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Yes-associated protein 1 (YAP1) regulates cell plasticity during liver injury, regeneration, and cancer, but its role in liver development is unknown. We detect YAP1 activity in biliary cells and in cells at the hepatobiliary bifurcation in single-cell RNA sequencing analysis of developing livers. Deletion of Yap1 in hepatoblasts does not impair Notch-driven SOX9+ ductal plate formation but does prevent the formation of the abutting second layer of SOX9+ ductal cells, blocking the formation of a patent intrahepatic biliary tree. Intriguingly, these mice survive for 8 months with severe cholestatic injury and without hepatocyte-to-biliary transdifferentiation. Ductular reaction in the perihilar region suggests extrahepatic biliary proliferation, likely seeking the missing intrahepatic biliary network. Long-term survival of these mice occurs through hepatocyte adaptation via reduced metabolic and synthetic function, including altered bile acid metabolism and transport. Overall, we show YAP1 as a key regulator of bile duct development while highlighting a profound adaptive capability of hepatocytes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The biliary system is routinely accessed for clinical purposes via endoscopic retrograde cholangiopancreatography (ERCP). We previously pioneered ERCP-mediated hydrodynamic injection in large animal models as an innovative gene delivery approach for monogenic liver diseases. However, the procedure poses potential safety concerns related mainly to liver or biliary tree injury. Here, we sought to further define biliary hydrodynamic injection parameters that are well-tolerated in a human-sized animal model. ERCP was performed in pigs, and hydrodynamic injection carried out using a novel protocol to reduce duct wall stress. Each pig was subjected to multiple repeated injections to expedite testing and judge tolerability. Different injection parameters (volume, flow rate) and injection port diameters were tested. Vital signs were monitored throughout the procedure, and liver enzyme panels were collected pre- and post-procedure. Pigs tolerated repeated biliary hydrodynamic injections with only occasional, mild, isolated elevation in aspartate aminotransferase (AST), which returned to normal levels within one day post-injection. All other liver tests remained unchanged. No upper limit of volume tolerance was reached, which suggests the biliary tree can readily transmit fluid into the vascular space. Flow rates up to 10 mL/sec were also tolerated with minimal disturbance to vital signs and no anatomic rupture of bile ducts. Measured intrabiliary pressure was up to 150 mmHg, and fluid-filled vesicles were induced in liver histology at high flow rates, mimicking the changes in histology observed in mouse liver after hydrodynamic tail vein injection. Overall, our investigations in a human-sized pig liver using standard clinical equipment suggest that ERCP-guided hydrodynamic injection will be safely tolerated in patients. Future investigations will interrogate if higher flow rates and pressure mediate higher DNA delivery efficiencies., Competing Interests: Conflict of Interest: R.L.K, Y.H., V.K., and F.M.S. have a patent application filed on the work. All other authors do not have conflict of interests. There are no other licensing or consulting interests; this does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: Chronic liver diseases are frequently accompanied with activation of biliary epithelial cells (BECs) that can differentiate into hepatocytes and cholangiocytes, providing an endogenous back-up system. Functional studies on BECs often rely on isolations of an BEC cell population from healthy and/or injured livers. However, a consensus on the characterization of these cells has not yet been reached. The aim of this study was to compare the publicly available transcriptome profiles of human and mouse BECs and to establish gene signatures that can identify quiescent and activated human and mouse BECs., Methods: We used publicly available transcriptome data sets of human and mouse BECs, compared their profiles and analyzed co-expressed genes and pathways. By merging both human and mouse BEC-enriched genes, we obtained a quiescent and activation gene signature and tested them on BEC-like cells and different liver diseases using gene set enrichment analysis. In addition, we identified several genes from both gene signatures to identify BECs in a scRNA sequencing data set., Results: Comparison of mouse BEC transcriptome data sets showed that the isolation method and array platform strongly influences their general profile, still most populations are highly enriched in most genes currently associated with BECs. Pathway analysis on human and mouse BECs revealed the KRAS signaling as a new potential pathway in BEC activation. We established a quiescent and activated BEC gene signature that can be used to identify BEC-like cells and detect BEC enrichment in alcoholic hepatitis, non-alcoholic steatohepatitis (NASH) and peribiliary sclerotic livers. Finally, we identified a gene set that can distinguish BECs from other liver cells in mouse and human scRNAseq data., Conclusions: Through a meta-analysis of human and mouse BEC gene profiles we identified new potential pathways in BEC activation and created unique gene signatures for quiescent and activated BECs. These signatures and pathways will help in the further characterization of this progenitor cell type in mouse and human liver development and disease., Competing Interests: The authors declare no conflict of interest.

Two distinct stem/progenitor cell populations of biliary origin have been identified in the adult liver and biliary tree. Hepatic Stem/progenitor Cells (HpSCs) are bipotent progenitor cells located within the canals of Hering and can be differentiated into mature hepatocytes and cholangiocytes; Biliary Tree Stem/progenitor Cells (BTSCs) are multipotent stem cells located within the peribiliary glands of large intrahepatic and extrahepatic bile ducts and able to differentiate into hepatic and pancreatic lineages. HpSCs and BTSCs are endowed in a specialized niche constituted by supporting cells and extracellular matrix compounds. The actual contribution of these stem cell niches to liver and biliary tree homeostatic regeneration is marginal; this is due to the high replicative capabilities and plasticity of mature parenchymal cells (i.e., hepatocytes and cholangiocytes). However, the study of human liver and biliary diseases disclosed how these stem cell niches are involved in the regenerative response after extensive and/or chronic injuries, with the activation of specific signaling pathways. The present review summarizes the contribution of stem/progenitor cell niches in human liver diseases, underlining mechanisms of activation and clinical implications, including fibrogenesis and disease progression., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Aim: To elucidate the role of STAT3 in hepatocarcinogenesis and biliary ductular proliferation following chronic liver injury., Methods: We investigated thioacetamide (TAA)-induced liver injury, compensatory hepatocyte proliferation, and hepatocellular carcinoma (HCC) development in hepatic STAT3-deficient mice. In addition, we evaluated TAA-induced biliary ductular proliferation and analyzed the activation of sex determining region Y-box9 (SOX9) and Yes-associated protein (YAP), which regulate the transdifferentiation of hepatocytes to cholangiocytes., Results: Both compensatory hepatocyte proliferation and HCC formation were significantly decreased in hepatic STAT3-deficient mice as compared with control mice. STAT3 deficiency resulted in augmentation of hepatic necrosis and fibrosis. On the other hand, biliary ductular proliferation increased in hepatic STAT3-deficient livers as compared with control livers. SOX9 and YAP were upregulated in hepatic STAT3-deficient hepatocytes., Conclusion: STAT3 may regulate hepatocyte proliferation as well as transdifferentiation into cholangiocytes and serve as a therapeutic target for HCC inhibition and biliary regeneration., Competing Interests: Conflict-of-interest statement: The authors have no conflicts of interest.

