Biliary Bicarbonate Secretion Research Articles

Active enterohepatic cycling is not required for the choleretic actions of 24-norUrsodeoxycholic acid in mice.

The pronounced choleretic properties of 24-norUrsodeoxycholic acid (norUDCA) to induce bicarbonate-rich bile secretion have been attributed to its ability to undergo cholehepatic shunting. The goal of this study was to identify the mechanisms underlying the choleretic actions of norUDCA and the role of the bile acid transporters. Here, we show that the apical sodium-dependent bile acid transporter (ASBT), organic solute transporter-α (OSTα), and organic anion transporting polypeptide 1a/1b (OATP1a/1b) transporters are dispensable for the norUDCA stimulation of bile flow and biliary bicarbonate secretion. Chloride channels in biliary epithelial cells provide the driving force for biliary secretion. In mouse large cholangiocytes, norUDCA potently stimulated chloride currents that were blocked by siRNA silencing and pharmacological inhibition of calcium-activated chloride channel transmembrane member 16A (TMEM16A) but unaffected by ASBT inhibition. In agreement, blocking intestinal bile acid reabsorption by coadministration of an ASBT inhibitor or bile acid sequestrant did not impact norUDCA stimulation of bile flow in WT mice. The results indicate that these major bile acid transporters are not directly involved in the absorption, cholehepatic shunting, or choleretic actions of norUDCA. Additionally, the findings support further investigation of the therapeutic synergy between norUDCA and ASBT inhibitors or bile acid sequestrants for cholestatic liver disease.

Secretin alleviates biliary and liver injury during late-stage primary biliary cholangitis via restoration of secretory processes.

Primary biliary cholangitis (PBC) is characterised by ductopenia, ductular reaction, impairment of anion exchanger 2 (AE2) and the 'bicarbonate umbrella'. Ductulo-canalicular junction (DCJ) derangement is hypothesised to promote PBC progression. The secretin (Sct)/secretin receptor (SR) axis regulates cystic fibrosis transmembrane receptor (CFTR) and AE2, thus promoting choleresis. We evaluated the role of Sct/SR signalling on biliary secretory processes and subsequent injury in a late-stage PBC mouse model and human samples. At 32 weeks of age, female and male wild-type and dominant-negative transforming growth factor beta receptor II (late-stage PBC model) mice were treated with Sct for 1 or 8 weeks. Bulk RNA-sequencing was performed in isolated cholangiocytes from mouse models. Biliary Sct/SR/CFTR/AE2 expression and bile bicarbonate levels were reduced in late-stage PBC mouse models and human samples. Sct treatment decreased bile duct loss, ductular reaction, inflammation, and fibrosis in late-stage PBC models. Sct reduced hepatic bile acid levels, modified bile acid composition, and restored the DCJ and 'bicarbonate umbrella'. RNA-sequencing identified that Sct promoted mature epithelial marker expression, specifically anterior grade protein 2 (Agr2). Late-stage PBC models and human samples exhibited reduced biliary mucin 1 levels, which were enhanced by Sct treatment. Loss of Sct/SR signalling in late-stage PBC results in a faulty 'bicarbonate umbrella' and reduced Agr2-mediated mucin production. Sct restores cholangiocyte secretory processes and DCJ formation through enhanced mature cholangiocyte phenotypes and bile duct growth. Sct treatment may be beneficial for individuals with late-stage PBC. Secretin (Sct) regulates biliary proliferation and bicarbonate secretion in cholangiocytes via its receptor, SR, and in mouse models and human samples of late-stage primary biliary cholangitis (PBC), the Sct/SR axis is blunted along with loss of the protective 'bicarbonate umbrella'. We found that both short- and long-term Sct treatment ameliorated ductular reaction, immune cell influx, and liver fibrosis in late-stage PBC mouse models. Importantly, Sct treatment promoted bicarbonate and mucin secretion and hepatic bile acid efflux, thus reducing cholestatic and toxic bile acid-associated injury in late-stage PBC mouse models. Our work perpetuates the hypothesis that PBC pathogenesis hinges on secretory defects, and restoration of secretory processes that promote the 'bicarbonate umbrella' may be important for amelioration of PBC-associated damage.

