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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asked that I describe some of the personal factors that influenced the development of my career in an effort to provide historical reference and possibly some insights into the excitement and unpredictability of the evolution of a career as a physician scientist.

Early Days

  1. Top of page
  2. Early Days
  3. Mechanisms of Bile Secretion and Cholestasis
  4. In Vitro Model Systems
  5. Molecular Biology
  6. Clinical Studies and PBC
  7. From Bedside to Bench and Back Again
  8. Acknowledgements
  9. References

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.

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Figure 1. Early days in India.

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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.

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Figure 2. Partially recanalized organized thrombus within an intrahepatic portal vein in a patient with idiopathic portal hypertension. Adapted from Annals of Internal Medicine.2

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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.

Mechanisms of Bile Secretion and Cholestasis

  1. Top of page
  2. Early Days
  3. Mechanisms of Bile Secretion and Cholestasis
  4. In Vitro Model Systems
  5. Molecular Biology
  6. Clinical Studies and PBC
  7. From Bedside to Bench and Back Again
  8. Acknowledgements
  9. References

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.

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Figure 3. Early studies of the isolated perfused rat liver (∼1970).

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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.

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Figure 4. 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.

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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 HCOmath image 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

In Vitro Model Systems

  1. Top of page
  2. Early Days
  3. Mechanisms of Bile Secretion and Cholestasis
  4. In Vitro Model Systems
  5. Molecular Biology
  6. Clinical Studies and PBC
  7. From Bedside to Bench and Back Again
  8. Acknowledgements
  9. References

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 HCOmath image 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+/HCOmath image symport, and Cl/HCOmath image exchange and that they should be capable of transepithelial H+/HCOmath image 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/HCOmath image exchanger, probably by activating Cl channels via the intracellular messenger cyclic adenosine monophosphate. This in turn depolarized the cell and stimulated electrogenic Na+/HCOmath image symport. The cell depolarization induced by Cl channel activation is thought to enhance HCOmath image entrance through electrogenic Na+/HCOmath image symport, which in turn stimulates the Cl/HCOmath image 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 HCOmath image 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

Molecular Biology

  1. Top of page
  2. Early Days
  3. Mechanisms of Bile Secretion and Cholestasis
  4. In Vitro Model Systems
  5. Molecular Biology
  6. Clinical Studies and PBC
  7. From Bedside to Bench and Back Again
  8. Acknowledgements
  9. References

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 for their function69 and provided genetic tools for diagnosis.

As the discovery of the molecular determinants of the bile secretory process unfolded (see Fig. 5; for reviews, see Trauner et al.69 and Trauner and Boyer70), our laboratory was one of the first to study the effects of cholestasis on hepatic transporter expression and function in rodent models of cholestasis, including common bile duct ligation, estrogen treatment, lithocholic acid, and administration of lipopolysaccaride.71, 72 More limited studies in patients with various cholestatic disorders have supported the findings in animal models, with few exceptions. From these investigations, the following paradigm has emerged:

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Figure 5. Molecular basis of bile formation as viewed in 1998. Abbreviations: AE2, anion exchanger 2; BS, bile salts; BSEP, bile salt export pump; MDR1, multidrug resistance protein 1; MDR3, multidrug resistance protein 3; MRP2, multidrug resistance protein 2; OA, organic anion; OATP, organic anion transporting polypeptide; OC+, organic cations; NTCP, sodium taurocholate cotransporting polypeptide; PL, phospholipid. Adapted from the New England Journal of Medicine.69

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Determinants of bile secretion undergo an adaptive response during cholestasis which tend to minimize hepatic injury. This adaptation occurs by: (a) limiting hepatic uptake of bile acids and other organic solutes, (b) reducing bile acid synthesis, (c) accelerating bile acid detoxification and (d) up-regulating alternative pathways for excretion of bile salts and other solutes in liver, kidney and intestine.76

Current interest in this field now focuses on understanding how this adaptive regulation occurs at both the transcriptional and posttranscriptional levels. Important breakthroughs made by others in this field have included the recognition that bile acids and bilirubin function as ligands for nuclear receptors73 and thus feed back to regulate the expression of some of the adaptive responses that then help to minimize their retention in the cholestatic liver (see Zollner et al.74 and Stahl et al.75 for details in current reviews). Hydrophobic bile acids are ligands for the farnesoid X receptor (FXR; see Fig. 6), which binds with its heterodimeric partner, the retinoid X receptor, to the promoters of a number of genes that regulate bile acid transport. Bile acids also regulate the transcription of another nuclear receptor, the small heterodimer partner, which is involved with the inhibition of hepatic bile acid uptake by organic anion transporting polypeptide 1B1 and contributes to the inhibition of cholesterol 7 alpha-hydroxylase (CYP7A1).76 FXR also regulates the expression of fibroblast growth factor 15/19 (FGF15/19) in the distal intestine, which functions as a hormone by circulating back to the liver, where it binds to the FGF receptor, leading to inhibition of CYP7A1.77 Recent evidence suggests that this may be the dominant mechanism regulating bile acid synthesis. Together, FXR, constitutive androstane nuclear receptor (CAR), and pregnane X receptor play pivotal roles in the transcriptional response to cholestatic liver injury (see also Koster and Karpen,78 Zollner et al.79 for recent reviews).