The treatment of common bile duct (CBD) disorders, such as biliary atresia or ischemic strictures, is restricted by the lack of biliary tissue from healthy donors suitable for surgical reconstruction. Here we report a new method for the isolation and propagation of human cholangiocytes from the extrahepatic biliary tree in the form of extrahepatic cholangiocyte organoids (ECOs) for regenerative medicine applications. The resulting ECOs closely resemble primary cholangiocytes in terms of their transcriptomic profile and functional properties. We explore the regenerative potential of these organoids in vivo and demonstrate that ECOs self-organize into bile duct-like tubes expressing biliary markers following transplantation under the kidney capsule of immunocompromised mice. In addition, when seeded on biodegradable scaffolds, ECOs form tissue-like structures retaining biliary characteristics. The resulting bioengineered tissue can reconstruct the gallbladder wall and repair the biliary epithelium following transplantation into a mouse model of injury. Furthermore, bioengineered artificial ducts can replace the native CBD, with no evidence of cholestasis or occlusion of the lumen. In conclusion, ECOs can successfully reconstruct the biliary tree, providing proof of principle for organ regeneration using human primary cholangiocytes expanded in vitro.

The complex architecture of the liver biliary network represents a structural prerequisite for the formation and secretion of bile as well as excretion of toxic substances through bile ducts. Disorders of the biliary tract affect a significant portion of the worldwide population, often leading to cholestatic liver diseases. Cholestatic liver disease is a condition that results from an impairment of bile formation or bile flow to the gallbladder and duodenum. Cholestasis leads to dramatic changes in biliary tree architecture, worsening liver disease and systemic illness. Recent studies show that the prevalence of cholestatic liver diseases is increasing. The availability of well characterized animal models, as well as development of visualization approaches constitutes a critical asset to develop novel pathogenetic concepts and new treatment strategies.

Bile, the central metabolic product of the liver, is transported by the bile canaliculi network. The impairment of bile flow in cholestatic liver diseases has urged a demand for insights into its regulation. Here, we developed a predictive 3D multi-scale model that simulates fluid dynamic properties successively from the subcellular to the tissue level. The model integrates the structure of the bile canalicular network in the mouse liver lobule, as determined by high-resolution confocal and serial block-face scanning electron microscopy, with measurements of bile transport by intravital microscopy. The combined experiment-theory approach revealed spatial heterogeneities of biliary geometry and hepatocyte transport activity. Based on this, our model predicts gradients of bile velocity and pressure in the liver lobule. Validation of the model predictions by pharmacological inhibition of Rho kinase demonstrated a requirement of canaliculi contractility for bile flow in vivo. Our model can be applied to functionally characterize liver diseases and quantitatively estimate biliary transport upon drug-induced liver injury., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Aberrant transcriptional regulation contributes to the pathogenesis of both congenital and adult forms of liver disease. Although the transcription factor RBPJ is essential for liver morphogenesis and biliary development, its specific function in the differentiation of hepatic progenitor cells (HPC) has not been investigated, and little is known about its role in adult liver regeneration. HPCs are bipotent liver stem cells that can self-replicate and differentiate into hepatocytes or cholangiocytes in vitro. HPCs are thought to play an important role in liver regeneration and repair responses. While the coordinated repopulation of both hepatocyte and cholangiocyte compartment is pivotal to the structure and function of the liver after regeneration, the mechanisms coordinating biliary regeneration remain vastly understudied. Here, we utilized complex genetic manipulations to drive liver-specific deletion of the Rbpj gene in conjunction with lineage tracing techniques to delineate the precise functions of RBPJ during biliary development and HPC-associated biliary regeneration after hepatectomy. Furthermore, we demonstrate that RBPJ promotes HPC differentiation toward cholangiocytes in vitro and blocks hepatocyte differentiation through mechanisms involving Hippo-Notch crosstalk. Overall, this study demonstrates that the Notch-RBPJ signaling axis critically regulates biliary regeneration by coordinating the fate decision of HPC and clarifies the molecular mechanisms involved.

Autosomal recessive polycystic kidney disease, an inherited disorder characterized by the formation of cysts in renal collecting ducts and biliary dysgenesis, is caused by mutations of the polycystic kidney and hepatic disease 1 (PKHD1) gene. Expression of PKHD1 is tissue specific and developmentally regulated. Here, we show that a 2.0-kb genomic fragment containing the proximal promoter of mouse Pkhd1 directs tissue-specific expression of a lacZ reporter gene in transgenic mice. LacZ is expressed in renal collecting ducts beginning during embryonic development but is not expressed in extrarenal tissues. The Pkhd1 promoter contains a binding site for the transcription factor hepatocyte nuclear factor (HNF)-1β, which is required for activity in transfected cells. Mutation of the HNF-1β-binding site abolishes the expression of the lacZ reporter gene in renal collecting ducts. Transgenes containing the 2.0-kb promoter and 2.7 kb of additional genomic sequence extending downstream to the second exon are expressed in the kidney, intrahepatic bile ducts, and male reproductive tract. This pattern overlaps with the endogenous expression of Pkhd1 and coincides with sites of expression of HNF-1β. We conclude that the proximal 2.0-kb promoter is sufficient for tissue-specific expression of Pkhd1 in renal collecting ducts in vivo and that HNF-1β is required for Pkhd1 promoter activity in collecting ducts. Additional genomic sequences located from exons 1-2 or elsewhere in the gene locus are required for expression in extrarenal tissues., (Copyright © 2014 the American Physiological Society.)