Role of the IgG4-related cholangitis autoantigen annexin A11 in cholangiocyte protection

Annexin A11 was identified as autoantigen in IgG4-related cholangitis (IRC), a B-cell driven disease. Annexin A11 modulates calcium-dependent exocytosis, a crucial mechanism for insertion of proteins into their target membranes. Human cholangiocytes form an apical 'biliary bicarbonate umbrella' regarded as defense against harmful hydrophobic bile acid influx. The bicarbonate secretory machinery comprises the chloride/bicarbonate exchanger AE2 and the chloride channel ANO1. We aimed to investigate the expression and function of annexin A11 in human cholangiocytes and a potential role of IgG1/IgG4-mediated autoreactivity against annexin A11 in the pathogenesis of IRC. Expression of annexin A11 in human liver was studied by immunohistochemistry and immunofluorescence. In human control and ANXA11 knockdown H69 cholangiocytes, intracellular pH, AE2 and ANO1 surface expression, and bile acid influx were examined using ratio microspectrofluorometry, cell surface biotinylation, and 22,23-3H-glycochenodeoxycholic acid permeation, respectively. The localization of annexin A11-mEmerald and ANO1-mCherry was investigated by live-cell microscopy in H69 cholangiocytes after incubation with IRC patient serum containing anti-annexin A11 IgG1/IgG4-autoantibodies or disease control serum. Annexin A11 was strongly expressed in human cholangiocytes, but not hepatocytes. Knockdown of ANXA11 led to reduced plasma membrane expression of ANO1, but not AE2, alkalization of intracellular pH and uncontrolled bile acid influx. High intracellular calcium conditions led to annexin A11 membrane shift and colocalization with ANO1. Incubation with IRC patient serum inhibited annexin A11 membrane shift and reduced ANO1 surface expression. Cholangiocellular annexin A11 mediates apical membrane abundance of the chloride channel ANO1, thereby supporting biliary bicarbonate secretion. Insertion is inhibited by IRC patient serum containing anti-annexin A11 IgG1/IgG4-autoantibodies. Anti-annexin A11 autoantibodies may contribute to the pathogenesis of IRC by weakening the 'biliary bicarbonate umbrella'. We previously identified annexin A11 as a specific autoantigen in immunoglobulin G4-related cholangitis (IRC), a B-cell driven disease affecting the bile ducts. Human cholangiocytes are protected against harmful hydrophobic bile acid influx by a defense mechanism referred to as the 'biliary bicarbonate umbrella'. We found that annexin A11 is required for the formation of a robust bicarbonate umbrella. Binding of patient-derived annexin A11 autoantibodies inhibits annexin A11 function, possibly contributing to bile duct damage by weakening the biliary bicarbonate umbrella in patients with IRC.

New treatments/targets for primary biliary cholangitis.

SummaryPrimary biliary cholangitis (PBC) is an autoimmune, cholestatic, chronic liver disease that ultimately progresses towards cirrhosis and liver failure if untreated. While ursodeoxycholic acid has been established as standard of care for PBC in the last few decades, significant advances in second-line treatment options have recently been made and new therapeutic developments are currently under evaluation. The purpose of this article is to provide the clinician with an overview of the current treatment options and future opportunities for patients with PBC.

Secretin/secretin receptor signaling mediates biliary damage and liver fibrosis in early-stage primary biliary cholangitis.

Primary biliary cholangitis (PBC) primarily targets cholangiocytes and is characterized by liver fibrosis and biliary proliferation. Activation of the secretin (Sct)/secretin receptor (SR) axis, expressed only by cholangiocytes, increases biliary proliferation, liver fibrosis, and bicarbonate secretion. We evaluated the effectiveness of SR antagonist treatment for early-stage PBC. Male and female dominant-negative TGF-β receptor II (dnTGF-βRII) (model of PBC) and wild-type mice at 12 wk of age were treated with saline or the SR antagonist, Sec 5-27, for 1 wk. dnTGF-βRII mice expressed features of early-stage PBC along with enhanced Sct/SR axis activation and Sct secretion. dnTGF-βRII mice had increased biliary proliferation or senescence, inflammation, and liver fibrosis. In dnTGF-βRII mice, there was increased microRNA-125b/TGF-β1/TGF-β receptor 1/VEGF-A signaling. Human early-stage PBC patients had an increase in hepatobiliary Sct and SR expression and serum Sct levels. Increased biliary Sct/SR signaling promotes biliary and hepatic damage during early-stage PBC.-Kennedy, L., Francis, H., Invernizzi, P., Venter, J., Wu, N., Carbone, M., Gershwin, M. E., Bernuzzi, F., Franchitto, A., Alvaro, D., Marzioni, M., Onori, P., Gaudio, E., Sybenga, A., Fabris, L., Meng, F., Glaser, S., Alpini, G. Secretin/secretin receptor signaling mediates biliary damage and liver fibrosis in early-stage primary biliary cholangitis.

