Functional expression of the apical Na+-dependent bile acid transporter in large but not small rat cholangiocytes (original) (raw)
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Journal of Clinical Investigation, 1997
Although bile acid transport by bile duct epithelial cells, or cholangiocytes, has been postulated, the details of this process remain unclear. Thus, we performed transport studies with [ 3 H]taurocholate in confluent polarized monolayers of normal rat cholangiocytes (NRC). We observed unidirectional (i.e., apical to basolateral) Na ϩ -dependent transcellular transport of [ 3 H]taurocholate. Kinetic studies in purified vesicles derived from the apical domain of NRC disclosed saturable Na ϩ -dependent uptake of [ 3 H]taurocholate, with apparent K m and V max values of 209 Ϯ 45 M and 1.23 Ϯ 0.14 nmol/mg/10 s, respectively. Reverse transcriptase PCR (RT-PCR) using degenerate primers for both the rat liver Na ϩdependent taurocholate-cotransporting polypeptide and rat ileal apical Na ϩ -dependent bile acid transporter, designated Ntcp and ASBT, respectively, revealed a 206-bp product in NRC whose sequence was identical to the ASBT. Northern blot analysis demonstrated that the size of the ASBT transcript was identical in NRC, freshly isolated cholangiocytes, and terminal ileum. In situ RT-PCR on normal rat liver showed that the message for ASBT was present only in cholangiocytes. Immunoblots using a well-characterized antibody for the ASBT demonstrated a 48-kD protein present only in apical membranes. Indirect immunohistochemistry revealed apical localization of ASBT in cholangiocytes in normal rat liver. The data provide direct evidence that conjugated bile acids are taken up at the apical domain of cholangiocytes via the ASBT, and are consistent with the notion that cholangiocyte physiology may be directly influenced by bile acids. ( J. Clin. Invest. 1997. 100:2714-2721.) Key words: biliary epithelia • taurocholate • transport • liver • plasma membrane vesicles Preliminary portions of this work were presented at the 47th meeting of the American Association for the Study of Liver Diseases, and have been published in abstract form (1996. Hepatology. 94:897 a ).
Bile acid interactions with cholangiocytes
World Journal of Gastroenterology, 2006
Cholangiocytes are exposed to high concentrations of bile acids at their apical membrane. A selective transporter for bile acids, the Apical Sodium Bile Acid Cotransporter (ASBT) (also referred to as Ibat; gene name Slc10a2) is localized on the cholangiocyte apical membrane. On the basolateral membrane, four transport systems have been identified (t-ASBT, multidrug resistance (MDR)3, an unidentified anion exchanger system and organic solute transporter (Ost) heteromeric transporter, Ostα-Ostβ. Together, these transporters unidirectionally move bile acids from ductal bile to the circulation. Bile acids absorbed by cholangiocytes recycle via the peribiliary plexus back to hepatocytes for re-secretion into bile. This recycling of bile acids between hepatocytes and cholangiocytes is referred to as the cholehepatic shunt pathway. Recent studies suggest that the cholehepatic shunt pathway may contribute in overall hepatobiliary transport of bile acids and to the adaptation to chronic cholestasis due to extrahepatic obstruction. ASBT is acutely regulated by an adenosine 3', 5'-monophosphate (cAMP)-dependent translocation to the apical membrane and by phosphorylation-dependent ubiquitination and proteasome degradation. ASBT is chronically regulated by changes in gene expression in response to biliary bile acid concentration and inflammatory cytokines. Another potential function of cholangiocyte ASBT is to allow cholangiocytes to sample biliary bile acids in order to activate intracellular signaling pathways. Bile acids trigger changes in intracellular calcium, protein kinase C (PKC), phosphoinositide 3-kinase (PI3K), mitogenactivated protein (MAP) kinase and extracellular signalregulated protein kinase (ERK) intracellular signals. Bile acids significantly alter cholangiocyte secretion, proliferation and survival. Different bile acids have differential effects on cholangiocyte intracellular signals, and in some instances trigger opposing effects on cholangiocyte secretion, proliferation and survival. Based upon these concepts and observations, the cholangiocyte has been proposed to be the principle target cell for bile acids in the liver.
