Activation of Cyclic AMP Signaling in Ae2-deficient Mouse Fibroblasts

Mardones, Pablo; Medina, Juan F.; Elferink, Ronald P.J. Oude

doi:10.1074/jbc.m710590200

Cited by 24 publications

(32 citation statements)

References 46 publications

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“…Intracellular pH was determined from the ratio of fluorescence intensity obtained at excitation at 490 nm over excitation at 440 nm. 37 Electron Microscopy of Cultured Cholangiocytes and Liver Sections. Cultured cells were fixed in McDowell fixative (4% paraformaldehyde, 1% glutaraldehyde, 0.1 M of cacodylate, and 4.4 mM of CaCl 2 ; pH 7.4), containing 0.2% ruthenium red (SigmaAldrich), and counterstained with 1% OsO 4 and 0.05% ruthenium red.…”

Section: Methodsmentioning

confidence: 99%

A Biliary HCO3 − Umbrella Constitutes A Protective Mechanism Against Bile Acid–Induced Injury in Human Cholangiocytes

et al. 2012

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Human cholangiocytes are continuously exposed to millimolar levels of hydrophobic bile salt monomers. We recently hypothesized that an apical biliary HCO À 3 umbrella might prevent the protonation of biliary glycine-conjugated bile salts and uncontrolled cell entry of the corresponding bile acids, and that defects in this biliary HCO À 3 umbrella might predispose to chronic cholangiopathies. Here, we tested in vitro whether human cholangiocyte integrity in the presence of millimolar bile salt monomers is dependent on (1) pH, (2) adequate expression of the key HCO À 3 exporter, anion exchanger 2 (AE2), and (3) an intact cholangiocyte glycocalyx. To address these questions, human immortalized cholangiocytes and cholangiocarcinoma cells were exposed to chenodeoxycholate and its glycine/taurine conjugates at different pH levels. Bile acid uptake was determined radiochemically. Cell viability and apoptosis were measured enzymatically. AE2 was knocked down by lentiviral short hairpin RNA. A cholangiocyte glycocalyx was identified by electron microscopy, was enzymatically desialylated, and sialylation was quantified by flow cytometry. We found that bile acid uptake and toxicity in human immortalized cholangiocytes and cholangiocarcinoma cell lines in vitro were pH and AE2 dependent, with the highest rates at low pH and when AE2 expression was defective. An apical glycocalyx was identified on cholangiocytes in vitro by electron microscopic techniques. Desialylation of this protective layer increased cholangiocellular vulnerability in a pH-dependent manner. Conclusion: A biliary HCO À 3 umbrella protects human cholangiocytes against damage by bile acid monomers. An intact glycocalyx and adequate AE2 expression are crucial in this process. Defects of the biliary HCO À 3 umbrella may lead to the development of chronic cholangiopathies.

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Section: Methodsmentioning

confidence: 99%

A Biliary HCO3 − Umbrella Constitutes A Protective Mechanism Against Bile Acid–Induced Injury in Human Cholangiocytes

et al. 2012

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“…19 These mice showed biochemical, serologic, histopathologic, and immunologic features compatible with PBC. 20 pH i assessment in lymphocytes 20 and fibroblasts 21 of Ae2 a,b Ϫ/Ϫ mice confirmed the expected intracellular alkalinization, while cholangiocytes isolated from these mice were found to have normal pH i values. 20 We therefore decided to analyze the mechanisms involved in HCO 3 Ϫ secretion in cultured Ae2 a,b ϩ/ϩ and Ae2 a,b Ϫ/Ϫ mouse cholangiocytes.…”

mentioning

confidence: 77%

“…4). Baseline NBC activity of these Ae2 a,b -deficient cells is surprisingly high in spite of elevated cAMP production (also observed in Ae2 a,b -deficient mouse fibroblasts), 21 but further increased cAMP levels after cAMP/SM stimulation may trigger the decreasing effect. In the case of wild-type mouse cholangiocytes, cAMP/SM administration leads to ϩ/ϩ mouse cholangiocytes has no effect on the recovering acidification after propionate removal and subsequent alkalinization.…”

Section: Biliary Hcomentioning

confidence: 99%

“…In this sense, cholangiocytes from Ae2 a,b -deficient mice appear to be more capable of maintaining their resting pH i 20 than Ae2 a,b Ϫ/Ϫ mouse fibroblasts, in which Slc4a4 is not expressed. 21 Stoichiometry of the NBC activity displayed by Slc4a4 is cell type-dependent; 1:3 stoichiometry runs for Na ϩ and HCO 3 Ϫ efflux (acid load), whereas 1:2 works for influx of these ions into the cell (acid extrusion). 26 Moreover, Slc4a4 stoichiometry may vary in the same cells, as reported for mouse kidney cells, in which protein kinase A-mediated phosphorylation in Ser 982 shifts stoichiometry from 1:3 to 1:2, leading to decreased HCO 3 Ϫ efflux.…”

