Different pathways of canalicular secretion of sulfated and non-sulfated fluorescent bile acids: a study in isolated hepatocyte couplets and TR− rats
Section snippets
Materials and Methods
CLF and LLF were synthesized as described previously, with minor modifications 22., 23.. Briefly, the synthetic method used excess N-ε-CBZ-1-lysine methyl ester hydrochloride and either cholic acid or lithocholic acid via N-ethoxycarbonyl-2 ethoxy-1, 2-dihydroquinolone (EEDQ) to give cholyl-lysine or lithocholyl-lysine. CLF or LLF was then prepared using equimolar amounts of choly-lysine or lithocholyl-lysine and fluorescein isothiocyanate (FITC), in bicarbonate buffer, pH 9.5. Purities of
Results
Fig. 2 shows cVA of CLF, LLF and sLLF in isolated hepatocyte couplets. The kinetic behaviors of non-sulfated (CLF and LLF) and sulfated (sLLF) bile acids were strikingly different. Whereas a plateau in cVA of non-sulfated bile acids was reached at 20–40 min of bile acid exposure, the sulfated bile acid studied did not appear to approach maximal canalicular accumulationuntil 90 min.
Representative examples of couplets treated with CLF, LLF and sLLF are shown in Fig. 3. Whereas sLLF exhibited a
Discussion
There have been numerous attempts to conjugate bile acids to fluorescent probes without affecting their physiological properties as bile acids 29., 30., 31.. We have synthesized LFCBAA and have shown that CLF and LLF have similar physical properties to their natural bile salt congeners 22., 23.. Moreover, CLF has been shown to have similar properties to cholylglycine when used as a probe in various transport studies 16., 17., 18., 19.. We have shown that CLF is closely similar to cholyglycine
Acknowledgements
Dr P. Milkiewicz was supported by an EASL Fellowship and the Midlands Gastroenterological Society. Dr M.G. Roma was supported by a Royal Society/CONICET Fellowship.
Portions of this work have been presented previously in abstract form at the 32nd EASL Meeting in London, UK, April, 1997.
References (32)
- et al.
Substrate specificity of sinusoidal bile acid and organic anion uptake systems in rat and human liver
Hepatology
(1997) - et al.
Hepatobiliary secretion of organic compounds; molecular mechanisms of membrane transport
Biochim Biophys Acta
(1995) - et al.
cDNA cloning of the hepatocyte canalicular isoform of the multidrug resistance protein, cMrp, reveals a novel conjugate export pump deficient in hyperbilirubinemic mutant rats
J Biol Chem
(1996) - et al.
Hepatocellular transport of bile acids: evidence for distinct subcellular localisations of electrogenic and ATP dependent taurocholate transport in rat hepatocytes
J Biol Chem
(1994) - et al.
Transport characteristics of three fluorescent conjugated bile acid analogs in isolated rat hepatocytes and couplets
Hepatology
(1995) - et al.
Fluorescent bile acid derivatives: relationship between chemical structure and hepatic and intestinal transport in the rat
Hepatology
(1997) - et al.
Biliary excretion of fluorescent cholephiles in hepatocyte couplets: an in vitro model for hepatobiliary and hepatotoxicity studies
Toxicol in Vitro
(1990) - et al.
Plasma clearance of cholyl-lysyl-fluorescein: a pilot study in humans
J Hepatol
(1997) - et al.
Hepatobiliary excretion of organic anions in double-mutant rats with a combination of defective canalicular transport and uridine 5′-diphosphate-glucuronyltransferase deficiency
Gastroenterology
(1987) - et al.
Cholyl lysyl fluorescein: synthesis, biliary excretion in vivo and during single-pass perfusion of isolated perfused rat liver
Biochim Biophys Acta
(1991)
Synthesis, physical and biological properties of lithocholyl lysyl fluorescein: a fluorescent monohydroxy bile salt analogue with cholestatic properties
Biochim Biophys Acta
Synthesis of sulfate esters of lithocholic acid, glycolithocholic acid and taurolithocholic acid with sulfur trioxide-triethylamine
J Lipid Res
An improved procedure for the synthesis of glycine and taurine conjugates of bile acids
J Lipid Res
Fluorescent derivatives of bile salts. III. Uptake of 7 beta-NBD-NCT into isolated hepatocytes by the transport system for cholyltaurine
J Lipid Res
Molecular aspects of hepatobiliary transport
Am J Physiol
Expression cloning of a rat liver Na(+)-independent organic anion transporter
Proc Natl Acad Sci
Cited by (0)
- *
Dr. C.O. Mills and Dr P. Milkiewicz contributed equally to this work.