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Figure 6. Hepatic transporters regulated by the nuclear receptor FXR by bile acids. Abbreviations: AE2, anion exchanger 2; BA, bile acid; BSEP, bile salt export pump; CYP7A1, cholesterol 7 alpha-hydroxylase; FIC1, familial intrahepatic cholestasis type 1; FXR, farnesoid X receptor; MDR3, multidrug resistance protein 3; MRP2, multidrug resistance protein 2; NTCP, sodium taurocholate cotransporting polypeptide; OATP-C, organic anion transporting polypeptide C; OST, organic solute transporter; SHP, small heterodimer partner. Reprinted with permission from Expert Opinion on Therapeutic Targets.105 Copyright 2006, Ingenta.

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Because FXR regulates so many steps in the hepatic synthesis and enterohepatic circulation of bile acids, the potential benefit of FXR agonist therapy in cholestasis has stimulated pharmaceutical and academic laboratories to develop more potent FXR ligands. Several examples include GW4064, 6-ethyl-chenodeoxycholic acid (6-ECDCA or INT-747), and fexaramine,80–82 which are 50 to 100 times more potent than chenodeoxycholic acid, the most effective endogenous ligand for FXR. The effectiveness of these compounds in treating rodent models of cholestasis has provided support for this approach.83, 84 GW4064 and 6-ECDCA have been shown to induce Bsep, Mrp2, and Mdr2 and to reverse ductular proliferation and necrosis in these models. However, for FXR agonist therapy to have any benefit, it seems likely that enhancement of the adaptive responses in transporter function observed in patients with cholestasis will need to occur early in the course of the disease before the development of irreversible liver injury. In addition, the agonist will need to have a strong safety profile because the treatment will need to be long-term. Phase II trials are currently underway, testing the efficacy and safety of 6-ECDCA in PBC.

Clinical Studies and PBC

  1. Top of page
  2. Early Days
  3. Mechanisms of Bile Secretion and Cholestasis
  4. In Vitro Model Systems
  5. Molecular Biology
  6. Clinical Studies and PBC
  7. From Bedside to Bench and Back Again
  8. Acknowledgements
  9. References

The long-term goal of our research and the field of hepatobiliary physiology and disease has been to understand the natural history of and find new treatments for cholestatic liver disorders such as PBC. Our early studies of the natural history of this disease were enabled by a patient population that had been assembled by Gerald Klatskin and immunologist Fred Kanter at Yale. Together with Joe Roll, we published a retrospective and prospective study to determine the life expectancy of patients with PBC in 1983.9 This study showed that the average length of survival for 243 symptomatic patients and 37 asymptomatic patients was nearly twice that previously reported, and over a 12-year period, the survival of the patients who were asymptomatic at diagnosis was similar to that of a control age-matched and sex-matched population (Fig. 7). A multivariate analysis of clinical features revealed that at the onset of disease, age, hepatomegaly, and elevated levels of serum bilirubin were independent discriminators of a poor prognosis. Liver biopsy findings of fibrosis limited to portal areas improved this discrimination and correlated with prolonged survival. This study was one of several reports at that time that examined prognostic factors for the disease.85, 86 The advantage of this study was that therapy with UDCA and liver transplantation were not confounding factors because they were not yet established therapies. We subsequently extended the study by 10 years to follow up 247 members of the original cohort (89%), which ranged up to 24 years from the time of the original diagnosis.87 Once again, the median predicted survival of patients who had presented without symptoms at the time of diagnosis was twice as long as that for patients who presented with symptoms. However, unlike the original study, the overall survival of asymptomatic patients was shorter than that predicted for an age-matched and sex-matched control group. This difference became apparent only after 11 years of follow-up, illustrating the problem with determining prognosis in a disease with such a long natural history. Median follow-up for the asymptomatic patients was 12.1 years, at which time 33% remained free of symptoms of liver disease. However, once symptoms developed, overall survival of this subgroup of patients with PBC was similar to those presenting originally with symptoms. Risk factors for diminished survival included elevated bilirubin, increasing age, ascites, advanced fibrosis, and the degree of portal bile stasis on liver biopsy. We were not able to determine which asymptomatic patients would remain symptom-free. This study will likely remain the last one to examine the natural history of PBC because of liver transplantation and UDCA therapy. UDCA is now the standard of care and appears to prolong survival if administered to patients early in the course of their disease, as represented by stage 1 or 2 fibrosis. Other more recent studies also have confirmed the differences in the prognosis of patients with earlier stages of fibrosis,88 including a multicenter national trial of UDCA and methotrexate in which we were participants.89 Interestingly, bile acid transporter expression is preserved in stage 1 and 2 disease90 but undergoes adaptive responses in stage 3 and 4 PBC.91

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Figure 7. Life-table analysis of survival of asymptomatic and symptomatic patients with primary biliary cirrhosis by the Kaplan-Meier method. Reprinted with permission from the New England Journal of Medicine.9 Copyright 1983, Massachusetts Medical Society. All rights reserved.

