Home Hydrophobic bile salts trigger ceramide formation through endosomal acidification
Article
Licensed
Unlicensed Requires Authentication

Hydrophobic bile salts trigger ceramide formation through endosomal acidification

  • Stephan Becker , Roland Reinehr , Susanne Grether-Beck , Andrea Eberle and Dieter Häussinger
Published/Copyright: January 29, 2007
Biological Chemistry
From the journal Volume 388 Issue 2

Abstract

Hydrophobic bile salts activate NADPH oxidase through a ceramide- and PKCζ-dependent pathway as an important upstream event of bile salt-induced hepatocyte apoptosis. The mechanisms underlying bile salt-induced ceramide formation have remained unclear to date and thus were studied in rat hepatocytes. Proapoptotic bile salts, such as taurolithocholylsulfate (TLCS), lowered the apparent pHves within seconds from 6.0 to 5.6 in an FITC-dextran-accessible endosomal compartment that also contains acidic sphingomyelinase. Simultaneously, a rapid decrease in N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) fluorescence was observed, suggestive of an increase in cytosolic [Cl-], which is known to activate vacuolar-type H+-ATPase. No vesicular acidification or increase in cytosolic [Cl-] was found in response to the non-apoptotic bile salt taurocholate or the anti-apoptotic bile salt tauroursodesoxycholate. Inhibition of TLCS-induced endosomal acidification by bafilomycin or 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid largely abolished the TLCS-induced ceramide-formation and downstream ceramide-dependent processes, such as p47phox-serine phosphorylation, NADPH oxidase activation, CD95 activation and apoptosis. These responses were also abolished after knockdown of acidic sphingomyelinase in rat hepatocytes. In conclusion, hydrophobic, proapoptotic bile salts stimulate ceramide formation through chloride-dependent acidification of endosomes, with subsequent activation of acidic sphingomyelinase. Our data suggest that changes in ion homeostasis underlie the stimulation of ceramide formation in response to hydrophobic bile acids as an important upstream event of bile salt-induced apoptosis.

:

Corresponding author

References

Anwer, M.S., Branson, A.U., and Atkinson, J.M. (1991). Mechanism of inhibition of hepatic bile acid uptake by amiloride and 4,4′-diisothiocyano-2,2′-disulfonic stilbene (DIDS). Biochem. Pharmacol.42, S135–S141.Search in Google Scholar

Bataller, R., Schwabe, R.F., Choi, Y.H., Yang, L., Paik, Y.H., Lindquist, J., Qian, T., Schoonhoven, R., Hagedorn, C.H., Lemasters, J.J., and Brenner, D.A. (2003). NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis. J. Clin. Invest.112, 1383–1394.10.1172/JCI18212Search in Google Scholar

Bowman, E.J., Siebers, A., and Altendorf, K. (1988). Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc. Natl. Acad. Sci. USA85, 7972–7976.10.1073/pnas.85.21.7972Search in Google Scholar

Brock, R., Hamelers, I.H., and Jovin, T.M. (1999). Comparison of fixation protocols for adherent cultured cells applied to a GFP fusion protein of the epidermal growth factor receptor. Cytometry35, 353–362.10.1002/(SICI)1097-0320(19990401)35:4<353::AID-CYTO8>3.0.CO;2-MSearch in Google Scholar

Chieco, P., Romagnoli, E., Aicardi, G., Suozzi, A., Forti, G.C., and Roda, A. (1997). Apoptosis induced in rat hepatocytes by in vivo exposure to taurochenodeoxycholate. Histochem. J.29, 875–883.10.1023/A:1026446008712Search in Google Scholar

Crowley, C.L., Pyne, C.M., Bernstein, H., Bernstein, C., and Roe, D. (2000). The NAD+ precursors, nicotinic acid and nicotin-amide protect cells against apoptosis induced by a multiple stress inducer, deoxycholate. Cell Death Differ.7, 314–326.10.1038/sj.cdd.4400658Search in Google Scholar

Dickinson, M.E., Simbuerger, E., Zimmermann, B., Waters, C.W., and Fraser, S.E. (2003). Multiphoton excitation spectra in biological samples. J. Biomed. Opt.8, 329–338.10.1117/1.1583734Search in Google Scholar

Eberle, A., Reinehr, R., Becker, S., and Häussinger, D. (2005). Fluorescence resonance energy transfer analysis of proapoptotic CD95/EGF receptor interactions in Huh7 cells. Hepatology41, 315–326.10.1002/hep.20564Search in Google Scholar

Edwards, J.C. and Kapadia, S. (2000). Regulation of the bovine kidney microsomal chloride channel p64 by p59fyn, a Src family tyrosine kinase. J. Biol. Chem.275, 31826–31832.10.1074/jbc.M005275200Search in Google Scholar

