Biliary Bicarbonate Excretion Research Articles

When lightning strikes twice: The plot thickens for a dual role of the anion exchanger 2 (AE2/SLC4A2) in the pathogenesis and treatment of primary biliary cirrhosis

Cl(-)/HCO(3)(-) anion exchanger 2 (AE2) is involved in intracellular pH (pH(i)) regulation and transepithelial acid-base transport, including secretin-stimulated biliary bicarbonate excretion. AE2 gene expression was found to be reduced in liver biopsy specimens and blood mononuclear cells from patients with primary biliary cirrhosis (PBC), a disease characterized by chronic nonsuppurative cholangitis associated with antimitochondrial antibodies (AMA) and other autoimmune phenomena. In mice with widespread Ae2 gene disruption, we previously reported altered spermiogenesis and reduced gastric acid secretion. We now describe the hepatobiliary and immunologic changes observed in these Ae2(a,b)-deficient mice. In this murine model, splenocyte pH(i) and T-cell populations were studied by flow cytometry. CD3-stimulated cytokine secretion was estimated using cytokine arrays. AMA were evaluated by immunoblotting and proteomics. Hepatobiliary changes were assessed by immunohistopathology, flow cytometry, and serum biochemistry. Cholangiocyte gene expression was analyzed by real-time polymerase chain reaction. Ae2(a,b)(-/-) mice exhibit splenomegaly, elevated pH(i) in splenocytes, increased production of interleukin-12p70 and interferon gamma, expanded CD8(+) T-cell population, and under represented CD4(+)FoxP3(+)/regulatory T cells. Most Ae2(a,b)(-/-) mice tested positively for AMA, showing increased serum levels of immunoglobulin M and G, and liver-specific alkaline phosphatase. About one third of Ae2(a,b)(-/-) mice had extensive portal inflammation with CD8(+) and CD4(+) T lymphocytes surrounding damaged bile ducts. Cholangiocytes isolated from Ae2(a,b)(-/-) mice showed gene expression changes compatible with oxidative stress and increased antigen presentation. Ae2 deficiency alters pH(i) homeostasis in immunocytes and gene expression profile in cholangiocytes, leading to immunologic and hepatobiliary changes that resemble PBC.

Ae2 a,b-Deficient Mice Develop Antimitochondrial Antibodies and Other Features Resembling Primary Biliary Cirrhosis

Cl(-)/HCO(3)(-) anion exchanger 2 (AE2) is involved in intracellular pH (pH(i)) regulation and transepithelial acid-base transport, including secretin-stimulated biliary bicarbonate excretion. AE2 gene expression was found to be reduced in liver biopsy specimens and blood mononuclear cells from patients with primary biliary cirrhosis (PBC), a disease characterized by chronic nonsuppurative cholangitis associated with antimitochondrial antibodies (AMA) and other autoimmune phenomena. In mice with widespread Ae2 gene disruption, we previously reported altered spermiogenesis and reduced gastric acid secretion. We now describe the hepatobiliary and immunologic changes observed in these Ae2(a.b)-deficient mice. In this murine model, splenocyte pH(i) and T-cell populations were studied by flow cytometry. CD3-stimulated cytokine secretion was estimated using cytokine arrays. AMA were evaluated by immunoblotting and proteomics. Hepatobiliary changes were assessed by immunohistopathology, flow cytometry, and serum biochemistry. Cholangiocyte gene expression was analyzed by real-time polymerase chain reaction. Ae2(a,b)(-/-) mice exhibit splenomegaly, elevated pH(i) in splenocytes, increased production of interleukin-12p70 and interferon gamma, expanded CD8(+) T-cell population, and under represented CD4(+)FoxP3(+)/regulatory T cells. Most Ae2(a,b)(-/-) mice tested positively for AMA, showing increased serum levels of immunoglobulin M and G, and liver-specific alkaline phosphatase. About one third of Ae2(a,b)(-/-) mice had extensive portal inflammation with CD8(+) and CD4(+) T lymphocytes surrounding damaged bile ducts. Cholangiocytes isolated from Ae2(a,b)(-/-) mice showed gene expression changes compatible with oxidative stress and increased antigen presentation. Ae2 deficiency alters pH(i) homeostasis in immunocytes and gene expression profile in cholangiocytes, leading to immunologic and hepatobiliary changes that resemble PBC.