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Despite these studies, the natural history of asymptomatic patients remains controversial because studies from England have not confirmed our experience or that of patients in Canada92 or Scandinavia93 and report no difference between patients with PBC who present with or without symptoms.94 One explanation is that on average the patients in the British study were significantly older at diagnosis (early 60s versus late 40s) and there was an excess of non–liver-related deaths in the asymptomatic groups. In addition, the disease in symptomatic patients may not have been as severe, as few patients were jaundiced.

From Bedside to Bench and Back Again

  1. Top of page
  2. Early Days
  3. Mechanisms of Bile Secretion and Cholestasis
  4. In Vitro Model Systems
  5. Molecular Biology
  6. Clinical Studies and PBC
  7. From Bedside to Bench and Back Again
  8. Acknowledgements
  9. References

One of the gratifying experiences of being involved with a field over many years is to see molecular explanations develop for a previously studied condition. For example, why was phenobarbital therapy effective in those cholestatic patients that we treated more than 35 years ago? We now know that phenobarbital is a strong activator of CAR95 and that activated CAR enhances the transcription of transporters and enzymes involved in hepatic uptake of organic anions and bilirubin, bile acid conjugation reactions, and excretion of these substrates via MRP2 and MRP4.96 Similar effects can be attributed to the Asian remedy for neonatal jaundice, Yin Zhi Huang, which is a decoction of Yin Chin (Artemisia capillaris) and three other herbs.97 Recent studies have demonstrated that CAR is a key regulator of bilirubin clearance in the liver of mice, and the treatment of wild-type and humanized CAR transgenic mice with Yin Zhi Huang for 3 days enhanced the clearance of intravenously infused bilirubin; this effect was abrogated in CAR knockout animals.96 Transporters and enzymes involved with bilirubin clearance and metabolism were all enhanced by this CAR-dependent activity of Yin Zhi Huang.

Finally, where are we now and what lies ahead? For most investigators in the field, the current challenge is to figure out how to safely regulate transporter and metabolic pathway expression therapeutically in order to minimize, if not prevent, the long-term consequences of cholestatic liver injury, which otherwise often leads to death or liver transplantation. Our own efforts are focused primarily on understanding how the basolateral (sinusoidal membrane) hepatic transporters are up-regulated in cholestasis in an attempt to reduce the hepatic accumulation of toxic bile acids and other solutes. These bile acid transporters include MRP3, MRP4, and OSTα-β.76 I will conclude with a brief summary of the discovery and significance of OSTα-β because this story illustrates how a long-term commitment to comparative physiologic approaches in marine elasmobranches has led to findings that unexpectedly have resulted in clinically valuable results.

This story begins in 1990, when I spent a sabbatical year in Zurich and first learned the rudiments of molecular biology from Bruno Hagenbuch and Bruno Steiger, who were part of Peter Meier's Clinical Pharmacology Division at the University of Zurich. They were my mentors in this endeavor, and this was not an easy task with a 54-year-old student. I remain grateful for their patient guidance. At that time, we determined that nonmammalian vertebrates do not express sodium-dependent bile acid transporters, such as Ntcp, but take up organic anions into the liver by sodium-independent means.98

With this background information, Ned Ballatori and I began to search for novel sterol transporters using a comparative approach by screening a liver library that we had obtained from the little skate at the MDIBL in Maine. Ultimately, Ned's laboratory at the University of Rochester used an oocyte expression system to make the unique discovery that there are two separate pools of messenger RNA that need to be expressed together in order for significant uptake of radio-labeled bile acids or estrone sulfate to occur. They identified two genes, which were named Ostα and Ostβ.99 Ostα is a seven-membrane spanning domain protein consisting of 340 amino acids, whereas Ostβ consists of 128 amino acids and is a single-membrane spanning domain protein. Both subunits are required for transport to occur. OSTα-β is expressed in many different tissues, with certain species differences, but it is most abundant in the human small intestine, kidney, liver, testis, and adrenal gland.100 Substrates for this transporter include bile acids, steroids (estrone-3-sulfate, dehydroepiandrosterone 3-sulfate, and digoxin), and prostaglandin E2; this is consistent with a role for OSTα-β in the disposition of key cellular metabolites and signaling molecules. Transport is mediated by facilitated diffusion, so efflux or uptake depends on the electrical-chemical gradient.101

Originally, neither the endogenous substrate(s) nor the function of OSTα-β was clear. However, in 2005, when Paul Dawson was looking for candidate genes for the ileal basolateral bile acid transporter, he discovered that one of the candidate genes that fit this description in the Asbt−/− mouse was Ostα. Subsequent collaboration with Ned Ballatori led to the determination that Ostα-β was the basolateral ileal bile acid transporter in the mouse.66 Thus, the last remaining transporter link in the enterohepatic circulation of bile acids was established. Subsequent studies in Ostα knockout mice have provided strong confirming evidence for its role in bile acid and sterol homeostasis.102, 103

Our current interest in this transporter comes from the observation that Ostα-β is up-regulated in the liver in cholestasis in an Fxr-dependent manner in animal models and also in patients with PBC.104 Furthermore, preliminary data from our laboratory suggest that Ostα knockout mice are partially protected from cholestatic liver injury induced by bile duct ligation and that bile acid excretion in the urine is enhanced. Because Ostα-β is also normally highly expressed in the basolateral membrane of the proximal renal tubule,101 these findings provide the exciting possibility that pharmacologic strategies which inhibit the function of renal and intestinal OSTα-β might provide novel therapeutic approaches for cholestatic liver disease (Soroka et al., unpublished data, 2008). Thus, basic discoveries of a novel transporter in an evolutionary ancient marine elasmobranch liver have given rise unexpectedly to important insights into human biology and disease that would not otherwise have occurred. These studies emphasize the importance of basic research in nonmammalian species and the role of serendipity in obtaining clinically meaningful outcomes.