Stem/progenitors have been identified intrahepatically in the canals of Hering and extrahepatically in glands of the biliary tree. Glands of the biliary tree (peribiliary glands) are tubulo-alveolar glands with mucinous and serous acini, located deep within intrahepatic and extrahepatic bile ducts. We have shown that biliary tree stem/progenitors (BTSCs) are multipotent, giving rise in vitro and in vivo to hepatocytes, cholangiocytes or pancreatic islets. Cells with the phenotype of BTSCs are located at the bottom of the peribiliary glands near the fibromuscular layer. They are phenotypically heterogeneous, expressing transcription factors as well as surface and cytoplasmic markers for stem/progenitors of liver (e.g. SOX9/17), pancreas (e.g. PDX1) and endoderm (e.g. SOX17, EpCAM, NCAM, CXCR4, Lgr5, OCT4) but not for mature markers (e.g. albumin, secretin receptor or insulin). Subpopulations co-expressing liver and pancreatic markers (e.g. PDX1(+)/SOX17(+)) are EpCAM(+/-), and are assumed to be the most primitive of the BTSC subpopulations. Their descendants undergo a maturational lineage process from the interior to the surface of ducts and vary in the mature cells generated: pancreatic cells in hepatopancreatic ducts, liver cells in large intrahepatic bile ducts, and bile duct cells along most of the biliary tree. We hypothesize that there is ongoing organogenesis throughout life, with BTSCs giving rise to hepatic stem cells in the canals of Hering and to committed progenitors within the pancreas. The BTSCs are likely to be central to normal tissue turnover and injury repair and to be key elements in the pathophysiology of liver, pancreas and biliary tree diseases, including oncogenesis., (© 2011 The Authors. Journal of Anatomy © 2011 Anatomical Society.)

Animal model phenotyping, in utero exposure toxicity studies, and investigation into causes of embryonic, fetal, or perinatal deaths have required pathologists to recognize and diagnose developmental disorders in spontaneous and engineered mouse models of disease. In mammals, the liver is the main site of hematopoiesis during fetal development, has endocrine and exocrine functions important for maintaining homeostasis in fetal and adult life; and performs other functions including waste detoxification, production and removal of glucose, glycogen storage, triglyceride and fatty acid processing, and serum protein production. Due to its role in many critical functions, alterations in the size, morphology, or function(s) of the liver often lead to embryonic lethality. Many publications and websites describe individual aspects of hepatobiliary development at defined stages. However, no single resource provides a detailed histological evaluation of H&E-stained sections of the developing murine liver and biliary systems using high-magnification and high-resolution color images. The work herein provides a histology atlas of hepatobiliary development between embryonic days 9.5-18.5. Although the focus of this work is normal hepatobiliary development, common defects in liver development are also described as a reference for pathologists who may be asked to phenotype mice with congenital, inherited, or treatment-related hepatobiliary defects. Authors' note: All digital images can be viewed online at https://niehsimagesepl-inc.com with the username "ToxPathLiver" and the password "embryolivers."

The results of experimental investigation of peculiarities of morphological transformation course in autotissues under the influence of bile were adduced. There was proposed a choice of peritoneum as an optimal plastic material in conditions of possible deficiency of local tissues during biliary surgery performance.

The biliary tree system consists of two divisions: intrahepatic bile ducts and extrahepatic bile ducts. The development of the biliary tree, and secondarily the liver, shares a common origin with ventral pancreas. A common progenitor for liver, biliary duct system, and ventral pancreas exists at early stages of development, when the anterior definitive endoderm is forming the foregut. Several studies indicate that the biliary tree contains stem cell compartments for liver, pancreas and the bile duct system and persisting into adulthood. These stem cell compartments are present in the peribiliary glands and possibly give rise to committed progenitors in gallbladder that does not have peribiliary glands. The biliary tree stem/progenitors represent a new source of cells that can be used as tools for regenerative medicine of liver, bile duct and pancreas.

In the liver, adenosine triphosphate (ATP) is an extracellular signaling molecule that is released into bile and stimulates a biliary epithelial cell secretory response via engagement of apical P2 receptors. The molecular identities of the ion channels involved in ATP-mediated secretory responses have not been fully identified. Intermediate-conductance Ca(2+)-activated K(+) channels (IK) have been identified in biliary epithelium, but functional data are lacking. The aim of these studies therefore was to determine the location, function, and regulation of IK channels in biliary epithelial cells and to determine their potential contribution to ATP-stimulated secretion. Expression of IK-1 mRNA was found in both human Mz-Cha-1 biliary cells and polarized normal rat cholangiocyte (NRC) monolayers, and immunostaining revealed membrane localization with a predominant basolateral signal. In single Mz-Cha-1 cells, exposure to ATP activated K(+) currents, increasing current density from 1.6 +/- 0.1 to 7.6 +/- 0.8 pA/pF. Currents were dependent on intracellular Ca(2+) and sensitive to clotrimazole and TRAM-34 (specific IK channel inhibitors). Single-channel recording demonstrated that clotrimazole-sensitive K(+) currents had a unitary conductance of 46.2 +/- 1.5 pS, consistent with IK channels. In separate studies, 1-EBIO (an IK activator) stimulated K(+) currents in single cells that were inhibited by clotrimazole. In polarized NRC monolayers, ATP significantly increased transepithelial secretion which was inhibited by clotrimazole. Lastly, ATP-stimulated K(+) currents were inhibited by the P2Y receptor antagonist suramin and by the inositol 1,4,5-triphosphate (IP3) receptor inhibitor 2-APB. Together these studies demonstrate that IK channels are present in biliary epithelial cells and contribute to ATP-stimulated secretion through a P2Y-IP3 receptor pathway.

In adults, the hepatobiliary system, together with the kidney, constitute the main routes for the elimination of several endogenous and xenobiotic compounds into bile and urine, respectively. However, during intrauterine life the biliary route of excretion for cholephilic compounds, such as bile acids and biliary pigments, is very poor. Although very early in pregnancy the fetal liver produces bile acids, bilirubin and biliverdin, these compounds cannot be efficiently eliminated by the fetal hepatobiliary system, owing to the immaturity of the excretory machinery in the fetal liver. Therefore, the potentially harmful accumulation of cholephilic compounds in the fetus is prevented by their elimination across the placenta. Owing to the presence of detoxifying enzymes and specific transport systems at different locations of the placental barrier, such as the endothelial cells of chorionic vessels and trophoblast cells, this organ plays an important role in the hepatobiliary-like function during intrauterine life. The relevance of this excretory function in normal fetal physiology is evident in situations where high concentrations of biliary compounds are accumulated in the mother. This may result in oxidative stress and apoptosis, mainly in the placenta and fetal liver, which might affect normal fetal development and challenge the fate of the pregnancy. The present article reviews current knowledge of the mechanisms underlying the hepatobiliary function of the fetal-placental unit and the repercussions of several pathological conditions on this tandem.

