It is a special honor to be asked to contribute to this Masters in Hepatology section and to share with you my personal perspective of the highlights of the field of hepatobiliary secretory physiology and disease as it has unfolded over a period of more than 4 decades. Dr. Lindor has also asked that I describe some of the personal factors that influenced the development of my career in an effort to provide historical reference and possibly some insights into the excitement and unpredictability of the evolution of a career as a physician scientist. When I entered college, I thought that I wanted to be an engineer; however, a drafting course made me realize quickly that I wanted a career that provided more human contact. Encouraged by an uncle, I decided to major in biology because this had been my favorite subject in high school. There were only eight biology majors at Haverford, a small Quaker-founded liberal arts college outside Philadelphia. As students, we received a great deal of personal attention from the three faculty members in the department, who were all quite young and newly hired. The double helix structure for DNA had just been published by Watson and Crick, and we were exposed to the beginnings of molecular biology out of their graduate school notes because there were no textbooks yet in print. Each of us had his own independent research project during our senior year, and mine involved growing a thiosulfate-using bacteria (Thiobacillus novellus) on both organic and inorganic media to determine if the organism had the ability to adapt to either substrate. The project required constructing growth curves by sampling the defined media every 2 hours for 24 hours. This was my introduction to bench research. As stimulating as an independent research experience seemed at the time, nothing compared to the excitement of receiving a reprint from my faculty advisor during my first year in medical school with my name included in the authorship.1 However, it was a long 8 years before I was to experience this excitement again, and for many of those intervening years, I was not at all certain that I had the talent to pursue a career in science. Nevertheless, I continued along this path and spent two summers while in medical school working at the National Institutes of Health (NIH) in the Commissioned Officer Student Training Extern Program. Initially, I learned to measure enzyme kinetics involved with serotonin metabolism using a spectrophotometer, but during the second summer, I worked on the pathogenesis of fever. They were good experiences for a student, although they did not yield publishable results, and I learned that research could also be frustrating and not always bear fruit. This was the beginning of the Vietnam war, and all male physicians in training were subject to the draft. As an alternative, one could become a commissioned officer in the US Public Heath Service. I applied during my internship year to the Institute of Allergy and Infectious Disease at the NIH, hoping to follow up on my interest in infectious diseases. However, I was not accepted into the program. Despite my obvious disappointment, it led to other opportunities. Shortly thereafter, I learned about a new program at the NIH, the Office of International Research, which was a precursor of the Fogerty Institute. Previously, during the summer of my junior year in medical school, I had received a traveling fellowship from the American Association of Medical Colleges, funded by Smith Kline and French, to work in a developing country, and for 10 weeks I rotated through several rural hospitals in Thailand. Living and working in the Far East had been fascinating, and I knew that my medical school, Johns Hopkins, was involved in cholera research in Calcutta (now Kolkata). Thus, when I learned about the Office of International Research, I immediately went down to the NIH from New York Hospital, where I was an intern, and I somehow talked my way into the program. I not only received a US Public Health Service commission but also was assigned to the Hopkins International Center for Medical Research and Training in Calcutta with the intention of working on cholera research. Thus, my wife of just a year and I spent 18 months in India for what was to become a pivotal career-developing experience (Fig. 1). At this point, I was intent on pursing further training in infectious disease and was interested in trying to determine where vibrio cholera resided during interepidemic periods as man was the only known host for this organism. However, first I needed to complete my second year of residency in internal medicine. During this time, Frank Iber, the hepatologist at Johns Hopkins, tried to convince me that I should study the pathogenesis of idiopathic portal hypertension because there were many patients with this condition that were being seen in Calcutta and other parts of India and Japan, and no cause had been determined. Initially, I thought it