This journey into the mechanisms of bile secretion and cholestasis has been pursued continuously for more than 4 decades at Yale University, the University of Chicago, and the MDIBL and often with colleagues around the world. It continues to be exciting and to reveal new secrets. None of this would have been possible without the support of several different families. Foremost have been my NIH family and the program staff at the National Institute of Diabetes and Digestive and Kidney Diseases, who have funded this work continuously for most of this period. Space does not allow me to adequately acknowledge all of my trainee and colleague family, the many postdoctoral fellows, students, and staff that have carried out the work and most often have generated the new ideas and approaches. Ned Ballatori, Peter Meier-Abt, Mario Strazzabosco, and Michael Trauner have been particularly influential. I am especially grateful to my laboratory staff family, including Albert Mennone, who has assisted me in much of our animal-related work for 20 years, Carol Soroka, who has provided special expertise in imaging and confocal microscopy and critical advice as a colleague for 15 years, and Shi-Ying Cai, who has led many of our recent molecular biology initiatives. My thanks also go to my many Yale colleagues for their support and encouragement over the years, particularly Michael Nathanson, currently Chief of the Division of Digestive Disease. Finally, the support of my wife Phoebe and our two daughters, Phoebe and Annie, has been essential, particularity in the first decades of this journey. It would not have been possible without them.