This study compared the effects of soybean oil- versus olive oil-based lipid emulsions on hepatobiliary function and serum triacylglycerols in patients who required transient parenteral nutrition support for significant weight loss. Patients who received a parenteral ready-to-use industry admixture including either soybean oil- (n = 10) or olive oil-based lipid emulsion (n = 11) for 2 weeks were retrospectively analysed. Cholestatic and cytolytic enzymes, conjugated bilirubin and serum triacylglycerols were sampled before and 1 day after completing parenteral nutrition support. Significant deterioration of cholestatic enzymes occurred in five patients in the soybean oil group and in one in the olive oil group. Serum triacylglycerols significantly deteriorated in seven patients in the soybean oil group and in one patient in the olive oil group. No differences were recorded for cytolytic enzyme abnormalities. In conclusion, the olive oil-based emulsion induced abnormalities of cholestatic enzymes and serum triacylglycerols significantly less frequently than the soybean oil-based emulsion.

This paper reviews the progress made in understanding the mechanical behaviour of the biliary system. Gallstones and diseases of the biliary tract affect more than 10% of the adult population. The complications of gallstones, i.e. acute pancreatitis and obstructive jandice, can be lethal, and patients with acalculous gallbladder pain often pose diagnostic difficulties and undergo repeated ultrasound scans and oral cholecystograms. Moreover, surgery to remove the gallbladder in these patients, in an attempt to relieve the symptoms, gives variable results. Extensive research has been carried out to understand the physiological and pathological functions of the biliary system, but the mechanism of the pathogenesis of gallstones and pain production still remain poorly understood. It is believed that the mechanical factors play an essential role in the mechanisms of the gallstone formation and biliary diseases. However, despite the extensive literature in clinical studies, only limited work has been carried out to study the biliary system from the mechanical point of view. In this paper, we discuss the state of art knowledge of the fluid dynamics of bile flow in the biliary tract, the solid mechanics of the gallbladder and bile ducts, recent mathematical and numerical modelling of the system, and finally the future challenges in the area.

Cytosolic Ca2+ is an important second messenger in virtually every type of cell. Moreover, Ca2+ generally regulates multiple activities within individual cells. This article reviews the cellular machinery that is responsible for Ca2+ signaling in cholangiocytes. In addition, two Ca2+-mediated events in cholangiocytes are discussed: bicarbonate secretion and apoptosis. Finally, emerging evidence is reviewed that Ca2+ signaling is involved in the pathogenesis of diseases affecting the biliary tree and that Ca2+ signaling pathways can be manipulated to therapeutic advantage in the treatment of cholestatic disorders.

This essay will address areas of liver stem/progenitor cell studies in which consensus has emerged and in which controversy still prevails over consensus, but it will also highlight important themes that inevitably should be a focus of liver stem/progenitor cell investigations in coming years. Thus concepts regarding cell plasticity, the existence of a physiological/anatomic stem cell niche, and whether intrahepatic liver stem/progenitor cells comprise true stem cells or progenitor cells (or both) will be approached in some detail.

T cells infiltrating the inflamed liver express high levels of CXCR 3 and show enhanced migration to CXCR 3 ligands in chemotactic assays. Moreover, CXCR 3 ligands are up-regulated on hepatic endothelium at sites of T-cell infiltration in chronic hepatitis, and their presence correlates with outcome of inflammatory liver disease. We used a flow-based adhesion assay with human hepatic endothelium to investigate the function of CXCR 3 on lymphocyte adhesion to and transmigration through hepatic endothelium under physiological conditions of blood flow. To more accurately model the function of in vivo activated CXCR 3(high) lymphocytes, we isolated T cells from human liver tissue and studied their behavior in flow-based adhesion assays. We demonstrate that CXCR 3 not only promoted the adhesion of effector T cells to endothelium from flow but also drove transendothelial migration. Moreover, these responses could be stimulated either by endogenous CXCR 3 ligands secreted by the endothelium or by exogenous CXCR 3 ligands derived from other cell types and presented by the endothelium. This study thus demonstrates that activation of CXCR 3 promotes lymphocyte adhesion and transendothelial migration under flow and that human hepatic endothelium can present functionally active chemokines secreted by other cell types within the liver.

Hepatobiliary transport systems mediate hepatic uptake and biliary excretion of bile acids, bilirubin and other biliary constituents. Hereditary or acquired defects of these transporters may cause or maintain cholestasis and jaundice under various clinical conditions including progressive familial intrahepatic cholestasis (PFIC) 1-3 or its milder forms, benign recurrent intrahepatic cholestasis (BRIC) 1 and 2 , Dubin-Johnson syndrome, drug and inflammation-induced cholestasis and intrahepatic cholestasis of pregnancy. Moreover, induction of alternative efflux pumps for bile acids/bilirubin and phase I/II detoxifying enzymes may counteract hepatic accumulation of potentially toxic biliary constituents in cholestasis by providing alternative escape routes. Transcriptional and post-transcriptional regulation of hepatobiliary transporters in health and disease is mediated by multiple factors such as bile acids, proinflammatory cytokines, drugs and hormones. Ligand-activated nuclear receptors (NR) and hepatocyte-enriched transcription factors play a critical role in transcriptional transporter regulation. Many hepatobiliary transporter alterations in cholestatic liver disease can now be explained by ligand binding of accumulating cholephiles to NRs. Moreover, NR-mediated actions may be targeted by pharmacological ligands. Understanding the transcriptional mechanisms leading to transporter changes therefore not only represents a key for understanding the pathophysiology of the cholestatic liver disease, but also represents a prerequisite for designing novel therapeutic strategies.

Our understanding of biliary epithelial cells (BEC) in physiobiology and immunology has steadily expanded. BEC transports IgA as well as IgM into bile, synthesizes and secretes various chemokines, cytokines, and expresses adhesion molecules involved in cell interaction and signal transduction. These then suggest a myriad of potential roles for BEC in defense from invading microorganisms as well as the pathogenesis of diverse immunologically driven diseases such as primary biliary cirrhosis (PBC), graft-versus-host disease, and primary sclerosing cholangitis (PSC). Despite the progress, there still remain many areas of BEC biology that require further investigation. Most importantly, it remains to be clarified that the extent to which the immunologic activities observed in BEC represent a BEC response to tissue injury or whether BEC themselves are the active participants in the pathogenesis of various cholestatic immunological diseases, including PBC and PSC.