Promoter hypermethylation of the AE2/SLC4A2 gene in PBC.

Background & AimsPatients with primary biliary cholangitis (PBC) exhibit reduced AE2/SLC4A2 gene expression in the liver and peripheral blood mononuclear cells (PBMCs). AE2 encodes a Cl–/HCO3– exchanger involved in biliary bicarbonate secretion and intracellular pH regulation. Reduced AE2 expression in PBC may be pathogenic, as Ae2-knockout mice reproduce characteristic PBC features. Herein, we aimed to identify CpG-methylation abnormalities in AE2 promoter regions that might contribute to the reduced gene transcription in PBC livers and PBMCs.MethodsCpG-cytosine methylation rates were interrogated at 1-base pair resolution in upstream and alternate AE2 promoter regions through pyrosequencing of bisulphite-modified genomic DNA from liver specimens and PBMCs. AE2a and alternative AE2b1 and AE2b2 mRNA levels were measured by real-time PCR. Human lymphoblastoid-T2 cells were treated with 5-aza-2´-deoxycytidine for demethylation assays.ResultsAE2 promoters were found to be hypermethylated in PBC livers compared to normal and diseased liver specimens. Receiver operating characteristic (ROC) curve analysis showed that minimal CpG-hypermethylation clusters of 3 AE2a-CpG sites and 4 alternate-AE2b2-CpG sites specifically differentiated PBC from normal and diseased controls, with mean methylation rates inversely correlating with respective transcript levels. Additionally, in PBMCs a minimal cluster of 3 hypermethylated AE2a-CpG sites distinguished PBC from controls, and mean methylation rates correlated negatively with AE2a mRNA levels in these immune cells. Alternate AE2b2/AE2b1 promoters in PBMCs were constitutively hypermethylated, in line with absent alternative mRNA expression in diseased and healthy PBMCs. Demethylation assays treating lymphoblastoid-T2 cells with 5-aza-2´-deoxycytidine triggered AE2b2/AE2b1 expression and upregulated AE2a-promoter expression.ConclusionsDisease-specific hypermethylation of AE2 promoter regions and subsequent downregulation of AE2-gene expression in the liver and PBMCs of patients with PBC might be critically involved in the pathogenesis of this complex disease.Lay summaryPrimary biliary cholangitis (PBC) is a chronic immune-associated cholestatic liver disease with unclear complex/multifactorial etiopathogenesis affecting mostly middle-aged women. Patients with PBC exhibit reduced expression of the AE2/SLC4A2 gene. Herein, we found that AE2 promoter regions are hypermethylated in the liver and peripheral blood mononuclear cells of patients with PBC. This increased methylation is associated with downregulated AE2-gene expression, which might contribute to the pathogenesis of PBC. Therefore, novel epigenetic targets may improve treatment in patients with PBC who respond poorly to current pharmacological therapies.

Ursodeoxycholic Acid Ameliorates Intrahepatic Cholestasis Independent of Biliary Bicarbonate Secretion in Vil2kd/kd Mice.

Ursodeoxycholic acid (UDCA) is a hydrophilic bile acid that possesses many pharmacological effects, including increasing bile flow, changing the hydrophobicity of the bile acid pool, and modulation of the immune response. UDCA has been approved for treating cholestatic liver disease, such as primary biliary cholangitis. However, several unanticipated severe side effects of UDCA are observed in cholestatic patients, and its pharmacological benefits remain controversial. We reported that ezrin-knockdown (Vil2kd/kd) mice exhibited severe hepatic injury because of a functional disorder in bile duct fluidity and alkalinity regulation, resembling human intrahepatic cholestatic disease. Here we used Vil2kd/kd mice as a cholestatic model to investigate the pharmacological effects of UDCA. We investigated the effects of oral and parenteral administration of UDCA on Vil2kd/kd mice. In Vil2kd/kd mice, fed a 0.5% (w/w) UDCA diet for 3 weeks, hepatic injury was exacerbated, although oral administration of a lower dose of UDCA slightly improved hepatic function in Vil2kd/kd mice. On the other hand, intraperitoneal administration of UDCA (50 mg/kg/d) ameliorated hepatic function and markedly reduced periductal fibrosis and cholangiocyte proliferation in Vil2kd/kd mice although biliary pH and HCO3- concentration were not improved. The expression levels of inflammatory and profibrotic genes were also significantly decreased in these mice. Furthermore, UDCA prevented cholangiocytes from hydrophobic bile acid-induced cytotoxicity independent of extracellular pH in in vitro experiments. These results suggest that an appropriate dosage of UDCA can ameliorate the intrahepatic cholestasis in Vil2kd/kd mice without changing the biliary bicarbonate secretion.