Faubion, W.A., Guicciardi, M.E., Miyoshi, H., Bronk, S.F., Roberts, P.J., Svingen, P.A., Kaufmann, S.H., and Gores, G.J. (1999). Toxic bile salts induce rodent hepatocyte apoptosis via direct activation of Fas. J. Clin. Invest.103, 137–145.10.1172/JCI4765Search in Google Scholar

Faundez, V. and Hartzell, H.C. (2004). Intracellular chloride channels: determinants of function in the endosomal pathway. Sci. STKE233, re8.Search in Google Scholar

Folch, J., Lees, M., and Sloane-Stanley, G.H. (1957). A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem.226, 497–509.10.1016/S0021-9258(18)64849-5Search in Google Scholar

Fowler, S. (1969). Lysosomal localization of sphingomyelinase in rat liver. Biochim. Biophys. Acta191, 481–484.10.1016/0005-2744(69)90271-XSearch in Google Scholar

Garcia-Ruiz, C., Colell, A., Mari, M., Morales, A., Calvo, M., Enrich, C., and Fernandez-Checa, J.C. (2003). Defective TNF-α-mediated hepatocellular apoptosis and liver damage in acidic sphingomyelinase knockout mice. J. Clin. Invest.111, 197–208.10.1172/JCI16010Search in Google Scholar

Graf, J., Henderson, R.M., Krumpholz, B., and Boyer, J.L. (1987). Cell membrane and transepithelial voltages and resistances in isolated rat hepatocyte couplets. J. Membr. Biol.95, 241–254.10.1007/BF01869486Search in Google Scholar

Grether-Beck, S., Bonizzi, G., Schmitt-Brenden, H., Felsner, I., Timmer, A., Sies, H., Johnson, J.P., Piette, J., and Krutmann, J. (2000). Non-enzymatic triggering of the ceramide signalling cascade by solar UVA radiation. EMBO J.19, 5793–5800.10.1093/emboj/19.21.5793Search in Google Scholar

Gulbins, E., Bissonnette, R., Mahboubi, A., Martin, S., Nishioka, W., Brunner, T., Baier, G., Baier-Bitterlich, G., Byrd, C., Lang, F., et al. (1995). FAS-induced apoptosis is mediated via a ceramide-initiated RAS signaling pathway. Immunity2, 341–351.10.1016/1074-7613(95)90142-6Search in Google Scholar

Gupta, S., Natarajan, R., Payne, S.G., Studer, E.J., Spiegel, S., Dent, P., and Hylemon, P.B. (2004). Deoxycholic acid activates the c-Jun N-terminal kinase pathway via FAS receptor activation in primary hepatocytes. Role of acidic sphingomyelinase-mediated ceramide generation in FAS receptor activation. J. Biol. Chem.279, 5821–5828.10.1074/jbc.M310979200Search in Google Scholar

Hara-Chikuma, M., Yang, B., Sonawane, N.D., Sasaki, S., Uchida, S., and Verkman, A.S. (2005). ClC-3 chloride channels facilitate endosomal acidification and chloride accumulation. J. Biol. Chem.280, 1241–1247.10.1074/jbc.M407030200Search in Google Scholar

Jentsch, T.J., Poet, M., Fuhrmann, J.C., and Zdebik, A.A. (2005). Physiological functions of CLC Cl- channels gleaned from human genetic disease and mouse models. Annu. Rev. Physiol.67, 779–807.10.1146/annurev.physiol.67.032003.153245Search in Google Scholar

Kirschnek, S., Paris, F., Weller, M., Grassme, H., Ferlinz, K., Riehle, A., Fuks, Z., Kolesnick, R., and Gulbins, E. (2000). CD95-mediated apoptosis in vivo involves acidic sphingomyelinase. J. Biol. Chem.275, 27316–27323.10.1016/S0021-9258(19)61513-9Search in Google Scholar

Krähenbühl, S., Talos, C., Fischer, S., and Reichen, J. (1994). Toxicity of bile acids on the electron transport chain of isolated rat mitochondria. Hepatology19, 471–479.10.1002/hep.1840190228Search in Google Scholar PubMed

Lambeth, J.D. (2002). Nox/Duox family of nicotinamide adenine dinucleotide (phosphate) oxidases. Curr. Opin. Hematol.9, 11–17.10.1097/00062752-200201000-00003Search in Google Scholar

Leppa, S. and Bohmann, D. (1999). Diverse functions of JNK signaling and c-Jun in stress response and apoptosis. Oncogene18, 6158–6162.10.1038/sj.onc.1203173Search in Google Scholar

Lepple-Wienhues, A., Belka, C., Laun, T., Jekle, A., Walter, B., Wieland, U., Welz, M., Heil, L., Kun, J., Busch, G., et al. (1999). Stimulation of CD95 (Fas) blocks T lymphocyte calcium channels through sphingomyelinase and sphingolipids. Proc. Natl. Acad. Sci. USA96, 13795–13800.10.1073/pnas.96.24.13795Search in Google Scholar