The enterohepatic recirculation of bile salts exerts important regulatory effects on many hepatic, biliary and intestinal functions: such regulation is likely to depend, to a large extent, on the physical-chemical property of hydrophobicity of the recirculating pool. The present review summarizes the main experimental evidence carried out by our research group over the past two decades, in the attempt to investigate systematically the relationships between structural properties and biological effects of bile acids in humans. Hydrophobic bile acids (chenodeoxycholic acid, deoxycholic acid), but not hydrophilic acids (ursodeoxycholic acid), significantly suppressed hepatic activity of HMG-CoA reductase, the limiting step of cholesterol synthesis, and in vivo cholesterol 7alpha-hydroxylation, the limiting step of bile acid synthesis. The output of biliary cholesterol and phospholipid was also directly related to the hydrophobicity of the bile acid pool. Finally, treatment with chenodeoxycholic acid, but not with ursodeoxycholic acid, significantly decreased gall-bladder emptying rates. When turning to the in vitro model of HepG2 cells, hydrophobic bile acids were found to induce greater cytotoxic and pro-apoptotic effects. From this series of studies, we conclude that the regulatory effects of bile acids on the liver and biliary tract are largely dependent on the hydrophobic-hydrophilic balance of the recirculating bile acid pool.

Unlabelled: Opioids cause spasm of the sphincter of Oddi. Remifentanil is metabolized enzymatically throughout the body. Its context-sensitive half-time is 3-4 min. The effect of remifentanil on the sphincter of Oddi, is unknown. We studied, in six healthy adult volunteers, the effect of remifentanil on the flow of dye from the gall bladder into the duodenum. Control hepatobiliary imaging with 5 mCi of technetium-labeled derivatives of iminodiacetic acid was performed on each volunteer. The time from IV dye (radiopharmaceutical) injection until its appearance in the duodenum was determined by continuous scanning. Two weeks later, each volunteer received remifentanil, 0.1 microg x kg(-1) x min(-1) infused for 30 min IV before the same dose of technetium-labeled derivatives of iminodiacetic acid was injected, and for the time of their control scan plus 10 min after the injection. When the dye appeared in the duodenum, the total time from injection was compared with the control value. The time from stopping the infusion until the dye appeared in the duodenum was the "recovery time." Control scan time was 20.5+/-9.9 min (mean +/- SD; range 10-33 min). Total scan time during and after the remifentanil infusion was 50.3+/-17.3 min (range 30-81 min) (P < 0002). The recovery time was 19.8+/-12.4 min (range 5-40 min). We conclude that remifentanil delays the drainage of dye from the gall bladder into the duodenum, but the delay is shorter than that reported after other studied opioids., Implications: Radioactive dye was injected IV into healthy volunteers to determine the time it took for the dye to appear in the duodenum. This was repeated under the influence of a short-acting narcotic analgesic, remifentanil. Remifentanil caused a much shorter delay than previously reported after morphine or meperidine.

The secretion of bile requires the vectorial transport of organic and inorganic solutes from sinusoidal blood to the canalicular lumen. Hydrostatic forces cannot account for biliary secretion, because secretory pressures within bile ducts exceed that of blood within the sinusoidal space. Instead, the process of bile formation requires active transport across the basolateral membrane, transcellular movement through a variety of mechanisms, and then active transport into the canalicular space between hepatocytes. Separate hepatic and ductular transport mechanisms allow for rapid regulation of bile volume and composition required for changing physiologic needs. The array of transport proteins localized to both poles of the hepatocyte have been characterized physiologically and during development. Many have now been cloned and studied further in transgenic models. The recent identification and characterization of several genes that are mutated in inherited forms of cholestatic liver disease have provided new insight into the normal physiology of bile secretion, the pathophysiology of intrahepatic cholestasis, and an unexpected major role for a novel group of P-type ATPases in human biology and disease.

1. In the present study it was tested whether known P-glycoprotein (P-gp) substrates/MDR reversal agents interact with small (type 1) and bulky (type 2) cationic drugs at the level of biliary excretion in the rat isolated perfused liver model (IPRL). The studies were performed with model compounds tri-n-butylmethylammonium (TBuMA) (a relatively small type 1 organic cation), rocuronium (Roc) (a bulky type 2 organic cation) and the classical P-gp substrate doxorubicin (Dox). 2. Inhibitors were given in a 4 fold molar excess to the substrate studied. To minimize an interaction of the substrates at the hepatic uptake level, the competing compounds were added when over 55% to 85% of the administered dose of the model compounds had been removed from the perfusate and taken up by the liver. 3. We found a mutual interaction between TBuMA and procainamidethobromide (PAEB), both type 1 cationic compounds during biliary excretion. Interestingly, type 2 compounds, such as rocuronium, clearly inhibited type 1 cationic drugs as well as Dox secretion into bile, whereas type 1 compounds did not significantly inhibit type 2 drug excretion into bile. The type 1 cations PAEB and TBuMA only moderately inhibited Dox biliary excretion. Dox did not inhibit the biliary excretion of the type 2 agent rocuronium whereas rocuronium reduced Dox biliary excretion by 50% compared to controls. 4. MDR substrates/reversal agents like verapamil, quinine, quinidine and vinblastine strongly reduced both type 1 and type 2 organic cation excretion into bile. Dox secretion into bile was also profoundly reduced by these drugs, vinblastine being the most potent inhibitor in general. 5. The lack of mutual inhibition observed in some combinations of substrates may indicate that major differences in affinity of the substrates for a single excretory system exist. Alternatively, multiple organic cation transport systems with separate substrate specificities may be involved in the biliary excretion of amphiphilic drugs. Furthermore, the present study revealed a clear positive correlation between the lipophilicity of the potential inhibitors studied and their respective inhibitory activity on the biliary excretion of the model drugs investigated. 6. Our data are compatible with a potential involvement of P-glycoprotein in the hepatobiliary excretion of doxorubicin as well as of some type 1 and type 2 organic cations. Furthermore we postulate that the hydrophobic properties of the amphiphilic cationic drugs studied play a crucial role in the accommodation of these agents by P-glycoprotein and/or other potential cationic drug carrier proteins in the canalicular membrane.

