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. 1994 Oct 2;127(2):401–410. doi: 10.1083/jcb.127.2.401

Fluorescent choleretic and cholestatic bile salts take different paths across the hepatocyte: transcytosis of glycolithocholate leads to an extensive redistribution of annexin II

PMCID: PMC2120198  PMID: 7929584

Abstract

We have used fluorescent derivatives of the choleretic bile salts cholate and chenodeoxycholate, the cholestatic salt lithocholate, and the therapeutic agent ursodeoxycholate to visualize distinct routes of transport across the hepatocyte and delivery to the canalicular vacuole of isolated hepatocyte couplets. The cholate and chenodeoxycholate derivatives produced homogeneous intracellular fluorescence and were rapidly transported to the vacuole, while the lithocholate analogue accumulated more slowly in the canalicular vacuole and gave rise to punctate fluorescence within the cell. Fluorescent ursodeoxycholate showed punctate intracellular fluorescence against a high uniform background indicating use of both pathways. Inhibition of vesicular transport by treatment with colchicine and Brefeldin A had no effect on the uptake of any of the compounds used, but it dramatically impaired delivery of both the lithocholate and the ursodeoxycholate derivatives to the canalicular vacuole. We conclude that while the chenodeoxycholate and cholate analogues traverse the hepatocyte by a cytoplasmic route, lithocholate and ursodeoxycholate analogues are transported by vesicle-mediated transcytosis. Treatment of couplets with glycine derivatives of lithocholate and ursodeoxycholate, but not cholate or chenodeoxycholate, led to a marked relocalization of annexin II, which initially became concentrated at the basolateral membrane, then moved to a perinuclear distribution and finally to the apical membrane as the incubation progressed. This suggests that lithocholate and ursodeoxycholate treatment leads to a rapid induction of transcytosis and that annexin II exchange occurs upon membrane fusion at all stages of the hepatocyte transcytotic pathway. These results indicate that isolated hepatocyte couplets may provide an inducible model system for the study of vesicle-mediated transcytosis.