might be possible to pursue both projects; however, I was intrigued by the opportunity of discovering something new because, unlike cholera research, no one else was working on portal hypertension. I had also enjoyed taking care of patients with liver disease during medical residency, and so unknowingly at the time, the direction of my future career was beginning to emerge. Early days in India. Prior to traveling to India, I learned to perform hepatic vein catheterizations in patients undergoing cardiac catheterization at Johns Hopkins Hospital in Baltimore. The plan was to perform hemodynamic studies in patients who were being evaluated at the Institute of Postgraduate Medical Education and Research and Seth Sukh Karnani Memorial (SSKM) Hospital for portocaval shunt surgery to treat bleeding esophageal varices. I was to work with a team under the direction of the pathologist, Dr. B. K. Aikat. However, when I arrived in Calcutta, I was told that Dr. Aikat had just moved to a new position in Chandigarh in the northern part of India. Having traveled half-way around the world, I found this to be a very discouraging moment. However, the Chief of Cardiovascular Surgery, A. K. Basu, volunteered to supervise me. This turned out to be fortuitous because the patients with portal hypertension were admitted to his service for shunt surgery, and he saw to it that I was provided with nursing and resident assistants to enable me to perform the planned hemodynamic studies, which otherwise might not have been possible. However, for many months, I was not at all certain that we would obtain publishable information, and I began to wonder if I had made a major mistake and whether my goal of an academic career was realistic. By this time, I had to abandon the idea of also pursuing studies in patients with cholera because the distance between the infectious disease hospital and the SSKM where I was located was too great. In the middle of this 2-year appointment, I returned to Johns Hopkins and the NIH for an interim report and brought with me several autopsy specimens from patients with portal hypertension of unclear cause for special histologic stains that were not available at that time in Calcutta. These specimens revealed highly organized thrombi within branches of the intrahepatic portal veins, which provided a possible explanation for the development of portal hypertension in these patients (Fig. 2). Although we did not discover the cause of these thrombi (umbilical sepsis and arsenic toxicity were candidates), a full description of 21 cases with noncirrhotic portal hypertension with comparison to patients with extrahepatic portal vein thrombosis and those with cirrhosis was accepted for publication in the Annals of Internal Medicine.2 This experience taught me how important it is to have an adequate patient base to be able to do meaningful clinical research. Partially recanalized organized thrombus within an intrahepatic portal vein in a patient with idiopathic portal hypertension. Adapted from Annals of Internal Medicine.2 By this time, I was fully committed to becoming a hepatologist and was accepted into Gerald Klatskin's postdoctoral fellowship program in hepatology at Yale after a final year of medical residency at Yale New Haven Hospital. Klatskin had also spent 2 years in India during World War II while serving in the army and had developed his interest in liver disorders during that time. I often wondered if these historical parallels influenced his decision to accept me into his program. 6-ECDCA, 6-ethyl-chenodeoxycholic acid; ABC, ATP binding cassette; AE2, anion exchanger 2; ATP, adenosine triphosphate; BA, bile acid; BDE, bile duct epithelium; BSEP, bile salt export pump; CAR, constitutive androstane nuclear receptor; CYP7A1, cholesterol 7 alpha-hydroxylase; FGF, fibroblast growth factor; FIC1, familial intrahepatic cholestasis type 1; FXR, farnesoid X receptor; MDIBL, Mt. Desert Island Biological Laboratory; MDR1, multidrug resistance protein 1; MDR3, multidrug resistance protein 3; MRP2, multidrug resistance protein 2; NIH, National Institutes of Health; NTCP, sodium taurocholate cotransporting polypeptide; OATP, organic anion transporting polypeptide; OST, organic solute transporter; PBC, primary biliary cirrhosis; RHCSC, rat hepatocyte cultured in a collagen sandwich configuration; SHP, small heterodimer partner; UDCA, ursodeoxycholic acid. Except for 2 summers at the NIH, I had little formal training in bench research and was entirely clinically oriented. Nevertheless, in addition to a clinical project that I had undertaken with Klatskin on the prognostic value of bridging (subacute hepatic necrosis)3 (which was primarily a chart review and follow-up study of patients), I thought I should also gain some laboratory experience. Klatskin had two faculty associates in the Yale Liver Unit at that time: Robert Scheig, who was interested in lipids, and a biochemist, Nick Alexander. Nick taught me some biochemistry