Hepatology, 2001
Bile acids (BA) enter cholangiocytes by the Na ؉ -dependent apical BA transporter (ABAT). By this mechanism, taurocholate (TC) and taurolithocholate (TLC) increase cholangiocyte proliferation. No in vivo studies exist regarding the anatomical sites involved in BA-regulation of cholangiocyte growth. Specific cholangiocyte subpopulations participate in BA-regulated proliferation. Proliferation was assessed in liver sections by determining the number of proliferating cellular nuclear antigen (PCNA)-positive cholangiocytes and cytokeratin-19 (CK-19)-positive ducts. We isolated small and large cholangiocytes from rats fed for 1 week TC, TLC, or BA control diet and determined PCNA and ABAT expression and BA transport activity. We evaluated if TC and TLC induction of ABAT expression was dependent on activation of PKC alpha. DNA replication was active only in large normal cholangiocytes. TC and TLC feeding increased proliferation of large cholangiocytes, induced the de novo activation of proliferation of small cholangiocytes, overexpression of ABAT and BA transport activity in large cholangiocytes, and de novo expression of ABAT and BA transport activity in small cholangiocytes. BA-stimulated ABAT expression was dependent on PKC activation in cholangiocytes. TC and TLC stimulate proliferation of small and large cholangiocytes associated with PKC-dependent up-regulation of ABAT. (HEPATOLOGY 2001;34:868-876.)
Comprehensive Physiology, 2013
Cholangiocytes are epithelial cells that line the intra-and extrahepatic ducts of the biliary tree. The main physiologic function of cholangiocytes is modification of hepatocyte-derived bile, an intricate process regulated by hormones, peptides, nucleotides, neurotransmitters, and other molecules through intracellular signaling pathways and cascades. The mechanisms and regulation of bile modification are reviewed herein. Overview of Ductal Architecture and Developmental Origin The biliary tree is a complex, three-dimensional network of tubular conduits (collectively referred to as "ducts") of various sizes and properties. It can be divided grossly into two anatomically and functionally different compartments: intra-hepatic and extrahepatic (162, 221, 247, 256) (Fig. 1A and b). In humans, the nomenclature of the biliary conduits of the intrahepatic compartment is categorized by size and proximity (Fig. 1C). The smallest (< 15 m in luminal diameter) and most proximal of the conduits are the ductules, which emerge from the canals of Hering, specialized channels lined by both hepatocytes and cholangiocytes which represent the anatomic and physiologic transition from entirely hepatocyte-lined canaliculi to entirely cholangiocyte-lined ductules
Hepatology, 1997
cAMP concentrations increased both Na / -dependent fluidity and alkalinity absorbing and/or secreting fluid and Na / -independent Cl 0 /HCO 0 3 exchange activity. Stimand electrolytes, particularly HCO 0 3 and Cl 0 . Mechaulation of Cl 0 /HCO 0 3 exchange activity was not prenisms responsible for transepithelial H / /HCO 0 3 secretion vented by the Cl 0 channel inhibitor 5-nitro-2(2)-phein human cholangiocytes are largely unknown. Human nylpropyl-amino-benzoate(NPPB). In conclusion, hucholangiocytes isolated by enzymatic digestion and imman cholangiocytes possess two acid extruders (Na / /H / munomagnetic purification from normal liver tissue obexchanger and Na / -dependent Cl 0 /HCO 0 3 exchange) and tained from reduced grafts used for pediatric liver transan acid loader (Cl 0 /HCO 0 3 exchange), whereas no eviplantation were cultured in the presence of human dence was found for cAMP activated H / -ATPase. Bicarhepatocyte growth factor. Maintenance of cholangiocyte bonate influx is thus mainly mediated by Na-dependent phenotypic features was assessed using markers such Cl 0 /HCO 0 3 exchange, whereas Na / :HCO 0 3 cotransport is as cytokeratin 19, g-glutamyltranspeptidase, vimentin, not active in the physiological range of pHi. Stimulation factor VIII-related antigen, desmin, epithelial memof Na / -independent Cl 0 /HCO 0 3 exchanger by cAMP does brane antigen (EMA), and human epithelial antigen not require activation of Cl 0 conductances. These mech-(HEA) 125. Intracellular pH (pHi) transients were meaanisms may underlay hormone-regulated biliary HCO 0 3 sured microfluorimetrically 27-Bis(2-carboxyethyl)-5,6, secretion in the human biliary tree. (HEPATOLOGY carboxyfluorescein-acetossimethylester (BCECF). In 1997;25:976-985.) the absence of HCO 0 3 , pHi recovery from an intracellular acid load (ammonia pre-pulse technique) was Na / -dependent and amiloride-inhibitable. No Na / -independent Interest in cholangiocyte physiology and pathophysiology recovery was recorded even after stimulation with has been stimulated by the observation that the biliary epiagents raising intracellular cyclic adenosine monophosthelium is the primary target in several chronic cholestatic phate (cAMP) concentrations. In the presence of liver disorders, including primary biliary cirrhosis, primary HCO 0 3 , recovery from an intracellular acid load required sclerosing cholangitis, and liver allograft rejection. 