References

  1. Top of page
  2. Early Days
  3. Mechanisms of Bile Secretion and Cholestasis
  4. In Vitro Model Systems
  5. Molecular Biology
  6. Clinical Studies and PBC
  7. From Bedside to Bench and Back Again
  8. Acknowledgements
  9. References
  • 1
    Santer M, Boyer J, Santer U. Thiobacillus novellus. I. Growth on organic and inorganic media. J Bacteriol 1959; 78: 197202.
  • 2
    Boyer JL, Sen Gupta KP, Biswas SK, Pal NC, Basu Mallick KC, Iber FL, et al. Idiopathic portal hypertension—comparison with the portal hypertension of cirrhosis and extrahepatic portal vein obstruction. Ann Intern Med 1967; 66: 4168.
  • 3
    Boyer JL, Klatskin G. Pattern of necrosis in acute viral hepatitis. Prognostic value of bridging (subacute hepatic necrosis). N Engl J Med 1970; 283: 10631071.
  • 4
    Boyer JL, Klatskin G. Canalicular bile flow and bile secretory pressure: evidence for a non-bile salt dependent fraction in the isolated perfused rat liver. Gastroenterology 1970; 59: 853859.
  • 5
    Boyer JL. Canalicular bile formation in the isolated perfused rat liver. Am J Physiol 1971; 221: 11561163.
  • 6
    Boyer JL, Bloomer JR. Canalicular bile secretion in man: studies utilizing the biliary clearance of (14C) mannitol. J Clin Invest 1974; 54: 773781.
  • 7
    Bloomer J, Boyer JL. Phenobarbital effects in cholestatic liver disease. Ann Intern Med 1975; 82: 310317.
  • 8
    Boyer JL. Nuclear receptor ligands: rational and effective therapy for chronic cholestatic liver disease? Gastroenterology 2005; 129: 735740.
  • 9
    Roll J, Boyer JL, Barry D, Klatskin G. The prognostic importance of clinical and histological features in asymptomatic and symptomatic primary biliary cirrhosis. N Engl J Med 1983; 308: 17.
  • 10
    Blitzer BL, Boyer JL. Cytochemical localization of Na+,K+-ATPase in the rat hepatocyte. J Clin Invest 1978; 62: 11041108.
  • 11
    Anwer MS, Hegner D. Effect of Na on bile acid uptake by isolated rat hepatocytes. Evidence for a heterogeneous system. Hoppe-Seylers Z Physiol Chem 1978; 359: 181192.
  • 12
    Paumgartner G, Herz R, Sauter K, Schwarz HP. Taurocholate excretion and bile formation in the isolated perfused rat liver: an in vitro-in vivo comparison. Arch Pharmacol 1974; 285: 165174.
  • 13
    Boyer JL. New concepts of mechanisms of hepatocyte bile formation. Physiol Rev 1980; 60: 303326.
  • 14
    Duffy MC, Blitzer BL, Boyer JL. Direct determination of the driving forces for taurocholate uptake into rat liver plasma membrane vesicles. J Clin Invest 1983; 72: 14701481.
  • 15
    Meier PJ, Sztul ES, Reuben A, Boyer JL. Structural and functional polarity of canalicular and basolateral plasma membrane vesicles isolated in high yield from rat liver. J Cell Biol 1984; 98: 9911000.
  • 16
    Meier PJ, Boyer JL. Preparation of basolateral (sinusoidal) and canalicular plasma membrane vesicles for the study of hepatic transport processes. In: Fleischer S, Fleischer B, eds. Methods in Enzymology. New York, NY: Academic Press; 1990: 534545.
  • 17
    Boyer JL, Graf J, Meier PJ. Hepatic transport systems regulating pHi, cell volume, and bile secretion. Ann Rev Physiol 1992; 54: 415438.
  • 18
    Meier PJ, Meier-Abt AS, Barrett C, Boyer JL. Mechanisms of taurocholate transport in canalicular and basolateral rat liver plasma membrane vesicles. J Biol Chem 1984; 259: 1061410622.
  • 19
    Weinman SA, Graf J, Boyer JL. Voltage-driven, taurocholate-dependent secretion in isolated hepatocyte couplets. Am J Physiol 1989; 256: G826G832.
  • 20
    Nishida T, Gatmaitan Z, Che MX, Arias IM. Rat liver canalicular membrane vesicles containing an ATP-dependent bile acid transport system. Proc Natl Acad Sci 1991; 88: 65906594.
  • 21
    Muller M, Ishikawa T, Berger U, Klunemann C, Lucka L, Schreyer A, et al. ATP-dependent transport of taurocholate across the hepatocyte canalicular membrane mediated by a 110-kDa glycoprotein binding ATP and bile salt. J Biol Chem 1991; 266: 1892018926.
  • 22
    Oshio C, Phillips MJ. Contractility of bile canaliculi, implications of liver functions. Science 1981; 212: 10411042.
  • 23
    Gautam A, Ng O-C, Boyer JL. Isolated rat hepatocyte couplets in short-term culture: structural characteristics and plasma membrane reorganization. HEPATOLOGY 1987; 7: 216223.
  • 24
    Graf J, Gautam A, Boyer JL. Isolated rat hepatocyte couplets: a primary secretory unit for electrophysiologic studies of bile secretory function. Proc Natl Acad Sci U S A 1984; 81: 65166520.
  • 25
    Graf J, Henderson RM, Krumpholz B, Boyer JL. Cell membrane and transepithelial voltages and resistances in isolated rat hepatocyte couplets. J Membr Biol 1987; 95: 241254.
  • 26
    Henderson RM, Graf J, Boyer JL. Na-H exchange regulates intracellular pH in isolated rat hepatocyte couplets. Am J Physiol 1987; 252: G109G113.
  • 27
    Benedetti A, Strazzabosco M, Corasanti JG, Haddad P, Graf J, Boyer JL. Cl(-)-HCO3- exchanger in isolated rat hepatocytes: role in regulation of intracellular pH. Am J Physiol 1991; 261: G512G522.
  • 28