Oxygenated Hypothermic Machine Perfusion After Static Cold Storage Improves Hepatobiliary Function of Extended Criteria Donor Livers

The mechanism through which oxygenated hypothermic machine perfusion (HMP) improves viability of human extended criteria donor (ECD) livers is not well known. Aim of this study was to examine the benefits of oxygenated HMP after static cold storage (SCS). Eighteen ECD livers that were declined for transplantation underwent ex situ viability testing using normothermic (37 °C) machine perfusion (NMP) after traditional SCS (0 °C-4 °C) for 7 to 9 hours. In the intervention group (n = 6), livers underwent 2 hours of oxygenated HMP (at 12 °C) after SCS and before NMP. Twelve control livers underwent NMP without oxygenated HMP after SCS. During HMP, hepatic ATP content increased greater than 15-fold, and levels remained significantly higher during the first 4 hours of NMP in the HMP group, compared with controls. Cumulative bile production and biliary secretion of bilirubin and bicarbonate were significantly higher after HMP, compared with controls. In addition, the levels of lactate and glucose were less elevated after HMP compared with SCS preservation alone. In contrast, there were no differences in levels of hepatobiliary injury markers AST, ALT, LDH, and gamma-GT after 6 hours of NMP. Hepatic histology at baseline and after 6 hours of NMP revealed no differences in the amount of ischemic necrosis between both groups. Two hours of oxygenated HMP after traditional SCS restores hepatic ATP levels and improves hepatobiliary function but does not reduce (preexisting) hepatobiliary injury in ECD livers.

CD8+ T cells undergo activation and programmed death-1 repression in the liver of aged Ae2a,b-/- mice favoring autoimmune cholangitis.

Primary biliary cirrhosis (PBC) is a chronic cholestatic disease of unknown etiopathogenesis showing progressive autoimmune-mediated cholangitis. In PBC patients, the liver and lymphocytes exhibit diminished expression of AE2/SLC4A2, a Cl-/HCO3- anion exchanger involved in biliary bicarbonate secretion and intracellular pH regulation. Decreased AE2 expression may be pathogenic as Ae2a,b(-/-) mice reproduce hepatobiliary and immunological features resembling PBC. To understand the role of AE2 deficiency for autoimmunity predisposition we focused on the phenotypic changes of T cells that occur over the life-span of Ae2a,b(-/-) mice. At early ages (1-9 months), knockout mice had reduced numbers of intrahepatic T cells, which exhibited increased activation, programmed-cell-death (PD)-1 expression, and apoptosis. Moreover, young knockouts had upregulated PD-1 ligand (PD-L1) on bile-duct cells, and administration of neutralizing anti-PD-L1 antibodies prevented their intrahepatic T-cell deletion. Older (≥ 10 months) knockouts, however, showed intrahepatic accumulation of cytotoxic CD8(+) T cells with downregulated PD-1 and diminished apoptosis. In-vitro DNA demethylation with 5-aza-2'-deoxycytidine partially reverted PD-1 downregulation of intrahepatic CD8(+) T cells from aged knockouts. Early in life, AE2 deficiency results in intrahepatic T-cell activation and PD-1/PD-L1 mediated deletion. With aging, intrahepatic CD8+ T cells epigenetically suppress PD-1, and their consequential expansion and further activation favor autoimmune cholangitis.

Stimulation of nuclear receptor peroxisome proliferator-activated receptor-γ limits NF-κB-dependent inflammation in mouse cystic fibrosis biliary epithelium.