Li, X. and Weinman, S.A. (2002). Chloride channels and hepatocellular function: prospects for molecular identification. Annu. Rev. Physiol.64, 609–633.10.1146/annurev.physiol.64.090501.145429Search in Google Scholar

Marandi, N., Konnerth, A., and Garaschuk, O. (2002). Two-photon chloride imaging in neurons of brain slices. Pflüger's Arch.445, 357–365.10.1007/s00424-002-0933-7Search in Google Scholar

Mathias, S., Pena, L.A., and Kolesnick, R.N. (1998). Signal transduction of stress via ceramide. Biochem. J.335, 465–480.10.1042/bj3350465Search in Google Scholar

Mauricio, A.C. and Ferreira, K.T. (1999). Activation of ionic channels by deoxycholate in frog and human cell lines. Exp. Physiol.84, 489–499.10.1111/j.1469-445X.1999.01734.xSearch in Google Scholar

Meng, L.J., Wang, P., Wolkoff, A.W., Kim, R.B., Tirona, R.G., Hofmann, A.F., and Pang, K.S. (2002). Transport of the sulfated, amidated bile acid, sulfolithocholyltaurine, into rat hepatocytes is mediated by Oatp1 and Oatp2. Hepatology35, 1031–1040.10.1053/jhep.2002.32667Search in Google Scholar

Miyoshi, H., Rust, C., Roberts, P.J., Burgart, L.J., and Gores, G.J. (1999). Hepatocyte apoptosis after bile duct ligation in the mouse involves CD95. Gastroenterology117, 669–677.10.1016/S0016-5085(99)70461-0Search in Google Scholar

Moriyama, Y. and Nelson, N. (1987). The purified ATPase from chromaffin granule membranes is an anion-dependent proton pump. J. Biol. Chem.262, 9175–9180.10.1016/S0021-9258(18)48064-7Search in Google Scholar

Muallem, S., Blissard, D., Cragoe, E.J. Jr., and Sachs, G. (1988). Activation of the Na+/H+ and Cl-/HCO3- exchange by stimulation of acid secretion in the parietal cell. J. Biol. Chem.263, 14703–14711.10.1016/S0021-9258(18)68094-9Search in Google Scholar

Paris, F., Grassme, H., Cremesti, A., Zager, J., Fong, Y., Haimovitz-Friedman, A., Fuks, Z., Gulbins, E., and Kolesnick, R. (2001). Natural ceramide reverses Fas resistance of acid sphingomyelinase-/- hepatocytes. J. Biol. Chem.276, 8297–8305.10.1074/jbc.M008732200Search in Google Scholar

Pazoles, C.J., Creutz, C.E., and Pollard, H.B. (1980). Evidence for direct coupling of proton and anion transport in chromaffin granules. Ann. NY Acad. Sci.358, 354–355.10.1111/j.1749-6632.1980.tb15417.xSearch in Google Scholar

Piiper, A., Elez, R., You, S.J., Kronenberger, B., Loitsch, S., Roche, S., and Zeuzem, S. (2003). Cholecystokinin stimulates extracellular signal-regulated kinase through activation of the epidermal growth factor receptor, Yes, and protein kinase C. J. Biol. Chem.278, 7065–7072.10.1074/jbc.M211234200Search in Google Scholar

Reinehr, R., Graf, D., and Häussinger, D. (2003). Bile salt-induced hepatocyte apoptosis involves epidermal growth factor receptor-dependent CD95 tyrosine phosphorylation. Gastroenterology125, 839–853.10.1016/S0016-5085(03)01055-2Search in Google Scholar

Reinehr, R., Becker, S., Wettstein, M., and Häussinger, D. (2004). Involvement of the Src family kinase yes in bile salt-induced apoptosis. Gastroenterology127, 1540–1557.10.1053/j.gastro.2004.08.056Search in Google Scholar PubMed

Reinehr, R., Becker, S., Keitel, V., Eberle, A., Grether-Beck, S., and Häussinger, D. (2005). Bile salt-induced apoptosis involves NADPH oxidase isoform activation. Gastroenterology129, 2009–2031.10.1053/j.gastro.2005.09.023Search in Google Scholar PubMed

Reinehr, R., Becker, S., Braun, J., Eberle, A., Grether-Beck, S., and Häussinger, D. (2006). Endosomal acidification and activation of NADPH oxidase isoforms are upstream events in hyperosmolarity-induced hepatocyte apoptosis. J. Biol. Chem. 2006281, 23150–23166.10.1074/jbc.M601451200Search in Google Scholar PubMed