    Haddad P, Beck JS, Boyer JL, Graf J. Role of chloride ions in liver cell volume regulation. Am J Physiol 1991; 261: G340G348.
  • 29
    Bruck R, Haddad P, Graf J, Boyer JL. Regulatory volume decrease stimulates bile flow, bile acid excretion, and exocytosis in isolated perfused rat liver. Am J Physiol 1992; 262: G806G812.
  • 30
    Henderson RM, Graf J, Boyer JL. Inward-rectifying potassium channels in rat hepatocytes. Am J Physiol 1989; 256: G1028G1035.
  • 31
    Sellinger M, Weinman SA, Henderson RM, Zweifach A, Boyer JL, Graf J. Anion channels in rat liver canalicular plasma membranes reconstituted into planar lipid bilayers. Am J Physiol 1992; 262: G1027G1032.
  • 32
    Weinman SA, Graf J, Veith C, Boyer JL. Electroneutral uptake and electrogenic secretion of a fluorescent bile salt by rat hepatocyte couplets. Am J Physiol 1993; 264: G220G230.
  • 33
    Gautam A, Ng OC, Strazzabosco M, Boyer JL. Quantitative assessment of canalicular bile formation in isolated hepatocyte couplets using microscopic optical planimetry. J Clin Invest 1989; 83: 565573.
  • 34
    Boyer JL, Gautam A, Graf J. Mechanisms of bile secretion: insights from the isolated rat hepatocyte couplet. Semin Liver Dis 1988; 8: 308316.
  • 35
    Graf J, Boyer JL. The use of isolated rat hepatocyte couplets in hepatobiliary physiology. J Hepatol 1990; 10: 387394.
  • 36
    Boyer JL, Phillips JM, Graf J. Preparation and specific application of isolated hepatocyte couplets. In: Fleischer S, Fleischer B, eds. Methods in Enzymology. New York, NY: Academic Press; 1990: 501516.
  • 37
    Boyer JL. Isolated rat hepatocyte couplets: a model for the study of bile secretory function. In: Tavoloni N, Berk PD, eds. Hepatic Transport and Bile Secretion: Physiology and Pathophysiology. New York, NY: Raven Press; 1993: 597606.
  • 38
    Nagata J, Guerra MT, Shugrue CA, Gomes DA, Nagata N, Nathanson MH. Lipid rafts establish calcium waves in hepatocytes. Gastroenterology 2007; 133: 256267.
  • 39
    Perez LM, Milkiewicz P, Elias E, Coleman R, Sanchez Pozzi EJ, Roma MG. Oxidative stress induces internalization of the bile salt export pump, Bsep, and bile salt secretory failure in isolated rat hepatocyte couplets: a role for protein kinase C and prevention by protein kinase A. Toxicol Sci 2006; 91: 150158.
  • 40
    Crocenzi FA, Mottino AD, Cao J, Veggi LM, Pozzi EJ, Vore M, et al. Estradiol-17beta-D-glucuronide induces endocytic internalization of Bsep in rats. Am J Physiol Gastrointest Liver Physiol 2003; 285: G449G459.
  • 41
    Decaens C, Rodriguez P, Bouchaud C, Cassio D. Establishment of hepatic cell polarity in the rat hepatoma-human fibroblast hybrid WIF-B9. J Cell Sci 1996; 109: 16231635.
  • 42
    Ihrke G, Neufeld EB, Meads T, Shanks MR, Cassio D, Laurent M, et al. WIF-B cells: an in vitro model for studies of hepatocyte polarity. J Cell Biol 1993; 123: 17611775.
  • 43
    Liu X, LeCluyse EL, Brouwer KR, Gan L-S, Lemasters JJ, Stieger B, et al. Biliary excretion in primary rat hepatocytes cultured in a collagen-sandwich configuration. Am J Physiol 1999; 277: G12G21.
  • 44
    Chandra P, LeCluyse EL, Brouwer KL. Optimization of culture conditions for determining hepatobiliary disposition of taurocholate in sandwich-cultured rat hepatocytes. In Vitro Cell Dev Biol Anim 2001; 37: 380385.
  • 45
    Wang W, Soroka CJ, Mennone A, Rahner C, Harry K, Pypaert M, et al. Radixin is required to maintain apical canalicular membrane structure and function in rat hepatocytes. Gastroenterology 2006; 131: 878884.
  • 46
    Alpini G, Lenzi R, Sarkozi L, Tavoloni N. Biliary physiology in rats with bile ductular cell hyperplasia evidence for a secretory function of proliferated bile ductules. J Clin Invest 1988; 81: 569578.
  • 47
    Strazzabosco M, Sakisaka S, Hayakawa T, Boyer JL. Effect of UDCA on intracellular and biliary pH in isolated rat hepatocyte couplets and perfused livers. Am J Physiol 1991; 260: G58G69.
  • 48
    Alpini G, Lenzi R, Zhai WR, Liu MH, Slott PA, Paronetto F, et al. Isolation of a nonparenchimal liver cell fraction enriched in cells with biliary epithelial phenotypes. Gastroenterology 1989; 97: 12481260.
  • 49
    Mathis GH, Walls SA, Sirica AE. Biochemical characteristics of hyperplastic rat bile ductular epithelial cells cultured “on top” and “inside” different extracellular matrix substitutes. Cancer Res 1988; 48: 61456153.
  • 50
    Strazzabosco M, Mennone A, Boyer JL. Intracellular pH regulation in isolated rat bile duct epithelial cells. J Clin Invest 1991; 87: 15031512.
  • 51
    Argent BE, Arkle S, Cullen MJ, Green R. Morphological, biochemical and secretory studies on rat pancreatic ducts maintained in tissue culture. Q J Exp Physiol 1986; 71: 633648.
  • 52
    Roberts SK, Kuntz SM, Gores GJ, LaRusso NF. Regulation of bicarbonate-dependent ductular bile secretion assessed by lumenal micropuncture of isolated rodent intrahepatic bile ducts. Proc Natl Acad Sci U S A 1993; 90: 90809084.
  • 53