when Bob and I decided to pursue the question of the role of the liver in the excretion of very low density lipoproteins; this required setting up an isolated perfused rat liver preparation. It was necessary to cannulate the bile duct and measure the volume of bile over time as a determinant of the quality of the function of this isolated preparation. When I first saw this isolated organ secreting bile (Fig. 3), I immediately became fascinated with how this process was occurring. Almost nothing was known about the mechanism of bile formation at that time, other than that it was an energy-consuming process and not hydrostatic filtration as with urine. The chemistry of bile was known, and when bile acids, the major solutes in bile, were infused into the animal or added to the perfusate of the isolated perfused liver, they were taken up by the liver from portal blood and excreted into bile, where they stimulated secretion, presumably by osmotic mechanisms. It was also known that the hormone secretin could stimulate a bile salt–independent component, which was presumed to arise from the bile ducts. I immediately lost all interest in the lipid project and began to pursue the question of how bile was formed, using the isolated perfused rat liver as a model. Once again, I was venturing into an area about which little was known, and I also did not have a mentor experienced in this area; this is a risky approach that I would not necessarily recommend to trainees today. Early studies of the isolated perfused rat liver (∼1970). My initial observations demonstrated that bile was secreted against a hydrostatic pressure gradient and that secretion could be inhibited by mitochondrial poisons and by scillerin, a cardiac glycoside and potent inhibitor of Na+/K+-ATPase; this suggested that the sodium pump was somehow involved. However, working on my own, I needed advice as to whether this was a worthwhile pursuit. Help came at Yale from the prominent physiologist Peter Curran and Don Powell, then a postdoctoral fellow in his laboratory. Don suggested that I present my observations to their laboratory group, and although I was initially reluctant to do so because I was unsure of the significance of my data, this opportunity led Dr. Curran, who was editor of the gastrointestinal section of the American Journal of Physiology at the time, to start sending me articles for review that were related to this field. It was an enormous vote of confidence to be taken seriously by a well-known basic scientist, particularly at my level of training and experience, and did much to encourage me to continue with this research. Those of us in more senior positions sometimes forget the influence that we can have both positively and negatively on the careers of younger colleagues and how important constructive criticism and support can be. These initial studies were subsequently published in Gastroenterology and the American Journal of Physiology and demonstrated that there was an energy-dependent bile salt–independent component to canalicular bile production.4, 5 At this time, I became an assistant professor at Yale in the Department of Medicine and also began clinical studies on bile formation and cholestasis together with Joe Bloomer, who was a postdoctoral fellow with Klatskin. Using radio-labeled mannitol, the synthetic choleretic, sodium dehydrocholate, and secretin infusions in patients with t-tubes following cholecystectomy, we defined for the first time the basic components of bile in humans.6 We showed that of the ∼600 mL of bile secreted daily, two-thirds was canalicular bile, divided between bile salt–dependent and bile salt–independent fractions, and the remainder was secretin-stimulated duct secretion. My clinical interests also began to focus on chronic cholestatic liver diseases, particularly primary biliary cirrhosis (PBC). Pruritus was a prominent symptom, and we determined that phenobarbital therapy, which was known to be choleretic when administered to rodents, was often of some benefit for pruritus and lowered serum levels of both bilirubin and bile acids when given chronically (Fig. 4).7 Thirty years later, a molecular explanation for this phenomenon would be possible (see Boyer8). We also became interested in the natural history of PBC.9 Thus, when an offer came in 1971 to create my own liver unit within the gastrointestinal division at the University of Chicago, I was already heavily involved in both the basic physiology of bile production and the clinical studies of chronic cholestatic liver disease. Effects of phenobarbital in a patient with primary biliary cirrhosis. Reprinted with permission from Annals of Internal Medicine.7 Copyright 1975, American College of Physicians. Even at this early stage in my career, I recognized that sometimes one must venture out of the norm and explore novel research systems. Thus, just prior to my move to Chicago, I spent the month of August at the Mt. Desert Island Biological Laboratory (MDIBL) in search of a marine species that might have a bile canaliculus where micropuncture and sampling of the primary secretion might be possible. Unlike urine, bile is formed in small 1-μm spaces between adjacent hepatocytes sealed off by the structures of the tight junction; this prohibits direct access to the primary secretion. Therefore, when bile is collected in cannulated bile ducts, canalicular bile has been modified by the addition or subtraction of fluid and solutes distally in the bile duct epithelium (BDE). I was disappointed not to find such an animal as all vertebrate livers have approximately the same canalicular ultrastructural anatomy. Instead, I found a rich intellectual environment in a beautiful location with informal and close scientific interactions with nephrologists and other physiologists that were highly influential in guiding our research into the next decade. At that time, there was considerable interest by physiologists in the role of the sodium pump in epithelial transport. We and others wondered if the sodium pump was located on the apical canalicular membrane and whether it might account for bile salt–independent bile flow. Applying a histochemical assay that we learned at the MDIBL from David Ernst, postdoctoral fellow Bennet Blitzer determined that the reaction product of the enzyme was actually basolateral in location.10 This observation was initially surprising to us but resulted in a paradigm change in our thinking about transport function in hepatocytes because we now considered the hepatocyte to have an organization similar, with respect to membrane polarity, to that of a classic epithelium, in which the sodium pump is almost always located on the basolateral membrane. Michael Field, who also worked at the MDIBL in the summers and was a colleague at the University of Chicago, suggested that if bile acid uptake was coupled with sodium, then the role of the sodium pump would be to generate an electrical-chemical gradient that would provide the driving force for bile acid transport into the hepatocytes by analogy to sodium-coupled chloride transport in other epithelia. Others had recently demonstrated that sodium was required for bile acid uptake in isolated hepatocytes and perfused rat liver.11, 12 From these observations, the fundamental role of the sodium pump and sodium-coupled solute transport in the formation of bile salt–dependent secretion was proposed in an invited review.13 Several years later, we were able to confirm the presence of this coupled transport system directly using isolated plasma membrane vesicles from rat liver; this was another technique learned at the MDIBL from Rolf Kinne and David Miller.14 Knowledge in a field is most often advanced when new techniques come along that allow novel insights not previously discernable. By the early 1980s, it was clear that it was necessary to obtain access to purified preparations of the apical canalicular membrane from the hepatocytes in order to determine the driving forces for bile acid and other solutes across this membrane domain. We began by using membrane vesicle techniques developed by physiologists studying transport in the kidneys and intestine. However, the canalicular membrane represents less than 15% of the surface area of the hepatocyte, and early attempts at purification either were highly contaminated with intracellular organelles or were isolated in amounts too small to be useful for physiologic studies. Peter Meier-Abt joined my laboratory at that time as a postdoctoral research fellow from Switzerland. Peter had previous experience in using zonal rotors for purifying large amounts of hepatic microsomes. He set about to adapt earlier methods of apical membrane separation to obtain a higher and more purified yield and succeeded admirably in characterizing this fraction.15 Irwin Arias's group and Ann Hubbard's group also developed similar techniques for the preparation of highly purified basolateral (sinusoidal) and canalicular plasma membrane vesicles to study hepatic transport processes, which we compared in an article for Methods in Enzymology.16 These preparations were of enormous value in characterizing the basic transport processes that regulated not only bile acid transport but also cell volume and intracellular pH and the relationship of these processes to bile formation in hepatocytes.17 One of the holy grails in the field at that time was to discover the mechanism by which bile acids were transported against large concentration gradients into bile. We had been able to demonstrate that 3H-taurocholate efflux from rat canalicular membrane vesicles could be stimulated by a negative electric potential;18 these findings were also reproduced in hepatocyte couplets by a postdoctoral fellow, Steven Weinman, using fluorescent bile acids.19 However, we entirely missed the more important role that adenosine triphosphate (ATP) played in this process as our additions of ATP failed to stimulate