Roberts, L.R., Kurisawa, H., Bronk, S.F., Fesmier, P.J., Agellon, L.B., Leung, W.Y., Mao, F., and Gores, G.J. (1997). Cathepsin B contributes to bile salt-induced apoptosis of rat hepatocytes. Gastroenterology113, 1714–1726.10.1053/gast.1997.v113.pm9352877Search in Google Scholar PubMed

Schreiber, R. and Häussinger, D. (1995). Characterization of the swelling-induced alkalinization of endocytotic vesicles in fluorescein isothiocyanate-dextran-loaded rat hepatocytes. Biochem. J.309, 19–24.10.1042/bj3090019Search in Google Scholar PubMed PubMed Central

Schreiber, R., Stoll, B., Lang, F., and Häussinger, D. (1994). Effects of aniso-osmolarity and hydroperoxides on intracellular pH in isolated rat hepatocytes as assessed by (2′,7′)-bis(carboxyethyl)-5(6)-carboxyfluorescein and fluorescein isothiocyanate-dextran fluorescence. Biochem. J.303, 113–120.10.1042/bj3030113Search in Google Scholar

Schreiber, R., Zhang, F., and Häussinger, D. (1996). Regulation of vesicular pH in liver macrophages and parenchymal cells by ammonia and anisotonicity as assessed by fluorescein isothiocyanate-dextran fluorescence. Biochem. J.315, 385–392.10.1042/bj3150385Search in Google Scholar

Sodeman, T., Bronk, S.F., Roberts, P.J., Miyoshi, H., and Gores, G.J. (2000). Bile salts mediate hepatocyte apoptosis by increasing cell surface trafficking of CD95. Am. J. Physiol. Gastrointest. Liver Physiol.278, G992–G999.Search in Google Scholar

Sokol, R.J., Winklhofer-Roob, B.M., Devereaux, M.W., and McKim, J.M. Jr. (1995). Generation of hydroperoxides in isolated rat hepatocytes and hepatic mitochondria exposed to hydrophobic bile acids. Gastroenterology109, 1249–1256.10.1016/0016-5085(95)90585-5Search in Google Scholar

Thalhammer, T., Kaschnitz, R., Mittermayer, K., Haddad, P., and Graf, J. (1993). Organic solvents increase membrane fluidity and affect bile flow and K+ transport in rat liver. Biochem. Pharmacol.46, 1207–1215.10.1016/0006-2952(93)90469-DSearch in Google Scholar

Thomas, J.A., Buchsbaum, R.N., Zimniak, A., and Racker, E. (1979). Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry19, 2210–2218.10.1021/bi00578a012Search in Google Scholar PubMed

Timmer, A., Brammertz, D., and Grether-Beck, S. (2004). Quantification of signaling ceramides in primary keratinocytes. Planar Chromatogr. CSB93, 2–4.Search in Google Scholar

Vignais, P.V. (2002). The superoxide-generating NADPH oxidase: structural aspects and activation mechanism. Cell. Mol. Life Sci.59, 1428–1459.10.1007/s00018-002-8520-9Search in Google Scholar PubMed

Wehner, F. (1993). Taurocholate depolarizes rat hepatocytes in primary culture by increasing cell membrane Na+ conductance. Pflüger's Arch.424, 145–151.10.1007/BF00374605Search in Google Scholar PubMed

Weinman, S.A., Carruth, M.W., and Dawson, P.A. (1998). Bile acid uptake via the human apical sodium-bile acid cotransporter is electrogenic. J. Biol. Chem.273, 34691–34695.10.1074/jbc.273.52.34691Search in Google Scholar PubMed

Yerushalmi, B., Dahl, R., Devereaux, M.W., Gumpricht, E., and Sokol, R.J. (2001). Bile acid induced rat hepatocyte apoptosis is inhibited by antioxidants and blockers of the mitochondrial permeability transition. Hepatology33, 616–626.10.1053/jhep.2001.22702Search in Google Scholar PubMed

Yoshida, Y., Arimoto, K., Sato, M., Sakuragawa, N., Arima, M., and Satoyoshi, E. (1985). Reduction of acid sphingomyelinase activity in human fibroblasts induced by AY-9944 and other cationic amphiphilic drugs. J. Biochem.98, 1669–1679.10.1093/oxfordjournals.jbchem.a135438Search in Google Scholar PubMed

Zsembery, A., Strazzabosco, M., and Graf, J. (2000). Ca2+-activated Cl- channels can substitute for CFTR in stimulation of pancreatic duct bicarbonate secretion. FASEB J.14, 2345–2356.10.1096/fj.99-0509comSearch in Google Scholar PubMed

Published Online: 2007-01-29
Published in Print: 2007-02-01

©2007 by Walter de Gruyter Berlin New York

Downloaded on 26.9.2025 from https://www.degruyterbrill.com/document/doi/10.1515/BC.2007.021/html
Scroll to top button