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Selected References

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  1. Ali S. M., Geisow M. J., Burgoyne R. D. A role for calpactin in calcium-dependent exocytosis in adrenal chromaffin cells. Nature. 1989 Jul 27;340(6231):313–315. doi: 10.1038/340313a0. [DOI] [PubMed] [Google Scholar]
  2. Barr V. A., Hubbard A. L. Newly synthesized hepatocyte plasma membrane proteins are transported in transcytotic vesicles in the bile duct-ligated rat. Gastroenterology. 1993 Aug;105(2):554–571. doi: 10.1016/0016-5085(93)90734-t. [DOI] [PubMed] [Google Scholar]
  3. Clerc P., Sansonetti P. J. Entry of Shigella flexneri into HeLa cells: evidence for directed phagocytosis involving actin polymerization and myosin accumulation. Infect Immun. 1987 Nov;55(11):2681–2688. doi: 10.1128/iai.55.11.2681-2688.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Coleman R. Bile salts and biliary lipids. Biochem Soc Trans. 1987 Dec;15 (Suppl):68S–80S. [PubMed] [Google Scholar]
  5. Crawford J. M., Berken C. A., Gollan J. L. Role of the hepatocyte microtubular system in the excretion of bile salts and biliary lipid: implications for intracellular vesicular transport. J Lipid Res. 1988 Feb;29(2):144–156. [PubMed] [Google Scholar]
  6. Donaldson J. G., Kahn R. A., Lippincott-Schwartz J., Klausner R. D. Binding of ARF and beta-COP to Golgi membranes: possible regulation by a trimeric G protein. Science. 1991 Nov 22;254(5035):1197–1199. doi: 10.1126/science.1957170. [DOI] [PubMed] [Google Scholar]
  7. Drust D. S., Creutz C. E. Aggregation of chromaffin granules by calpactin at micromolar levels of calcium. Nature. 1988 Jan 7;331(6151):88–91. doi: 10.1038/331088a0. [DOI] [PubMed] [Google Scholar]
  8. Emans N., Gorvel J. P., Walter C., Gerke V., Kellner R., Griffiths G., Gruenberg J. Annexin II is a major component of fusogenic endosomal vesicles. J Cell Biol. 1993 Mar;120(6):1357–1369. doi: 10.1083/jcb.120.6.1357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Frimmer M., Ziegler K. The transport of bile acids in liver cells. Biochim Biophys Acta. 1988 Feb 24;947(1):75–99. doi: 10.1016/0304-4157(88)90020-2. [DOI] [PubMed] [Google Scholar]
  10. Gautam A., Ng O. C., Boyer J. L. Isolated rat hepatocyte couplets in short-term culture: structural characteristics and plasma membrane reorganization. Hepatology. 1987 Mar-Apr;7(2):216–223. doi: 10.1002/hep.1840070203. [DOI] [PubMed] [Google Scholar]
  11. Groothuis G. M., Hardonk M. J., Keulemans K. P., Nieuwenhuis P., Meijer D. K. Autoradiographic and kinetic demonstration of acinar heterogeneity of taurocholate transport. Am J Physiol. 1982 Dec;243(6):G455–G462. doi: 10.1152/ajpgi.1982.243.6.G455. [DOI] [PubMed] [Google Scholar]
  12. Handel S. E., Rennison M. E., Wilde C. J., Burgoyne R. D. Annexin II (calpactin I) in the mouse mammary gland: immunolocalization by light- and electron microscopy. Cell Tissue Res. 1991 Jun;264(3):549–554. doi: 10.1007/BF00319044. [DOI] [PubMed] [Google Scholar]
  13. Harder T., Gerke V. The subcellular distribution of early endosomes is affected by the annexin II2p11(2) complex. J Cell Biol. 1993 Dec;123(5):1119–1132. doi: 10.1083/jcb.123.5.1119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hayakawa T., Ng O. C., Ma A., Boyer J. L., Cheng O. Taurocholate stimulates transcytotic vesicular pathways labeled by horseradish peroxidase in the isolated perfused rat liver. Gastroenterology. 1990 Jul;99(1):216–228. doi: 10.1016/0016-5085(90)91251-z. [DOI] [PubMed] [Google Scholar]
  15. Haüssinger D. Nitrogen metabolism in liver: structural and functional organization and physiological relevance. Biochem J. 1990 Apr 15;267(2):281–290. doi: 10.1042/bj2670281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Helms J. B., Rothman J. E. Inhibition by brefeldin A of a Golgi membrane enzyme that catalyses exchange of guanine nucleotide bound to ARF. Nature. 1992 Nov 26;360(6402):352–354. doi: 10.1038/360352a0. [DOI] [PubMed] [Google Scholar]
  17. Hubbard A. L. Targeting of membrane and secretory proteins to the apical domain in epithelial cells. Semin Cell Biol. 1991 Dec;2(6):365–374. [PubMed] [Google Scholar]
  18. Hugentobler G., Meier P. J. Multispecific anion exchange in basolateral (sinusoidal) rat liver plasma membrane vesicles. Am J Physiol. 1986 Nov;251(5 Pt 1):G656–G664. doi: 10.1152/ajpgi.1986.251.5.G656. [DOI] [PubMed] [Google Scholar]
  19. Hunziker W., Whitney J. A., Mellman I. Selective inhibition of transcytosis by brefeldin A in MDCK cells. Cell. 1991 Nov 1;67(3):617–627. doi: 10.1016/0092-8674(91)90535-7. [DOI] [PubMed] [Google Scholar]
  20. Jungermann K., Katz N. Functional specialization of different hepatocyte populations. Physiol Rev. 1989 Jul;69(3):708–764. doi: 10.1152/physrev.1989.69.3.708. [DOI] [PubMed] [Google Scholar]
  21. Kaplan K. B., Swedlow J. R., Varmus H. E., Morgan D. O. Association of p60c-src with endosomal membranes in mammalian fibroblasts. J Cell Biol. 1992 Jul;118(2):321–333. doi: 10.1083/jcb.118.2.321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kast C., Stieger B., Winterhalter K. H., Meier P. J. Hepatocellular transport of bile acids. Evidence for distinct subcellular localizations of electrogenic and ATP-dependent taurocholate transport in rat hepatocytes. J Biol Chem. 1994 Feb 18;269(7):5179–5186. [PubMed] [Google Scholar]