    Mennone A, Alvaro D, Cho W, Boyer JL. Isolation of small polarized bile duct units. Proc Natl Acad Sci 1995; 92: 65276531.
  • 54
    Cho WK, Boyer JL. Vasoactive intestinal polypeptide is a potent regulator of bile secretion from rat cholangiocytes. Gastroenterology 1999; 117: 420428.
  • 55
    Cho WK, Boyer JL. Characterization of ion transport mechanisms involved in bombesin-stimulated biliary secretion in rat cholangiocytes. J Hepatol 1999; 30: 10451051.
  • 56
    Cho WK, Mennone A, Boyer JL. Intracellular pH regulation in bombesin-stimulated secretion in isolated bile duct units from rat liver. Am J Physiol 1998; 275: G1028G1036.
  • 57
    Cho WK, Mennone A, Rydberg SA, Boyer JL. Bombesin stimulates bicarbonate secretion from rat cholangiocytes: implications for neural regulation of bile secretion. Gastroenterology 1997; 113: 311321.
  • 58
    Alvaro D, Cho WK, Mennone A, Boyer JL. Effect of secretin on intracellular pH regulation in isolated rat bile duct epithelial cells. J Clin Invest 1993; 92: 13141325.
  • 59
    Alvaro D, Mennone A, Boyer JL. Effect of ursodeoxycholic acid on intracellular pH regulation in isolated rat bile duct epithelial cells. Am J Physiol 1993; 265: G783G791.
  • 60
    Cho WK, Mennone A, Boyer JL. Isolation of functional polarized bile duct units from mouse liver. Am J Physiol Gastrointest Liver Physiol 2001; 280: G241G246.
  • 61
    Fiorotto R, Spirli C, Fabris L, Cadamuro M, Okolicsanyi L, Strazzabosco M. Ursodeoxycholic acid stimulates cholangiocyte fluid secretion in mice via CFTR-dependent ATP secretion. Gastroenterology 2007; 133: 16031613.
  • 62
    Tietz PS, Chen XM, Gong AY, Huebert RC, Masyuk A, Masyuk T, et al. Experimental models to study cholangiocyte biology. World J Gastroenterol 2002; 8: 14.
  • 63
    Alpini G, McGill JM, LaRusso NF. The pathobiology of biliary epithelia. HEPATOLOGY 2002; 35: 12561268.
  • 64
    Lazaridis KN, Strazzabosco M, LaRusso NF. The cholangiopathies: disorders of biliary epithelia. Gastroenterology 2004; 127: 15651577.
  • 65
    Hagenbuch B, Stieger B, Foguet M, Lubbert H, Meier PJ. Functional expression cloning and characterization of the hepatocyte Na+/bile acid cotransport system. Proc Natl Acad Sci U S A 1991; 88: 1062910633.
  • 66
    Dawson PA, Hubbert M, Haywood J, Craddock AL, Zerangue N, Christian WV, et al. The heteromeric organic solute transporter alpha-beta, Ostα-Ostβ, is an ileal basolateral bile acid transporter. J Biol Chem 2005; 280: 69606968.
  • 67
    Kartner N, Riordan JR, Ling V. Cell surface P-glycoprotein associated with multidrug resistance in mammalian cell lines. Science 1983; 221: 12851288.
  • 68
    Kamimoto Y, Gatmaitan Z, Hsu J, Arias IM. The function of Gp170, the multidrug resistance gene product in rat liver canalicular membrane vesicles. J Biol Chem 1989; 264: 1169311698.
  • 69
    Trauner M, Meier PJ, Boyer JL. Molecular pathogenesis of cholestasis. N Engl J Med 1998; 339: 12171227.
  • 70
    Trauner M, Boyer JL. Bile salt transporters: molecular characterization, function and regulation. Physiol Rev 2003; 83: 633671.
  • 71
    Gartung C, Ananthanarayanan M, Rahman MA, Schuele S, Nundy S, Soroka CJ, et al. Down-regulation of expression and function of the rat liver Na+/bile acid cotransporter in extrahepatic cholestasis. Gastroenterology 1996; 110: 199209.
  • 72
    Trauner M, Arrese M, Soroka C, Ananthanarayanan M, Koeppel TA, Schlosser SF, et al. The rat canalicular conjugate export pump (Mrp 2) is down-regulated in intrahepatic and obstructive cholestasis. Gastroenterology 1997; 113: 255264.
  • 73
    Karpen SJ. Bile acids go nuclear! HEPATOLOGY 1999; 30: 11071109.
  • 74
    Zollner G, Trauner M. Mechanisms of cholestasis. Clin Liver Dis 2008; 12: 126, vii.
  • 75
    Stahl S, Davies MR, Cook DI, Graham MJ. Nuclear hormone receptor-dependent regulation of hepatic transporters and their role in the adaptive response in cholestasis. Xenobiotica 2008; 38: 725777.
  • 76
    Boyer JL. New perspectives for the treatment of cholestasis: lessons from basic science applied clinically. J Hepatol 2007; 46: 365371.
  • 77
    Kim I, Ahn SH, Inagaki T, Choi M, Ito S, Guo GL, et al. Differential regulation of bile acid homeostasis by the farnesoid X receptor in liver and intestine. J Lipid Res 2007; 48: 26642672.
  • 78
    Kosters A, Karpen SJ. Bile acid transporters in health and disease. Xenobiotica 2008; 38(7–8): 10431071.
  • 79
    Zollner G, Marschall HU, Wagner M, Trauner M. Role of nuclear receptors in the adaptive response to bile acids and cholestasis: pathogenetic and therapeutic considerations. Mol Pharm 2006; 3: 231251.
  • 80
    Kast HR, Goodwin B, Tarr PT, Jones SA, Anisfeld AM, Stoltz CM, et al. Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid X-activated receptor, and constitutive androstane receptor. J Biol Chem 2001; 277: 29082915.
  • 81
    Downes M, Verdecia MA, Roecker AJ, Hughes R, Hogenesch JB, Kast-Woelbern HR, et al. A chemical, genetic, and structural analysis of the nuclear bile acid receptor FXR. Mol Cell 2003; 11: 10791092.
  • 82