transport in the vesicles. What was lacking was an ATP-regenerating system to maintain ATP concentrations in the vesicles. When this was done, ATP-dependent bile acid transmembrane transport could be demonstrated; this was an exciting finding demonstrated first by Irwin Arias's group20 and then by Deitrich Keppler's group.21 A second major technical challenge to the field was the inability to access the bile canalicular space. In 1980, I was stimulated by a plenary presentation from James Phillips, a hepatic pathologist at the University of Toronto, at the annual meeting of the American Association for the Study of Liver Diseases in Chicago. Philips made the important observation that some rat hepatocytes retained their junctional contacts following collagenase digestion of liver and formed an expanded canalicular space when placed in short-term cultures.22 We later learned that the canalicular space expands because the junctional complexes between the two adjacent hepatocytes seal the space between the two adjacent cells and the unattached hemicanalicular remnants are retargeted to the remaining apical canalicular domain.23 These lumens expand to 5 to 10 μM; this is large enough that a micropipette might be able to enter this space. However, because I had no training in electrophysiologic techniques and a limited knowledge of biophysics, I sought assistance from the faculty in the Department of Physiology at Yale. Despite initial skepticism, I received support from Gerhard Giebisch, Sterling Professor of Physiology, who suggested collaborating with Jürg Graf from the University of Vienna. At that time, Jürg was probably the only person with expertise in electrophysiologic techniques who also was interested in the physiology of bile. He agreed to come to New Haven, and 2 weeks later, Jürg managed to insert a micropipette into the bile canalicular lumen of an hepatocyte couplet, confirm its location by electroporating fluorescein into the lumen, and record the electrical potential profile of the hepatocyte canalicular lumen. These experiments defined the electrical driving force across this apical membrane for the first time. The initial findings were published in the Proceedings of the National Academy of Sciences in an article communicated by Hans Popper24 and thus began a decade-long Vienna-Yale collaboration (see Boyer JL. Bile canalicular secretion—tales from Vienna and Yale. Wien Med Wochenschr 2008;158(12-20):534-538 for more details about these developments). Subsequent studies by postdoctoral fellow Robert Henderson, who joined us from Cambridge University, and Jürg defined the basic electrical properties of this primary bile secretory unit, including measurements of intracellular and canalicular electric potentials, sinusoidal and canalicular membrane resistances, tight junction resistances, and intracellular ion activities.25 During the next decade, the chemical and electrical driving forces for solute transport in hepatocytes were defined as well as the role of K+, Cl−, and HCO in intracellular pH and cell volume regulation.26-29 Inward rectifying potassium channels in the plasma membrane were characterized30 as well as chloride channels in isolated canalicular membranes reconstituted into lipid bilayers,31 in addition to the previously described effects of the electrical membrane potential on canalicular bile acid excretion and hepatic uptake.19, 32 During this period, the basic properties of the isolated hepatocyte couplet as the primary bile secretory unit were further defined. These units exhibited microtubule-dependent vesicle-mediated transcytosis and responded to choleretic stimuli detected by expansion of the canalicular lumen.33 Over time, the hepatocyte couplet model became a unique and powerful tool for studying the structural and functional aspects of bile secretion with video image analysis, confocal fluorescent microscopy, and electrophysiologic techniques.34-37 Although this model continues to be used by investigators today,38-40 it has limitations. Alternative cell culture preparations that maintain structural and secretory polarity include WIF-B cells41, 42 and isolated rat hepatocytes cultured in a collagen sandwich configuration (RHCCSCs).43, 44 RHCCSCs, originally described by Brower and colleagues, develop and maintain cell polarity and bile secretory function for 5 to 7 days after isolation and thus are useful for longer term experiments, including knockdown of proteins using small interfering RNA technology. Studies in the isolated perfused rat liver and hepatocyte couplets yielded much information about the mechanisms of bile secretion at the level of the hepatocyte, but few studies examined the role of the BDE in this process. Modern studies of cholangiocyte biology began when Gianfranco Alpini and colleagues demonstrated a secretory role for rat bile ducts by examining the effect of secretin after bile duct ligation where proliferation of the ducts had occurred.46 