Cystic fibrosis-associated liver disease is a chronic cholangiopathy that negatively affects the quality of life of cystic fibrosis patients. In addition to reducing biliary chloride and bicarbonate secretion, up-regulation of toll-like receptor 4/nuclear factor kappa light-chain-enhancer of activated B cells (NF-κB)-dependent immune mechanisms plays a major role in the pathogenesis of cystic fibrosis-associated liver disease and may represent a therapeutic target. Nuclear receptors are transcription factors that regulate several intracellular functions. Some nuclear receptors, including peroxisome proliferator-activated receptor-γ (PPAR-γ), may counterregulate inflammation in a tissue-specific manner. In this study, we explored the anti-inflammatory effect of PPAR-γ stimulation in vivo in cystic fibrosis transmembrane conductance regulator (Cftr) knockout mice exposed to dextran sodium sulfate and in vitro in primary cholangiocytes isolated from wild-type and from Cftr-knockout mice exposed to lipopolysaccharide. We found that in CFTR-defective biliary epithelium expression of PPAR-γ is increased but that this does not result in increased receptor activity because the availability of bioactive ligands is reduced. Exogenous administration of synthetic agonists of PPAR-γ (pioglitazone and rosiglitazone) up-regulates PPAR-γ-dependent genes, while inhibiting the activation of NF-κB and the secretion of proinflammatory cytokines (lipopolysaccharide-induced CXC chemokine, monocyte chemotactic protein-1, macrophage inflammatory protein-2, granulocyte colony-stimulating factor, keratinocyte chemoattractant) in response to lipopolysaccharide. PPAR-γ agonists modulate NF-κB-dependent inflammation by up-regulating nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor alpha, a negative regulator of NF-κB. Stimulation of PPAR-γ in vivo (rosiglitazone) significantly attenuates biliary damage and inflammation in Cftr-knockout mice exposed to a dextran sodium sulfate-induced portal endotoxemia. These studies unravel a novel function of PPAR-γ in controlling biliary epithelium inflammation and suggest that impaired activation of PPAR-γ contributes to the chronic inflammatory state of CFTR-defective cholangiocytes.

Up-regulation of microRNA 506 leads to decreased Cl−/HCO3 − anion exchanger 2 expression in biliary epithelium of patients with primary biliary cirrhosis

Cl(-) /HCO3- anion exchanger 2 (AE2) participates in intracellular pH homeostasis and secretin-stimulated biliary bicarbonate secretion. AE2/SLC4A2 gene expression is reduced in liver and blood mononuclear cells from patients with primary biliary cirrhosis (PBC). Our previous findings of hepatic and immunological features mimicking PBC in Ae2-deficient mice strongly suggest that decreased AE2 expression might be involved in the pathogenesis of PBC. Here, we tested the potential role of microRNA 506 (miR-506) - predicted as candidate to target AE2 mRNA - for the decreased expression of AE2 in PBC. Real-time quantitative polymerase chain reaction showed that miR-506 expression is increased in PBC livers versus normal liver specimens. In situ hybridization in liver sections confirmed that miR-506 is up-regulated in the intrahepatic bile ducts of PBC livers, compared with normal and primary sclerosing cholangitis livers. Precursor-mediated overexpression of miR-506 in SV40-immortalized normal human cholangiocytes (H69 cells) led to decreased AE2 protein expression and activity, as indicated by immunoblotting and microfluorimetry, respectively. Moreover, miR-506 overexpression in three-dimensional (3D)-cultured H69 cholangiocytes blocked the secretin-stimulated expansion of cystic structures developed under the 3D conditions. Luciferase assays and site-directed mutagenesis demonstrated that miR-506 specifically may bind the 3'untranslated region (3'UTR) of AE2 messenger RNA (mRNA) and prevent protein translation. Finally, cultured PBC cholangiocytes showed decreased AE2 activity, together with miR-506 overexpression, compared to normal human cholangiocytes, and transfection of PBC cholangiocytes with anti-miR-506 was able to improve their AE2 activity. miR-506 is up-regulated in cholangiocytes from PBC patients, binds the 3'UTR region of AE2 mRNA, and prevents protein translation, leading to diminished AE2 activity and impaired biliary secretory functions. In view of the putative pathogenic role of decreased AE2 in PBC, miR-506 may constitute a potential therapeutic target for this disease.