Cryptosporidium parvum infection in the immunosuppressed host is frequently complicated by biliary tract involvement. The recent production of human biliary epithelial cell lines was exploited to develop an in vitro model of biliary cryptosporidiosis. Infection with C. parvum oocysts was detected by IFA and ELISA and confirmed by transmission electron microscopy. Inoculation of monolayers with 10(4) to 5 X 10(5) oocysts/well resulted in a dose-dependent increase in infection. Time-course experiments showed that the number of parasitic stages was maximal at 18-24 h after inoculation. Infection was significantly enhanced by bile at concentrations of 50 and 100 microg/mL and inhibited by 400 microg/mL paromomycin. Infection of human biliary cells with C. parvum can be consistently achieved and monitored by use of IFA or ELISA. This system will be of use in evaluating mechanisms of C. parvum infection and response to therapeutic agents in biliary cryptosporidiosis.

In this review, we have emphasized the importance of cholangiocytes in biology and pathology and highlighted recent advances in experimental approaches to study these increasingly important cells. It is anticipated that major advances will continue to be made in our understanding of how these cells function and the processes which result in their dysfunction. The techniques are now available to allow major inroads in our understanding of the pathobiology of cholangiocytes.

It has been reported that interdigestive motor activities occur in the gallbladder and sphincter of Oddi as well as in the gastroduodenal tract and truncal vagus nerves modulate the gastroduodenal motility pattern. In the present study, the vagal branches which influence on the interdigestive motor activity of these organs were determined in conscious dogs. In the normal dog, interdigestive motor activities closely related to interdigestive migrating motor complex (IMC) in the gastric antrum and descending duodenum were recorded in the gallbladder and sphincter of Oddi. In dogs whose celiac branches of the vagus nerve were chronically resected, cycle periods of interdigestive motor activity in the stomach, duodenum, gallbladder and sphincter of Oddi significantly prolonged as compared with those in the normal dog but the interrelation of them persisted. Quiescent period in motility prolonged and the active period shortened. Resection of the pyloric branches had no influence on the interdigestive motor activities in all organs. The results imply that celiac branches of the vagus nerve modulate the interdigestive motor activity in the stomach, descending duodenum, gallbladder and sphincter of Oddi.

At one time it was thought that biliary epithelial cells simply formed the lining to the tubular conduits which constitute the biliary tract. Development of in vitro systems for culturing biliary epithelial cells has enabled functional studies which increasingly show that this is far from true, and that biliary epithelial cells do have important functional roles. Disruption of these functions may be involved in the generation of pathology. Most functional studies to date have utilised cells isolated from rat liver. Increasingly, variations are being found between human and animal cells both in terms of function and phenotype. The relevance of animal cells in the study of human disease therefore remains obscure. Human biliary tract disease has to date been studied almost exclusively by examination of histological sections. The development of improved methods for isolating highly pure biliary epithelial cells from human liver provides a new technology with which to investigate directly the dynamics of human biliary epithelial cell biology and pathobiology. It is predicted that further progress will now be made in dissecting the biology and physiology of human biliary epithelium.

Many xenobiotics cause hepatobiliary toxicity and cholestasis in the rat. Initial assessment of hepatobiliary damage in rats can be accomplished by measuring serum concentrations of bile acids and bilirubin, serum activities of liver-associated enzymes such as 5'-nucleotidase, alkaline phosphatase, gamma-glutamyltranspeptidase, and plasma clearances of dyes [e.g., bromosulfophthalein (BSP)] excreted primarily through the bile. More detailed evaluation of hepatobiliary disturbances involves cannulation of the bile duct of anesthetized rats and subsequent measurement of rates of bile flow, bile acid excretion, and bile composition. Canalicular bile flow can be estimated from clearances of nonmetabolized sugars (i.e., erythritol) which enter bile via paracellular transport. Tight junction permeability also can be assessed by either biliary excretion of such a marker as horseradish peroxidase or sucrose following portal vein infusion or via retrograde biliary infusion. Subsequent morphologic evaluation of the liver provides information on damage to cells which may contribute to hepatobiliary dysfunction (i.e., bile duct obstruction). Isolated perfused livers offer the ability to measure all of the above mentioned parameters as well as to make a more accurate determination of the effects of xenobiotics on bile acid-dependent and -independent bile flow. A good example of the advantage of combining techniques as well as following complete time courses of changes in hepatobiliary function is provided by using studies of alpha-naphthylisothiocyanate-induced hepatotoxicity.

Although muscarinic receptor subtypes in the biliary system have been characterized, those in the vagal routes have not. This study was performed to characterize the muscarinic receptors in the vagal routes to the biliary system. The effects of four types of muscarinic antagonists, pirenzepine, AF-DX 116, p-F-HHSiD, and atropine, on excitatory responses in the gallbladder and sphincter of Oddi induced by electrical stimulation of the dorsal motor nucleus of the vagus (DMV) were studied in cervical cord-transected anesthetized dogs. Atropine 10 micrograms/kg and over and p-F-HHSiD 50 micrograms/kg and over significantly decreased the excitatory responses in both effectors, but it required a high dose of AF-DX 116 (1-2 mg/kg) to reduce those responses. Pirenzepine (50-300 micrograms/kg) significantly decreased the excitatory response in the gallbladder, but not that in the sphincter of Oddi. A high dose of atropine (0.5 mg/kg) abolished the gallbladder response, whereas a slight excitatory response remained in the sphincter. With the exception of pirenzepine, there was no significant difference in the effects of the three muscarinic blockers on the gallbladder response and sphincter response after reduction of the mean value of the atropine resistant responses from those of the sphincter responses after the muscarinic blockers. These results indicate that the excitatory vagal routes to the gallbladder include at least M1 receptors and those to the sphincter of Oddi may include M3 receptors.

Postnatal response of the gall bladder to feeding was studied in 28 preterm infants (gestational age 24-37 weeks) by ultrasonography immediately before feeding and one hour after feeding. Nineteen of the infants were studied once during the first week of life, and nine infants were studied weekly from a postconceptional age of 27-31 until a postprandial gall bladder contraction was seen. A contraction index was calculated as a percentage decrement of the surface area of the gall bladder from its initial value. All preterm infants of more than 33 weeks' gestation showed a gall bladder response to feeding with a contraction index of at least 50%. Very preterm infants (gestational age 27-32 weeks) showed no postprandial gall bladder contraction or the contraction index was under 50%. In a follow up study of nine very preterm infants, the contraction index exceeded 50% at a postconceptional age of 29 to 32 weeks. One infant with prolonged feeding problems had no postprandial gall bladder response before the postconceptional age of 38 weeks. The contraction index increase was dependent on gestational age at birth and on the bolus volume of feeds.