Combination of ursodeoxycholic acid and glucocorticoids upregulates the AE2 alternate promoter in human liver cells

Primary biliary cirrhosis (PBC) is a cholestatic disease associated with autoimmune phenomena and alterations in both biliary bicarbonate excretion and expression of the bicarbonate carrier AE2. The bile acid ursodeoxycholic acid (UCDA) is currently used in treatment of cholestatic liver diseases and is the treatment of choice in PBC; however, a subset of PBC patients respond poorly to UDCA monotherapy. In these patients, a combination of UDCA and glucocorticoid therapy appears to be beneficial. To address the mechanism of this benefit, we analyzed the effects of UDCA and dexamethasone on AE2 gene expression in human liver cells from hepatocyte and cholangiocyte lineages. The combination of UDCA and dexamethasone, but not UDCA or dexamethasone alone, increased the expression of liver-enriched alternative mRNA isoforms AE2b1 and AE2b2 and enhanced AE2 activity. Similar effects were obtained after replacing UDCA with UDCA conjugates. In in vitro and in vivo reporter assays, we found that a UDCA/dexamethasone combination upregulated human AE2 alternate overlapping promoter sequences from which AE2b1 and AE2b2 are expressed. In chromatin immunoprecipitation assays, we demonstrated that combination UCDA/dexamethasone treatment induced p300-related interactions between HNF1 and glucocorticoid receptor on the AE2 alternate promoter. Our data provide a potential molecular explanation for the beneficial effects of the combination of UDCA and glucocorticoids in PBC patients with inadequate response to UDCA monotherapy.

Cholangiocyte anion exchange and biliary bicarbonate excretion

Primary canalicular bile undergoes a process of fluidization and alkalinization along the biliary tract that is influenced by several factors including hormones, innervation/neuropeptides, and biliary constituents. The excretion of bicarbonate at both the canaliculi and the bile ducts is an important contributor to the generation of the so-called bile-salt independent flow. Bicarbonate is secreted from hepatocytes and cholangiocytes through parallel mechanisms which involve chloride efflux through activation of Cl- channels, and further bicarbonate secretion via AE2/SLC4A2-mediated Cl-/HCO3- exchange. Glucagon and secretin are two relevant hormones which seem to act very similarly in their target cells (hepatocytes for the former and cholangiocytes for the latter). These hormones interact with their specific G protein-coupled receptors, causing increases in intracellular levels of cAMP and activation of cAMP-dependent Cl- and HCO3- secretory mechanisms. Both hepatocytes and cholangiocytes appear to have cAMP-responsive intracellular vesicles in which AE2/SLC4A2 colocalizes with cell specific Cl- channels (CFTR in cholangiocytes and not yet determined in hepatocytes) and aquaporins (AQP8 in hepatocytes and AQP1 in cholangiocytes). cAMP-induced coordinated trafficking of these vesicles to either canalicular or cholangiocyte lumenal membranes and further exocytosis results in increased osmotic forces and passive movement of water with net bicarbonate-rich hydrocholeresis.

Hydrogen Sulfide as an Endogenous Modulator of Biliary Bicarbonate Excretion in the Rat Liver

Cystathionine gamma-lyase (CSE) is an enzyme catalyzing cystathionine and cysteine to yield cysteine and hydrogen sulfide (H(2)S), respectively. This study aimed to examine if H(2)S generated from the enzyme could serve as an endogenous regulator of hepatobiliary function. Gas chromatographic analyses indicated that, among rat organs herein examined, liver constituted one of the greatest components of H(2)S generation in the body, at 100 mumol/g of tissue, comparable to that in kidney and 1.5-fold greater than that in brain, where roles of the gas in the regulation of neurotransmission were reported previously. At least half of the gas amount in the liver appeared to be derived from CSE, because blockade of the enzyme by propargylglycine suppressed it by 50%. Immunohistochemistry revealed that CSE occurs not only in hepatocytes, but also in bile duct. In livers in vivo, as well as in those perfused ex vivo, treatment with the CSE inhibitor induced choleresis by stimulating the basal excretion of bicarbonate in bile samples. Transportal supplementation of NaHS at 30 mumol/L, but not that of N-acetylcysteine as a cysteine donor, abolished these changes elicited by the CSE inhibitor in the perfused liver. The changes elicited by the CSE blockade did not coincide with alterations in hepatic vascular resistance, showing little involvement of vasodilatory effects of the gas in these events, if any. These results first provided evidence that H(2)S generated through CSE modulates biliary bicarbonate excretion and is thus a determinant of bile salt-independent bile formation in the rat liver.

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.