  23. Knutton S., Baldwin T., Williams P. H., McNeish A. S. Actin accumulation at sites of bacterial adhesion to tissue culture cells: basis of a new diagnostic test for enteropathogenic and enterohemorrhagic Escherichia coli. Infect Immun. 1989 Apr;57(4):1290–1298. doi: 10.1128/iai.57.4.1290-1298.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lake J. R., Licko V., Van Dyke R. W., Scharschmidt B. F. Biliary secretion of fluid-phase markers by the isolated perfused rat liver. Role of transcellular vesicular transport. J Clin Invest. 1985 Aug;76(2):676–684. doi: 10.1172/JCI112021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lowe P. J., Kan K. S., Barnwell S. G., Sharma R. K., Coleman R. Transcytosis and paracellular movements of horseradish peroxidase across liver parenchymal tissue from blood to bile. Effects of alpha-naphthylisothiocyanate and colchicine. Biochem J. 1985 Jul 15;229(2):529–537. doi: 10.1042/bj2290529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Meier P. J. The bile salt secretory polarity of hepatocytes. J Hepatol. 1989 Jul;9(1):124–129. doi: 10.1016/0168-8278(89)90085-8. [DOI] [PubMed] [Google Scholar]
  27. Mills C. O., Rahman K., Coleman R., Elias E. Cholyl-lysylfluorescein: synthesis, biliary excretion in vivo and during single-pass perfusion of isolated perfused rat liver. Biochim Biophys Acta. 1991 Dec 6;1115(2):151–156. doi: 10.1016/0304-4165(91)90024-b. [DOI] [PubMed] [Google Scholar]
  28. Moldéus P., Högberg J., Orrenius S. Isolation and use of liver cells. Methods Enzymol. 1978;52:60–71. doi: 10.1016/s0076-6879(78)52006-5. [DOI] [PubMed] [Google Scholar]
  29. Müller M., Ishikawa T., Berger U., Klünemann C., Lucka L., Schreyer A., Kannicht C., Reutter W., Kurz G., Keppler D. ATP-dependent transport of taurocholate across the hepatocyte canalicular membrane mediated by a 110-kDa glycoprotein binding ATP and bile salt. J Biol Chem. 1991 Oct 5;266(28):18920–18926. [PubMed] [Google Scholar]
  30. Nishida T., Gatmaitan Z., Che M., Arias I. M. Rat liver canalicular membrane vesicles contain an ATP-dependent bile acid transport system. Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6590–6594. doi: 10.1073/pnas.88.15.6590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Phillips M. J., Oshio C., Miyairi M., Katz H., Smith C. R. A study of bile canalicular contractions in isolated hepatocytes. Hepatology. 1982 Nov-Dec;2(6):763–768. doi: 10.1002/hep.1840020603. [DOI] [PubMed] [Google Scholar]
  32. Ruetz S., Fricker G., Hugentobler G., Winterhalter K., Kurz G., Meier P. J. Isolation and characterization of the putative canalicular bile salt transport system of rat liver. J Biol Chem. 1987 Aug 15;262(23):11324–11330. [PubMed] [Google Scholar]
  33. Sakisaka S., Ng O. C., Boyer J. L. Tubulovesicular transcytotic pathway in isolated rat hepatocyte couplets in culture. Effect of colchicine and taurocholate. Gastroenterology. 1988 Sep;95(3):793–804. doi: 10.1016/s0016-5085(88)80030-1. [DOI] [PubMed] [Google Scholar]
  34. Sarafian T., Pradel L. A., Henry J. P., Aunis D., Bader M. F. The participation of annexin II (calpactin I) in calcium-evoked exocytosis requires protein kinase C. J Cell Biol. 1991 Sep;114(6):1135–1147. doi: 10.1083/jcb.114.6.1135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Stieger B., O'Neill B., Meier P. J. ATP-dependent bile-salt transport in canalicular rat liver plasma-membrane vesicles. Biochem J. 1992 May 15;284(Pt 1):67–74. doi: 10.1042/bj2840067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Stolz A., Takikawa H., Ookhtens M., Kaplowitz N. The role of cytoplasmic proteins in hepatic bile acid transport. Annu Rev Physiol. 1989;51:161–176. doi: 10.1146/annurev.ph.51.030189.001113. [DOI] [PubMed] [Google Scholar]
  37. Weinman S. A., Graf J., Boyer J. L. Voltage-driven, taurocholate-dependent secretion in isolated hepatocyte couplets. Am J Physiol. 1989 May;256(5 Pt 1):G826–G832. doi: 10.1152/ajpgi.1989.256.5.G826. [DOI] [PubMed] [Google Scholar]
  38. Wilton J. C., Chipman J. K., Lawson C. J., Strain A. J., Coleman R. Periportal- and perivenous-enriched hepatocyte couplets: differences in canalicular activity and in response to oxidative stress. Biochem J. 1993 Jun 15;292(Pt 3):773–779. doi: 10.1042/bj2920773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wilton J. C., Coleman R., Lankester D. J., Chipman J. K. Stability and optimization of canalicular function in hepatocyte couplets. Cell Biochem Funct. 1993 Sep;11(3):179–185. doi: 10.1002/cbf.290110305. [DOI] [PubMed] [Google Scholar]
  40. Zimmerli B., Valantinas J., Meier P. J. Multispecificity of Na+-dependent taurocholate uptake in basolateral (sinusoidal) rat liver plasma membrane vesicles. J Pharmacol Exp Ther. 1989 Jul;250(1):301–308. [PubMed] [Google Scholar]

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