담관 결찰 쥐의 간세포와 담관세포의 anion exchanger와 CFTR 발현

Cystic fibrosis transmembrane conductance regulator (CFTR) gene and sodium-independent Cl?/HCO₃? anion exchanger (AE) genes are expressed in a wide variety of mammalian tissues including cholangiocytes. They play an important role in the regulation of intracellular pH (pHi) as well as in transepithelial acid/base transport necessary for biliary bicarbonate secretion. The aim of this study was to examine the expression level of CFTR gene and AE genes (AE1, AE2 and AE3) in the cholangiocytes and the hepatocytes, and also measure AE2 gene expression level after bile duct ligation (BDL). As we previously described, isolated hepatocytes and cholangiocytes from the liver of normal and BDL rats were prepared and gene expression levels were measured by using RT-PCR. We found that AE1, AE2, and AE3 genes were expressed in both hepatocytes and cholangiocytes, but CFTR was only in cholangiocytes. AE2 gene expression level was higher in the BDL hepatocytes than normal hepatocytes, which was significantly different between two groups. AE2 gene expression level was lower in the BDL cholangiocytes than normal cholangiocytes. However, AE2 gene expression level in both hepatocytes and cholangiocytes were not changed with a longer duration of BDL. These results suggest that CFTR and AE2 may play an important role in the pathogenetic mechanism of biliary cholestatic liver disease.

Importance of Each Residue within Secretin for Receptor Binding and Biological Activity

Secretin is a linear 27-residue peptide hormone that stimulates pancreatic and biliary ductular bicarbonate and water secretion by acting at its family B G protein-coupled receptor. While, like other family members, the carboxyl-terminal region of secretin is most important for high affinity binding and its amino-terminal region is most important for receptor selectivity and receptor activation, determinants for these activities are distributed throughout the entire length of this peptide. In this work, we have systematically investigated changing each residue within secretin to alanine and evaluating the impact on receptor binding and biological activity. The residues most critical for receptor binding were His1, Asp3, Gly4, Phe6, Thr7, Ser8, Leu10, Asp15, Leu19, and Leu23. The residues most critical for biological activity included His1, Gly4, Thr7, Ser8, Glu9, Leu10, Leu19, Leu22, and Leu23, with Asp3, Phe6, Ser11, Leu13, Asp15, Leu26, and Val27 also contributing. While the importance of residues in positions analogous to His1, Asp3, Phe6, Thr7, and Leu23 is conserved for several closely related members of this family, Leu19 is uniquely important for secretin. We, therefore, have further studied this residue by molecular modeling and molecular dynamics simulations. Indeed, the molecular dynamics simulations showed that mutation of Leu19 to alanine was destabilizing, with this effect greater than that observed for the analogous position in the other close family members. This could reflect reduced contact with the receptor or an increase in the solvent-accessible surface area of the hydrophobic residues in the carboxyl terminus of secretin as bound to its receptor.

Biliary secretion of S‐nitrosoglutathione is involved in the hypercholeresis induced by ursodeoxycholic acid in the normal rat†

Ursodeoxycholic acid (UDCA) induces bicarbonate-rich hypercholeresis by incompletely defined mechanisms that involve the stimulation of adenosine triphosphate (ATP) release from cholangiocytes. As nitric oxide (NO) at a low concentration can stimulate a variety of secretory processes, we investigated whether this mediator could be implicated in the choleretic response to UDCA. Our in vivo experiments with the in situ perfused rat liver model in anesthetized rats, showed that UDCA infusion increased the biliary secretion of NO derivatives, hepatic inducible NO synthase expression, and NO synthase activity in liver tissue. UDCA also stimulated NO release by isolated rat hepatocytes. In contrast to UDCA, cholic acid was a poor inducer of NO secretion, and tauroursodeoxycholic acid showed no effect on NO secretion. Upon UDCA administration, NO was found in bile as low-molecular-weight nitrosothiols, of which S-nitrosoglutathione (GSNO) was the predominant species. UDCA-stimulated biliary NO secretion was abolished by the inhibition of inducible NO synthase with N(omega)-nitro-L-arginine methyl ester in isolated perfused livers and also in rats whose livers were depleted of glutathione with buthionine sulfoximine. Moreover, the biliary secretion of NO species was significantly diminished in UDCA-infused transport mutant [ATP-binding cassette C2 (ABCC2)/multidrug resistance-associated protein 2 (Mrp2)-deficient] rats, and this finding was consistent with the involvement of the glutathione carrier ABCC2/Mrp2 in the canalicular transport of GSNO. It was particularly noteworthy that in cultured normal rat cholangiocytes, GSNO activated protein kinase B, protected against apoptosis, and enhanced UDCA-induced ATP release to the medium; this effect was blocked by phosphoinositide 3-kinase inhibition. Finally, retrograde GSNO infusion into the common bile duct increased bile flow and biliary bicarbonate secretion. UDCA induces biliary secretion of GSNO, which contributes to stimulating ductal secretion.