1-5 Inter-Na / , but was only partly inhibited by amiloride. In these estingly, in cystic fibrosis, a disease of ion transporting epiconditions H / extrusion was inhibited by 4,4,-diisothiothelia, hepatobiliary complications occur in approximately cyan atostilben-2,2,-disulfonic acid (DIDS) and by intra-30% of cases 6 and ultrastructural bile duct cell damage can cellular Cl 0 depletion. Acute removal of extracellular Cl 0 be shown well before hepatocellular damage. 7 In this condiinduced a pHi alkalinization that was inhibited by DIDS. tion, low rates of ductular HCO 0 3 and Cl 0 secretion may lead pHi recovery from an intracellular alkaline load (isohyto increased viscosity of bile and ''ductular'' cholestasis. 7,8 dric CO 2 changes) was Cl 0 -dependent and DIDS-inhibit-Hepatocellular bile is, in fact, extensively modified while flowing through the biliary tree. The epithelial cells lining the intrahepatic bile ducts (cholangiocytes) modulate bile fluidity Abbreviations: cAMP, cyclic adenosine monophosphate; NPPB, 5-nitro-2(2)-phenylproand alkalinity absorbing or secreting fluid and electrolytes, pyl-amino-benzoate; Hepes, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; DIDS, 4,4,-diisothiocyanatostilben-2,2,-disulfonic acid; DBcAMP, N 6 ,2-O-dibutyryladenosine particularly HCO 0 3 and Cl 0 , under the influence of digestive 3:5-cyclic monophosphate; IBMX, 3-isobutyl-1-methyl-xanthine; BCECF-AM, 2:7-Bis(2-hormones such as secretin and somatostatin. 1,3-5,9,10 Studies carboxyethyl)-5,6, carboxyfluorescein-acetossimethylester; HEA, human epithelial antigen; in short-term cultured intrahepatic rat cholangiocytes have g-GT, g-glutamyltranspeptidase; pHi, intracellular measurements; Bi, intrinsic buffering characterized a number of membrane carriers and ion chanpower; KRB, bicarbonate buffered ringer. From the HCO 0 3 exchange, whereas HCO 0 3 entry through the basolat-Sponsored by grant 94.02908.CT04 and ''Progetto Nazionale Invecchiamento'' from Consiglio Nazionale delle Ricerche (Italy) and by ''Centro per lo studio dell'invecchiamento'', eral membrane was mediated by the electrogenic Na / : University of Padova. The financial support of Telethon-Italy (Grant MD.E-430) is gratefully HCO 0 3 cotransport. 11 On the other hand, studies in pig cholacknowledged. Dr. À kos Zsembery was a recipient of an International Fellowship for the angiocytes suggested a role for cyclic adenosine mono-University of Padova. phosphate (cAMP)-activated H / -ATPase in the generation of Part of this work has been presented at the 95th Annual Meeting of the American Gastroenterological Association (New Orleans, LA, 1994) and published in Abstract form intracellular HCO 0 3 . in Gastroenterology 106:A990, 1994. Because of the presence of a number of species-specific vari-Address reprint requests to: Mario Strazzabosco, M.D., Institute of Internal Medicine, ations in bile physiology 13,14 the relevance of the above trans-
World Journal of Gastroenterology, 2008
The formation of bile depends on the structural and functional integrity of the bile-secretory apparatus and its impairment, in different situations, results in the syndrome of cholestasis. The structural bases that permit bile secretion as well as various aspects related with its composition and flow rate in physiological conditions will first be reviewed. Canalicular bile is produced by polarized hepatocytes that hold transporters in their basolateral (sinusoidal) and apical (canalicular) plasma membrane. This review summarizes recent data on the molecular determinants of this primary bile formation. The major function of the biliary tree is modification of canalicular bile by secretory and reabsorptive processes in bileduct epithelial cells (cholangiocytes) as bile passes through bile ducts. The mechanisms of fluid and solute transport in cholangiocytes will also be discussed. In contrast to hepatocytes where secretion is constant and poorly controlled, cholangiocyte secretion is regulated by hormones and nerves. A short section dedicated to these regulatory mechanisms of bile secretion has been included. The aim of this revision was to set the bases for other reviews in this series that will be devoted to specific issues related with biliary physiology and pathology.