    Guo GL, Lambert G, Negishi M, Ward JM, Brewer JrHB, Kliewer SA, et al. Complementary roles of farnesoid X receptor, pregnane X receptor, and constitutive androstane receptor in protection against bile acid toxicity. J Biol Chem 2003; 278: 4506245071.
  • 83
    Fiorucci S, Clerici C, Antonelli E, Orlandi S, Goodwin B, Sadeghpour BM, et al. Protective effects of 6-ethyl chenodeoxycholic acid, a farnesoid X receptor ligand, in estrogen-induced cholestasis. J Pharmacol Exp Ther 2005; 313: 604612.
  • 84
    Liu Y, Binz J, Numerick MJ, Dennis S, Luo G, Desai B, et al. Hepatoprotection by the farnesoid X receptor agonist GW4064 in rat models of intra- and extrahepatic cholestasis. J Clin Invest 2003; 112: 16781687.
  • 85
    Christensen E, Crowe J, Doniach D, Popper H, Ranek L, Rodes J, et al. Clinical pattern and course of disease in primary biliary cirrhosis based on an analysis of 236 patients. Gastroenterology 1980; 78: 236246.
  • 86
    Sherlock S. Treatment and prognosis of primary biliary cirrhosis. Semin Liver Dis 1981; 1: 354364.
  • 87
    Mahl TC, Shockcor W, Boyer JL. Primary biliary cirrhosis survival of a large cohort of symptomatic and asymptomatic patients followed for 24 years. J Hepatol 1994; 20: 707713.
  • 88
    Corpechot C, Carrat F, Bahr A, Chretien Y, Poupon RE, Poupon R. The effect of ursodeoxycholic acid therapy on the natural course of primary biliary cirrhosis. Gastroenterology 2005; 128: 297303.
  • 89
    Combes B, Emerson SS, Flye NL, Munoz SJ, Luketic VA, Mayo MJ, et al. Methotrexate (MTX) plus ursodeoxycholic acid (UDCA) in the treatment of primary biliary cirrhosis. HEPATOLOGY 2005; 42: 11841193.
  • 90
    Zollner G, Fickert P, Zenz R, Fuchsbichler A, Stumptner C, Kenner L, et al. Hepatobiliary transporter expression in percutaneous liver biopsies of patients with cholestatic liver diseases. HEPATOLOGY 2001; 33: 633646.
  • 91
    Zollner G, Fickert P, Silbert D, Fuchsbichler A, Marschall H-U, Zatloukal K, et al. Adaptive changes in hepatobiliary transporter expression in primary biliary cirrhosis. J Hepatol 2003; 38: 717727.
  • 92
    Springer J, Cauch-Dudek K, O'Rourke K, Wahless IR, Heathcote EJ. Asymptomatic primary biliary cirrhosis: a study of its natural history and prognosis. Am J Gastroenterol 1999; 94: 4753.
    Direct Link:
  • 93
    Nyberg A, Loof L. Primary biliary cirrhosis: clinical features and outcome, with special reference to asymptomatic disease. Scand J Gastroenterol 1989; 24: 5764.
  • 94
    Prince M, Chetwynd A, Newman W, Metcalf JV, James OF. Survival and symptom progression in a geographically based cohort of patients with primary biliary cirrhosis: follow-up for up to 28 years. Gastroenterology 2002; 123: 10441051.
  • 95
    Wei P, Zhang J, Egan-Hafley M, Liang S, Moore DD. The nuclear receptor CAR mediates specific xenobiotic induction of drug metabolism. Nat (Lond) 2000; 407: 920923.
  • 96
    Huang W, Zhang Z, Chua SS, Qatanani M, Han Y, Granata R, et al. Induction of bilirubin clearance by the constitutive androstane receptor (CAR). Proc Natl Acad Sci U S A 2003; 100: 41564161.
  • 97
    Huang W, Zhang J, Moore DD. A traditional herbal medicine enhances bilirubin clearance by activating the nuclear receptor CAR. J Clin Invest 2004; 113: 137143.
  • 98
    Boyer JL, Hagenbuch B, Ananthanarayanan M, Suchy F, Stieger B, Meier PJ. Phylogenic and ontogenic expression of hepatocellular bile acid transport. Proc Natl Acad Sci 1993; 90: 435438.
  • 99
    Wang W, Seward DJ, Li L, Boyer JL, Ballatori N. Expression cloning of two genes that together mediate organic solute and steroid transport in the liver of a marine vertebrate. Proc Natl Acad Sci U S A 2001; 98: 94319436.
  • 100
    Seward DJ, Koh AS, Boyer JL, Ballatori N. Functional complementation between a novel mammalian polygenic transport complex and an evolutionarily ancient organic solute transporter, OSTα-OSTβ. J Biol Chem 2003; 278: 2747327482.
  • 101
    Ballatori N, Christian WV, Lee JY, Dawson Pa, Soroka CJ, Boyer JL, et al. OSTα-OSTβ: a major basolateral bile acid and steroid transporter in human intestinal, renal, and biliary epithelia. HEPATOLOGY 2005; 42: 12701279.
  • 102
    Rao A, Haywood J, Craddock AL, Belinsky MG, Kruh GD, Dawson PA. The organic solute transporter alpha-beta, Ostalpha-Ostbeta, is essential for intestinal bile acid transport and homeostasis. Proc Natl Acad Sci U S A 2008; 105: 38913896.
  • 103
    Ballatori N, Fang F, Christian WV, Li N, Hammond CL. Ost{alpha}-Ost{beta} is required for bile acid and conjugated steroid disposition in the intestine, kidney, and liver. Am J Physiol Gastrointest Liver Physiol 2008; 295: G179G186.
  • 104
    Boyer JL, Trauner M, Mennone A, Soroka CJ, Cai SY, Moustafa T, et al. Up-regulation of a basolateral FXR-dependent bile acid efflux transporter, OST{alpha}-OST{beta}, in cholestasis in humans and rodents. Am J Physiol Gastrointest Liver Physiol 2006; 290: G1124G1130.
  • 105
    Cai SY, Boyer JL. FXR: a target for cholestatic syndromes? Expert Opin Ther Targets 2006; 10: 409421.