HNF1α upregulates the human AE2 anion exchanger gene ( SLC4A2) from an alternate promoter

The human AE2 gene ( SLC4A2) is transcribed in a widespread fashion from the upstream promoter, the resultant full-length transcript AE2a being encountered in most tissues. Moreover, alternate promoter sequences within intron 2 may drive tissue-restricted expression of variants AE2b 1 and AE2b 2, mainly in liver and kidney. AE2b 2 proximal promoter sequences are highly active in transfected liver-derived HepG2 cells and contain an HNF1 motif. Mutation-disruption of this motif dramatically decreased alternate promoter activity in HepG2 cells but not in prostate-derived PC-3 cells. Electromobility shift and supershift assays indicated that HNF1α from HepG2 nuclear extracts binds the HNF1 sequence. Transactivation studies in PC-3 cells showed enhanced activity of the wild-type construct upon cotransfection with an HNF1α expression plasmid, while activity of the HNF1-mutated construct remained unaffected. Since liver AE2 is putatively involved in the biliary secretion of bicarbonate, HNF1α may have a role in increasing bicarbonate secretion in response to certain stimuli.

Defective regulation of cholangiocyte Cl −/HCO [overthree] and Na +/H + exchanger activities in primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is a disorder of unknown origin with autoimmune features. Recently, impaired biliary secretion of bicarbonate has been shown in patients with PBC. Here we have investigated whether bile duct epithelial cells isolated from PBC patients exhibit defects in transepithelial bicarbonate transport by analyzing the activities of 2 ion exchangers, Cl −/HCO[overthree] anion exchanger 2 (AE2) and Na +/H + exchanger (NHE) in isolated cholangiocytes. AE2 and NHE activities were studied in basal conditions and after stimulation with cyclic adenosine monophosphate (cAMP) and extracellular adenosine triphosphate (ATP), respectively. Cholangiocytes were grown from needle liver biopsies from 12 PBC patients, 8 normal controls, and 9 patients with other liver diseases. Also, intrahepatic cholangiocytes were cultured after immunomagnetic isolation from normal liver tissue (n = 6), and from recipients undergoing liver transplantation for end-stage PBC (n = 9) and other forms of liver disease (n = 8). In needle-biopsy cholangiocytes, basal AE2 activity was significantly decreased in PBC as compared with normal livers and disease controls. In addition, we observed that though cAMP increased AE2 activity in cholangiocytes from both normal and non-PBC livers, this effect was absent in PBC cholangiocytes. Similarly, though in cholangiocytes from normal and disease control livers extracellular ATP induced a marked enhancement of NHE activity, cholangiocytes from PBC patients failed to respond to purinergic stimulation. In conclusion, our findings provide functional evidence that PBC cholangiocytes exhibit a widespread failure in the regulation of carriers involved in transepithelial H +/HCO[overthree] transport, thus, providing a molecular basis for the impaired bicarbonate secretion in this cholestatic syndrome. (H EPATOLOGY 2002;35:1513-1521.)

Corticosteroids modulate the secretory processes of the rat intrahepatic biliary epithelium

We investigated the expression of glucocorticoid receptors (GcRs) in the intrahepatic biliary epithelium and the role of corticosteroids in the regulation of cholangiocyte secretion. GcR was studied by immunohistochemistry, reverse-transcription polymerase chain reaction, and Western blots. The effects of dexamethasone and budesonide on biliary bicarbonate excretion and H+/HCO3- transport processes were investigated in bile fistula rats, isolated intrahepatic bile duct units (IBDUs), and purified cholangiocytes. GcRs were expressed by rat cholangiocytes. Although acute administration of corticosteroids showed no effect, treatment for 2 days with dexamethasone or budesonide increased (P < 0.05) biliary bicarbonate concentration and secretion, which were blocked by the specific GcR antagonist, RU-486. IBDUs isolated from rats treated with dexamethasone or budesonide showed an increased (P < 0.05) activity of the Na+/H+ exchanger (NHE1 isoform) and Cl-/HCO3- exchanger (AE2 member), which was blocked by RU-486. Protein expression of NHE1 and AE2 and messenger RNA for NH1 but not AE2 were increased (P < 0.05) in isolated cholangiocytes by dexamethasone treatment. The intrahepatic biliary epithelium expresses GcR and responds to corticosteroids by increasing bicarbonate excretion in bile. This is caused by corticosteroid-induced enhanced activities and protein expression of transport processes driving bicarbonate excretion in the biliary epithelium.