Bile duct epithelia regulate biliary bicarbonate excretion in the isolated bivascularly perfused rat liver

Bile duct epithelia regulate biliary bicarbonate excretion in the isolated bivascularly perfused rat liver

Bile duct epithelia regulate biliary bicarbonate excretion in the isolated bivascularly perfused rat liver

Bile duct epithelia regulate biliary bicarbonate excretion in the isolated bivascularly perfused rat liver

The function of alkaline phosphatase in the liver: Regulation of intrahepatic biliary epithelium secretory activities in the rat

We studied the effects of alkaline phosphatase (AP) on the secretory processes of the rat intrahepatic biliary epithelium as well as the role of the intrahepatic biliary epithelium in the uptake and biliary secretion of exogenous AP. The effects of acute and chronic administration of AP on bile secretory parameters were investigated in vivo in normal and bile duct ligated (BDL) rats and in vitro in isolated rat bile duct units (IBDU).In vivo, acute AP administration decreased bile flow and biliary bicarbonate excretion and abolished secretin choleresis in BDL rats but not in normal rats. On the contrary, the AP inhibitor, levamisole, increased in BDL rat bile flow and biliary bicarbonate excretion. In vitro, basal and secretin-stimulated Cl−/HCO3− exchanger activity in IBDU was immediately inhibited by AP intraluminal microinjection (apical exposure) but only after a prolonged exposure to the basolateral pole. Levamisole increased the Cl−/HCO3− exchanger activity of IBDU. A significant basolateral uptake of AP occurs in IBDU with a progressive transport to the apical domain. AP chronic treatment increased AP and γ-glutamyltranspeptidase (γ-GT) activities in the intrahepatic bile ducts and hepatocyte canalicular pole, promoted enlargement of bile canaliculi, and decreased bile flow and biliary bicarbonate excretion. In conclusion, the intrahepatic biliary epithelium plays a role in the uptake and biliary secretion of serum AP. AP inhibits the secretory processes of the intrahepatic biliary epithelium and induces features of intrahepatic cholestasis after chronic administration. These findings indicate that AP plays an active role in down-regulating the secretory activities of the intrahepatic biliary epithelium. (Hepatology 2000;32:174-184.)

Effects of secretin on bile production in two kinds of cholestatic models by choledocho-caval fistula and bile duct ligation in rats.

Secretin is known to stimulate bile flow from the bile duct epithelium. To investigate the effects of secretin in cholestasis, we studied the response of the bile flow and the excretion of biliary components to secretin using two cholestatic models with or without damage to the bile duct epithelium. The model without bile duct epithelial damage was a choledocho-caval (CC) fistula over a 24-hour period, and the model with bile duct damage was a bile duct ligation over a 48-hour period. Secretin was administered by intravenous infusion for 30 minutes and bile was collected for 120 minutes. Controls were given saline similarly. The bile flow and biliary bicarbonate excretion rate were significantly increased after secretin infusion in the CC fistula rats when compared with the control rats, but no stimulation by secretin was observed in the ligated rats. These data indicate that secretin-induced bile production was enhanced under cholestatic conditions with no bile duct epithelial disturbance.

The Ursodeoxycholate Dose–Dependent Formation of Ursodeoxycholate–Glucuronide in the Rat and the Choleretic Potencies

The reason for the discrepancy between bile flow and biliary bile acid excretion during ursodeoxycholate infusion in rats is unknown. We found that ursodeoxycholate-glucuronide is formed during ursodeoxycholate infusion at higher doses. Ursodeoxycholate infusion (1 to 3 mumol/min/100 gm body weight) for 90 min caused marked hypercholeresis, and the previously reported discrepancy between bile flow and biliary bile acid excretion was observed when bile acid concentrations were measured by regular enzymatic methods. However, the appearance of ursodeoxycholate-glucuronide was observed on thin-layer chromatography analysis and up to 30% of the ursodeoxycholate in bile was found to be glucuronidated when determined by the enzymatic method after beta-glucuronidase treatment. The choleretic activity of ursodeoxycholate-glucuronide (25.2 microliters/mumol) was about 3 times higher than that of ursodeoxycholate (8.9 microliters/mumol) when infused at 0.25 mumol/min/100 gm body weight and ursodeoxycholate-glucuronide also stimulated higher biliary bicarbonate excretion than ursodeoxycholate. These results indicate that the discrepancy between bile flow and biliary bile acid excretion caused by high-dose infusion of ursodeoxycholate into rats can be explained by glucuronide conjugation of ursodeoxycholate that cannot be detected by the regular enzymatic method. The glucuronidation of ursodeoxycholate might also be important in the ursodeoxycholate-induced increase in biliary bicarbonate excretion.