This study compared the effect of ketorolac tromethamine with that of morphine and placebo on biliary tract pressure. Intraoperatively, 31 anesthetized patients received either ketorolac (30 mg IV, n = 16) or morphine (5 mg IV, n = 15) after a cholecystectomy or gallstone removal. Intrabiliary tract pressure was measured 5 min after dosing. Postoperatively, 11 patients who had undergone biliary tract surgery received 10 mg of ketorolac or placebo, according to a randomized crossover design on 2 consecutive days. Intraoperatively, the biliary tract pressure did not change significantly from baseline after ketorolac administration. In the morphine group, there was significant increase in pressure over baseline. Postoperatively, there was no significant difference between ketorolac and placebo. We conclude that ketorolac has little or no effect on biliary tract dynamics; therefore, ketorolac may be a logical choice for analgesia in those situations in which spasm of the biliary tract is undesirable.

Peripheral routes of the vagus nerves to the biliary system were studied in anesthetized dogs using various selective vagal stimulation. Efferent stimulation of the gastric, hepatic, or celiac vagal branch as well as the cervical or thoracic vagal trunk induced gallbladder and Oddi's sphincter contractions, but those induced by hepatic vagal stimulation were rather small. The contraction responses in the gallbladder and sphincter of Oddi induced by thoracic vagal stimulation were greatly reduced after an external ligation around the pyloric sphincter. After administration of sympathetic blockers and atropine, vagally-induced gallbladder contractions were completely abolished and slight relaxation was seen in some animals. On the other hand, relaxation or transient relaxation followed by enhanced contractions was elicited in the sphincter of Oddi by vagal stimulation after atropine and sympathetic blockers. The relaxation response in the gallbladder after atropine and sympathetic blockers was abolished and that in the sphincter of Oddi was greatly reduced after the ligation around the pyloric sphincter. Stimulation of a ventral part of the anterior hypothalamic area induced gallbladder contraction and simultaneous relaxation of the sphincter of Oddi. These responses were completely abolished by the ligation around the pyloric sphincter in six dogs, while a slight relaxation response in the sphincter of Oddi remained in two dogs. These results suggest that the vagal fibers passing across the pyloric sphincter region are important for regulation of canine biliary motility and that extragastric vagal routes play a minor role in the nervous control of canine bile evacuation.

The present study concerns short- and long-term effects of interruption of the enterohepatic circulation (EHC) on hepatic cholesterol metabolism and biliary secretion in rats. For this purpose, we employed a technique that allows reversible interruption of the EHC, during normal feeding conditions, and excludes effects of anaesthesia and surgical trauma. [3H]Cholesteryl oleate-labelled human low-density lipoprotein (LDL) was injected intravenously in rats with (1) chronically (8 days) interrupted EHC, (2) interrupted EHC at the time of LDL injection and (3) intact EHC. During the first 3 h after interruption of the EHC, bile flow decreased to 50% and biliary bile acid, phospholipid and cholesterol secretion to 5%, 11% and 19% of their initial values respectively. After 8 days of bile diversion, biliary cholesterol output and bile flow were at that same level, but bile acid output was increased 2-3-fold and phospholipid output was about 2 times lower. The total amount of cholesterol in the liver decreased after interruption of the EHC, which was mainly due to a decrease in the amount of cholesteryl ester. Plasma disappearance of LDL was not affected by interruption of the EHC. Biliary secretion of LDL-derived radioactivity occurred 2-4 times faster in chronically interrupted rats as compared with the excretion immediately after interruption of the EHC. Radioactivity was mainly in the form of bile acids under both conditions. This study demonstrates the very rapid changes that occur in cholesterol metabolism and biliary lipid composition after interruption of the EHC. These changes must be taken into account in studies concerning hepatic metabolism of lipoprotein cholesterol and subsequent secretion into bile.

The catabolism of streptokinase (SK) and polyethylene glycol derivatives of SK (PEG-SK) were studied in mice. The clearance and catabolism of SK:plasmin (SK:Pm) and PEG-SK:Pm activator complexes were also investigated. Native 125I-SK cleared rapidly (t1/2 = 15 minutes) from the circulation, with the majority of the ligand accumulating in the liver and gastrointestinal (GI) tract and a substantial fraction also localizing in the kidneys. SK, which was removed from the plasma by the liver, was secreted into bile and then the GI tract. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that 125I-SK recovered from liver and bile was homogeneous and of the same molecular weight (mol wt approximately 50,200) as native SK. PEG-125I-SK cleared slowly (t1/2 greater than 200 minutes), with more than 80% of the preparation localizing in liver and GI tract. The PEG-125I-SK secreted into the bile was also intact. The bile containing 125I-SK was incubated with stoichiometric amounts of plasminogen and electrophoresed under nondenaturing conditions. This study demonstrated that the secreted SK was able to form SK:Pg complexes. SDS-PAGE also showed activation of 125I-Pg that was incubated with recovered bile containing the SK. 125I-SK:Pm catabolism was also studied. In these experiments, the mol wt approximately 42,000 fragment obtained when SK is cleaved by plasmin was found in the bile. This fragment of 125I-SK was not recovered as part of a complex with plasmin, consistent with our previous observations that catabolism of SK:Pm involves transfer of the plasmin to plasma proteinase inhibitors while SK is catabolized independently. By contrast, when PEG-125I-SK:Pm was injected into mice, only intact PEG-125I-SK was found in the bile, consistent with our previous observations that the PEG derivatization blocks its degradation by plasmin.

Intravital microscopy, a new in vivo technique, documented age-dependent changes in choledochoduodenal junction motility in male guinea pigs. In the guinea pig, the choledochoduodenal junction served as a pump that actively emptied its luminal contents into the duodenum. In the neonates (less than or equal to 1 wk old), this choledochoduodenal junction pump was not fully developed. Unlike the older guinea pigs, some neonates had an incompetent sphincter ductus choledochi (SDC) allowing retrograde flow of bile during ampullary contractions. While fasting, neonates had decreased frequency of SDC (1.2 +/- 0.4 contractions/min) and ampullary (0.1 +/- 0.1 contractions/min) contractions as compared to juveniles (4-6 wk old) (SDC = 6.4 +/- 1.0; ampulla = 1.2 +/- 0.2 contractions/min) and adults (greater than 1 yr old) (SDC = 6.7 +/- 1.6; 0.8 +/- 0.2 contractions/min). Following a meal (Ensure), unlike older guinea pigs, the neonate did not have a significant increased duration and decreased frequency of SDC contractions. Altered neonatal SDC motility correlated with an incompletely developed SDC including decreased muscle mass and mucosal thickness. By 4 wk of age, choledochoduodenal junction motility was similar to that of the adult. These developmental alterations in junctional motility and structure may affect the flow of bile into the duodenum contributing to physiologic cholestasis and decreased intraduodenal bile acids seen in neonates.