Section: Biliary Hcomentioning

confidence: 99%

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1120 BICARBONATE SECRETION OF MOUSE CHOLANGIOCYTES INVOLVES Na+–HCO3− COTRANSPORT IN ADDITION TO Na+-INDEPENDENT Cl−/HCO3− EXCHANGE

Uriarte¹,

Sáez²,

Arenas³

et al. 2010

Journal of Hepatology

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Uriarte, I.; Banales, J.M.; Sáez, E.; Arenas, F.; Oude Elferink, R.P.J.; Prieto, J.; Medina, J.F. Published in: Hepatology DOI:10.1002/hep.23403 Link to publication Citation for published version (APA):Uriarte, I., Banales, J. M., Sáez, E., Arenas, F., Oude Elferink, R. P. J., Prieto, J., & Medina, J. F. (2010). Bicarbonate Secretion of Mouse Cholangiocytes Involves Na+-HCO3-Cotransport in Addition to Na+-Independent Cl-/HCO3-Exchange. Hepatology, 51(3), 891-902. DOI: 10.1002/hep.23403 General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. 3 ؊ secretion not observed in control rat cholangiocytes. Propionate-based maneuvers indicated that this supplemental Na ؉ -driven HCO 3 ؊ -secreting activity is Cl ؊ -independent, consistent with a Na ؉ -HCO 3 ؊ cotransport (NBC). NBC activity is greater in

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“…Obviously the deranged homeostasis of intracellular pH would cause cellular distress and reduce the threshold to apoptosis, but a few observations and evidence suggest that sAC, an evolutionarily conserved bicarbonate sensor, might mediate the sensitization to apoptosis. First, fibroblasts from AE2 a,b -/- mice have an alkalinized intracellular pH, higher cellular cAMP and higher expression of sAC at both the messenger RNA level and protein level [31]. Second, the expression of sAC is highest in the testes, and AE2 a,b -/- mice are infertile due to increased apoptosis in seminiferous tubules and epididymis [32].…”

Section: Sac As a Novel Regulator Of Bile Salt-induced Apoptosismentioning

confidence: 99%

The Emerging Role of Soluble Adenylyl Cyclase in Primary Biliary Cholangitis

Chang

Beuers²,

Elferink³

2017

Dig Dis

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Background: Primary biliary cholangitis (PBC; previously referred to as primary biliary cirrhosis) is a chronic fibrosing cholangiopathy with the signature of an autoimmune disease and features of intrahepatic cholestasis. Immunosuppressing treatments are largely unsuccessful. Responsiveness to ursodeoxycholic acid and reduced expression of anion exchanger 2 (AE2) on canalicular membranes and small bile ducts underline the importance of bicarbonate transportation in its disease mechanism. Soluble adenylyl cyclase (sAC; ADCY10) is an evolutionarily conserved bicarbonate sensor that regulates apoptosis, barrier function and TNF signaling. Key Messages: The biliary epithelium defends against the toxic bile by bicarbonate secretion and by maintaining a tight barrier. Passive diffusion of weak acid conjugates (e.g. bile salts and other toxins) across plasma membrane is pH-dependent. Reduced AE2 expression results in both reduced bicarbonate secretion and accumulation of bicarbonate in the cells. Increased intracellular bicarbonate leads to increased sAC activity, which regulates bile salt-induced apoptosis. Reduced bicarbonate secretion causes more bile salts to enter cells, which further increase sAC activity by releasing intracellular Ca²⁺ store. In vitro studies demonstrate that inhibition of sAC not only corrects sensitization to bile salt-induced apoptosis as a result of AE2 down-regulation but also prevents bile salt-induced apoptosis altogether. Targeting sAC is also likely to slow down disease progression by strengthening the barrier function of biliary epithelia and by reducing oxidative stress as a result of chronic inflammation. Conclusions: sAC is a potential therapeutic target for PBC. More in vitro and in vivo studies are needed to understand how sAC regulates bile salt-induced apoptosis and to establish its therapeutic value in PBC and other cholestatic cholangiopathies.

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Activation of Cyclic AMP Signaling in Ae2-deficient Mouse Fibroblasts

Cited by 24 publications

References 46 publications

A Biliary HCO3 − Umbrella Constitutes A Protective Mechanism Against Bile Acid–Induced Injury in Human Cholangiocytes

A Biliary HCO3 − Umbrella Constitutes A Protective Mechanism Against Bile Acid–Induced Injury in Human Cholangiocytes

1120 BICARBONATE SECRETION OF MOUSE CHOLANGIOCYTES INVOLVES Na+–HCO3− COTRANSPORT IN ADDITION TO Na+-INDEPENDENT Cl−/HCO3− EXCHANGE

The Emerging Role of Soluble Adenylyl Cyclase in Primary Biliary Cholangitis

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