Gastroenterology, 2002
Background & Aims: We tested the hypothesis that during bile duct obstruction, increased biliary bile acids trigger cholangiocyte proliferation and secretion by a phosphatidylinositol 3-kinase (PI3-K)-dependent pathway. Methods: In bile duct-incannulated (BDI) rats, bile duct obstruction present for 7 days was relieved for 24 hours by external bile drainage. During the 24-hour drainage period, animals received either Krebs Ringer Henseleit (the bile-depleted group), or sodium taurocholate (the bile-depleted, taurocholate-infused group). We evaluated cholangiocyte proliferation and secretinstimulated ductal secretion. Apical bile acid transporter (ABAT) expression and bile acid transport activity was determined. In pure preparations of cholangiocytes, we examined the effect of taurocholate (in the absence or presence of wortmannin or PI 3,4-bisphosphate the lipid product of PI3-K) on cholangiocyte proliferation and secretin-stimulated cyclic adenosine 3,5-monophosphate (cAMP) levels. Results: Bile depletion reduced cholangiocyte proliferation and secretin-stimulated ductal secretion and ABAT expression and bile acid transport activity compared with 1-week BDI control rats. In bile-depleted, taurocholate-infused rats, cholangiocyte proliferation and secretion and ABAT expression and bile acid transport activity were maintained at levels similar to those seen in BDI control rats. In vitro, taurocholate stimulation of DNA replication and secretin-stimulated cAMP levels was blocked by wortmannin. The inhibitory effect of wortmannin on taurocholate stimulation of cholangiocyte proliferation and secretion was prevented by PI 3,4-bisphosphate. Conclusions: Bile acid uptake by ABAT and the PI3-K pathway are important for bile acids to signal cholangiocyte proliferation. In bile duct obstruction, increased biliary bile acid concentration and ABAT expression initiate increased cholangiocyte proliferation and secretion.
Gastroenterology, 1996
The molecular regulation of headenosine triphosphate-dependent and potential-driven patic bile acid transporters during cholestasis is largely bile acid transporters, 4-9 although a recent study suggests unknown. Cloning of complementary DNAs for the sinuthat the latter system may be confined to the endoplasmic soidal sodium-dependent taurocholate cotransporting reticulum. 10 polypeptide (ntcp), the cytosolic bile acid-binding pro-Recently, complementary DNAs (cDNAs) encoding tein 3a-hydroxysteroid dehydrogenase (3a-HSD), and a for a sodium-dependent taurocholate cotransporting putative canalicular bile acid transporter Ca 2/ , Mg 2/polypeptide have been cloned from rat (ntcp) and human ecto-adenosine triphosphatase, now facilitates such liver. 11,12 Expression of the rat liver ntcp in oocytes, transtudies. Methods: Protein mass, steady-state messensiently transfected COS cells, or stably transfected CHO ger RNA (mRNA) levels, and gene transcription were cells resulted in a strictly sodium-dependent saturable assessed in rat livers after common bile duct ligation uptake of taurocholate with a similar Michaelis constant (CBDL) from 1-7 days, and taurocholate uptake was (K m ) of 30-40 mmol/L as previously determined in isodetermined in isolated hepatocytes. Results: After CBDL, Na / -dependent taurocholate uptake (V max ) delated hepatocytes and basolateral plasma membrane prepclined by 70%. The levels of ntcp protein were reduced Abbreviations used in this paper: CBDL, common bile duct ligation;
Calcium signaling and the secretory activity of bile duct epithelia
Cell Calcium, 2014
Cytosolic calcium (Ca i 2+) is a second messenger that is important for the regulation of secretion in many types of tissues. Bile duct epithelial cells, or cholangiocytes, are polarized epithelia that line the biliary tree in liver and are responsible for secretion of bicarbonate and other solutes into bile. Ca i 2+ signaling plays an important role in the regulation of secretion by cholangiocytes, and this review discusses the machinery involved in the formation of Ca 2+ signals in cholangiocytes, along with the evidence that these signals regulate ductular secretion. Finally, this review discusses the evidence that impairments in cholangiocyte Ca 2+ signaling play a primary role in the pathogenesis of cholestatic disorders, in which hepatic bile secretion is impaired.