Studies utilizing balloon-occludable T or duodenal tubes in subjects with and without gallbladders were undertaken to identify the contribution of the gallbladder, the sphincter of Oddi, and bile salts to the delivery of bile to the duodenum. Patients with and without a functional sphincter of Oddi and with and without a gallbladder were compared. The presence of a functional sphincter of Oddi in duodenal tube patients reduced bile salt output by more than 67% from that observed in T-tube patients. When cholecystectomized and normal patients were compared using the duodenal tube, peak bile salt output was significantly increased in normal subjects, reflecting gallbladder contraction, but total bile salt output was not significantly increased suggesting that the gallbladder has a minor role in bile delivery. Exogenous infusion of cholecystokinin produced much more stable bile secretion than did endogenous release of cholecystokinin by intraduodenal infusion of essential amino acids. This rhythmic release of bile after endogenous cholecystokinin release was related to the concentration of bile salts in the intestinal lumen. Thus, delivery of bile to the duodenum is wave-like and is predominantly controlled by the sphincter of Oddi.

We studied 115 healthy adult volunteers, fasting overnight, to establish the normal scintigraphic variability. Five Tc-99m IDA agents with liver excretion half-times ranging from 18 to 108 min were used. The time of appearance of the common bile duct correlated directly with the liver's excretion half-time. The appearance of the gallbladder and the small intestine were independent of the excretion half-time and showed a reciprocal relationship suggesting a major role for the sphincter of Oddi. In 22% of the subjects, the sphincter tonus was tight enough to divert all of the hepatic bile into the gallbladder, allowing none into the intestine. All of such subjects showed normal dynamic response to intravenous cholecystokinin. The pattern of the bile drainage from the two lobes differed, resulting in asymmetry of the right and left hepatic ducts. It is concluded that the selection of a Tc-99m IDA agent should be based on the clinical problem at hand and that a clinician's understanding of the scintigraphic variability in normal subjects is critical before attempting diagnosis.

Evolutionary processes have not yet developed specific and safe ways to detoxify all chemical species new to our environment. Indeed, some are transformed and/or conjugated by the liver into more toxic species. Environmental factors can modulate hepatic enzyme systems. Particularly responsive are the mixed function oxidases, which initiate the transformation of many xenobiotics to excretable species via reactions which generate electrophilic intermediates such as free radicals, epoxides and aldehydes. Unless these reactive metabolites are rapidly removed by subsequent detoxification reactions or by endogenous defense systems, destructive cytotoxic reactions can be triggered or cell constitutents "attacked" thereby causing either acute injury and/or more latent molecular injury to long chain biopolymers resulting in chromatin damage, or tumors. In vitro systems using purified, specialized cell fractions may be of considerable value in defining metabolic processes, but the results must be relevant to in vivo conditions. Although human liver is peculiarly resistant to tumorigenesis, liver microsomes (isolated endoplasmic reticulum) are extensively used as biological activators for in vitro mutagenicity test systems. The in vivo defense system of liver cells must be exceptionally efficient! Reactive metabolites generated in liver may be stable enough to migrate and cause injury to other tissues or organ systems. It is important to characterize metabolic pathways of toxic xenobiotics, subsequent molecular sites or modes of injury, and factors which depress or augment cellular defense systems including the biliary system responsible for the excretion of many xenobiotics. Only then can techniques or treatments be developed to screen individuals for risk to specific groups of xenobiotics, to protect those exposed, and to treat those injured.

The membrane potentials of the smooth muscle cells of the sphincter of Oddi, gall bladder or common bile duct of the guinea pig was --56, --40 or --54 mV, respectively. The length constant of the tissue of the sphincter of Oddi was 1.7 mm and this value was larger than that measured in hypertonic solution. The smooth muscle cells of the gall bladder and common bile duct were always spontaneously active, while this was not the case with cells from the sphincter of Oddi, nevertheless, electrical stimulation consistently produced spike generation. Acetylcholine depolarized the membrane and increased the spike frequency in muscle cells of the sphincter of Oddi, gall bladder and common bile duct. However, noradrenaline produced alpha-adrenergic inhibitory action in the sphincter of Oddi and common bile duct, and alpha-adrenergic excitatory action in the gall bladder. Isoprenaline, a beta-adrenergic agent, induced an inhibitory action in the above three regions. Prostaglandin E1 (PGE1) suppressed the membrane activity in the sphincter of Oddi, and this response was similar to that observed in the circular muscle of the intestine. In the gall bladder, PGE1 accelerated the membrane activity. Gastrin and secretin did not affect the smooth muscle activity of the gall bladder but these agents slightly suppressed electrical and mechanical activities in the sphincter of Oddi. On the other hand, cholecystokinin markedly accelerated the electrical and mechanical activities in the gall bladder but these actions were weak in the sphincter of Oddi. Muscle properties of the smooth muscle cells of the biliary system are discussed in relation to the mechanism of bile expulsion through the sphincter of Oddi.

Despite wide variations in bile secretion and biliary tract capacitance, the pressure in the bile ducts is fairly constant. Recent studies have demonstrated that both inhibitory and excitatory nerves regulate the activity of the sphincter of Oddi. In the present study, it was consistently found that the resistance by the choledochoduodenal junction to a constant flow, within the physiologic range of hepatic bile output, is reduced when the hydrostatic pressure in the gallbladder and bile ducts is increased from 0 to 10, 0 to 15, and 0 to 20 cmH2O. This response was eliminated by tetrodotoxin or infiltration of the junction between the common bile duct and the cystic duct by mepivacaine, a local anesthetic. The results suggest a homeostatic mechanism during the interprandial periods, when the activity of the sphincter of Oddi is regulated by the distending pressure in the biliary tract. This reflex regulation is mediated by modulation of the activity of inhibitory nerves running along the common bile duct.