Secretin in plasma: Ups and downs

The establishment of a reliable radioimmunoassay for secretin in plasma enabled studies on the physiology of secretin. It was shown that secretin, i.e. the heptacosapeptide isolated by Jorpes and Mutt, is in fact responsible for the phenomena observed by Starling and Bayliss in 1902 when studying the stimulation of pancreatic bicarbonate secretion in response to duodenal acidification. Secretin is released in amounts considerably lower than anticipated, but these amounts are nevertheless sufficient to drive pancreatic and biliary secretion of bicarbonate. Whereas secretin in the fasting state is the most important stimulus to pancreatic secretion of water and bicarbonate, other hormones and nervous factors are essential for the majority of pancreatic postprandial secretion.

Sodium, Hydrogen exchange type 1 and bile ductular secretory activity in the guinea pig

Biliary epithelial cells (BECs) express different Na(+), H(+) exchange (NHE) isoforms. In this study, the potential role of NHE in ductular bile secretion is assessed. Experiments were performed in guinea pig perfused livers and isolated BECs. Inhibition of NHE was achieved by hypotonic stress and by using the unspecific NHE inhibitor, amiloride, or the specific NHE 1 inhibitor, cariporide (HOE 642). Hypotonic stress inhibited basal bile flow by 46% and prevented secretin stimulation of bile flow by reducing biliary bicarbonate output by 50%. Secretin increased bile flow from 3.7 +/- 0.8 microL/min/g to 4.78 microL/min/g (P <.01); subsequent exposure to hypotonic stress decreased secretin-stimulated bile flow by 35% and biliary bicarbonate secretion by approximately 50%. Inhibition of NHE by amiloride or cariporide resulted in a similar reduction of secretin-stimulated bile flow and bicarbonate secretion. Basal bile flow was unaffected by the NHE inhibitors. In isolated guinea pig BECs, regulatory volume decrease and inhibition of NHE was demonstrated after hypotonic stress under basal and secretin-stimulated conditions. In contrast, hypotonic exposure inhibited Cl(-), HCO(3)(-) exchange activity in isolated BECs only during basal conditions but incompletely after secretin stimulation. Our study shows that hypotonic stress inhibits basal bile flow in the guinea pig by inhibition of Cl(-), HCO(3)(-) exchange. NHE1 is not involved in basal bile formation. Increased choleresis after ductular stimulation by secretin depends on intact NHE1 activity. These data indicate that BEC volume changes have profound effects on biliary secretory function.

Assessment of biliary bicarbonate secretion in humans by positron emission tomography

Background & Aims: Positron emission tomography (PET) allows imaging and quantitative analysis of organ functions in basal and stimulated conditions. We have applied this method to the study of biliary bicarbonate secretion in humans. Methods: PET was performed in 5 healthy subjects and 13 patients with hepatobiliary disorders after intravenous injection of NaH 11CO 3. In each case the study was performed in basal conditions and after secretin stimulation. Positron emission from the hepatic area was scanned, and normalized uptake values for parenchymal and hilar regions were estimated. Results: In healthy individuals, the injection of NaH 11CO 3 resulted in a peak uptake of the label in parenchymal and hilar regions 2–3 minutes after the injection. In both normal and cirrhotic subjects, secretin administration increased bicarbonate uptake in the parenchymal region, followed by accumulation of the label in the perihilar area. Normal basal uptake with absent response to secretin was registered in extrahepatic biliary obstruction and in untreated primary biliary cirrhosis (PBC). The secretin response was present in patients with PBC undergoing treatment with ursodeoxycholic acid. Conclusions: PET allows investigation of biliary bicarbonate secretion in humans. An impaired response to secretin was observed in cholestatic conditions. Preliminary data suggest that ursodeoxycholic acid might improve the response to secretin in PBC. GASTROENTEROLOGY 1999;117:167-172