Interest in our laboratory began when Mario Strazzabosco, then a postdoctoral fellow from Padova, Italy, wanted to look at the mechanism of bicarbonate choleresis produced by ursodeoxycholic acid (UDCA). He determined that UDCA acts as a weak acid in the hepatocyte and appears to alkalinize bile by protonation within the bile duct lumen. We thought that H+-UDCA might then diffuse back across the biliary epithelium, leaving an HCO anion to be secreted.47 The obvious need for pure preparations of cholangiocytes led Mario to modify procedures previously described for isolating bile duct cells from bile duct–obstructed and alpha-naphthyl isothiocyanate–treated rats48, 49 and to successfully isolate normal cholangiocytes from rat liver and determine their basic characteristics. These data indicated that BDE cells possess mechanisms for Na+/H+ exchange, Na+/HCO symport, and Cl−/HCO exchange and that they should be capable of transepithelial H+/HCO transport.50 Despite their usefulness in characterizing ion transport systems in cholangiocytes, these preparations were not polarized, and so it was not possible to study transport systems across the luminal apical membrane. To address this need, we adapted techniques that had been used to enzymatically digest pancreatic acini51 and those that had been recently developed to micropuncture larger bile ducts.52 We succeeded in isolating a fraction that was derived from ducts ∼50 to 100 μM in diameter and that represented the major sites of hormone-stimulated secretion. After 24 to 48 hours in culture, these preparations formed closed secretory units,53 which permitted the direct determination of the effects of secretagogues such as secretin, bombesin, and vasoactive intestinal peptide on fluid excretion.54-57 Subsequently, Dominico Alvaro, also a postdoctoral fellow, demonstrated that secretin stimulated the activity of the Cl−/HCO exchanger, probably by activating Cl− channels via the intracellular messenger cyclic adenosine monophosphate. This in turn depolarized the cell and stimulated electrogenic Na+/HCO symport. The cell depolarization induced by Cl− channel activation is thought to enhance HCO entrance through electrogenic Na+/HCO symport, which in turn stimulates the Cl−/HCO exchange. Together, these mechanisms appear to account for secretin-stimulated bicarbonate secretion in bile.58 Alvaro et al. also demonstrated that UDCA does not stimulate HCO excretion from isolated rat BDE cells but modifies pHi in BDE cells as a weak acid. Subsequent adaptation of these isolation techniques to bile duct units from the mouse60 led 16 years later to work by Mario Strazzabosco and his colleagues,61 who showed that UDCA stimulates secretion by releasing ATP, which activates purinergic receptors that stimulate chloride secretion via the cystic fibrosis transmembrane conductance regulator and calcium-regulated mechanisms. These and other new developments, including the adaptation of isolated bile duct units for in vitro perfusion by others,62 has led to a new field of cholangiocyte physiology and pathophysiology, which others have pursued.63, 64 Meanwhile, the successful cloning of bile acid transporters and the completion of the human genome were about to revolutionize the entire hepatobiliary transport field. This era began with the use of the oocyte expression system for the cloning of the rat sodium taurocholate cotransporting polypeptide (Ntcp) responsible for the hepatic uptake of bile acids65 and continued until just recently with the discovery of the basolateral bile acid transporter in the ileum, the organic solute transporter α and β (Ostα-Ostβ), a heteromeric bile acid and sterol transporter that represents the last remaining link in the transport proteins that regulate the enterohepatic circulation of bile acids.66 The completion of the human genome and the field of cancer biology had a great deal to do with this revolution. The rate-limiting step in the hepatic clearance of bile acids and other biliary constituents is their export from the hepatocyte into bile. Many of these transporters are members of the ATP binding cassette (ABC) superfamily of transporters, for which there are 50 known members in humans. Multidrug resistance protein 1 (MDR1) or P-glycoprotein, initially found to be a cause of drug resistance in cancer cells,67 was the first ABC transporter to be found at the canalicular domain.68 Other ABC transporters at the canalicular membrane were soon identified, including the phospholipid export pump, the multidrug resistance protein 3 (MDR3), the multidrug resistance protein 2 (MRP2), the bile salt export pump (BSEP), the breast cancer resistance protein (BCRP), and the sitosterol and cholesterol export pumps sterolin 1 and sterolin 2 (ABCG5/G8). The discovery that there were mutations in several of these transporters (MRP2, MDR3, and BSEP) and in FIC1 that produced jaundice and different forms of cholestatic liver disease provided dramatic proof of principal