Transport of fluorescent bile acids by the isolated perfused rat liver: Kinetics, sequestration, and mobilization

Abstract

Hepatocyte transport of six fluorescent bile acids containing nitrobenzoxadiazolyl (NBD) or a fluorescein derivative on the side chain was compared with that of natural bile acids using the single-pass perfused rat liver. Compounds were infused at 40 nmol/g liver min for 15 minutes; hepatic uptake and biliary recovery were measured; fractional extraction, intrinsic basolateral clearance, and sequestration (nonrecovery after 45 minutes of additional perfusion) were calculated. Fluorescent bile acids were efficiently extracted during the first 3 minutes (70%-97%), but net extraction decreased with time mostly because of regurgitation into the perfusate. For cholylglycine and ursodeoxycholylglycine (UDC-glycine), extraction was 94% to 99%, and regurgitation did not occur. Intrinsic hepatic clearance of fluorescent bile acids (2-7 mL/g liver x min) was lower than that of cholylglycine (9.0 +/- 0.6; mean +/- SD) and UDC-glycine (21.4 +/- 0.4). Sequestration at 60 minutes was 8% to 26% for fluorescent bile acids with a cholyl moiety (cholylglycylaminofluorescein [CGamF], cholyllysylfluorescein [C-L-F], cholyl-[N epsilon-NBD]-lysine [C-L-NBD], and cholylaminofluorescein [CamF]), 32% for ursodeoxycholylaminofluorescein (UDCamF), and 88% for ursodeoxycholyl-(N epsilon-NBD)lysine (UDC-L-NBD). Cholylglycine and UDC-glycine had <3% retention. Biliary secretion of sequestered UDCamF, but not of UDC-L-NBD, was induced by adding dibutyryl cyclic adenosine monophosphate (DBcAMP) to the perfusate, possibly by translocation to the canaliculus of pericanalicular vesicles containing fluorescent bile acids. Biliary secretion of UDC-L-NBD, but not of UDCamF, was induced by adding cholyltaurine or UDC-taurine, possibly by inhibition of binding to intracellular constituents or of transport into organelles. It is concluded that fluorescent bile acids are efficiently transported across the basolateral membrane, but in contrast to natural conjugated bile acids, are sequestered in the hepatocyte (UDC derivatives > cholyl derivatives). Two modes of hepatic sequestration of fluorescent bile acids were identified. Fluorescent bile acids may be useful to characterize sequestration processes during bile acid transport through the hepatocyte.

Small hepatocytes in culture develop polarized transporter expression and differentiation

“…Ntcp) and bile-salt secretory (i.e. Bsep) systems at the basolateral and canalicular surface domains, respectively (Holzinger et al, 1998;Holzinger et al, 1997). Hence, the data are fully consistent with the development of a functionally competent transport polarization in longterm-cultured small hepatocytes.…”

“…2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) was from Fluka (Buchs, Switzerland). The fluorescent bile salt cholylglycyl-fluorescein (Holzinger et al, 1998;Holzinger et al, 1997) was a gift from A. F. Hofmann (University of California, San Diego, CA). All other chemicals and reagents were of analytical grade and were readily available from commercial sources.…”

Interaction of HIV protease inhibitors with OATP1B1, 1B3, and 2B1

“…In this context, we recently reported on the characterization of the hepatic disposition of the bile salt analog cholyl-glycylamido-fluorescein (CGamF) in animal and human hepatocytes (Ye et al, 2008;Ye et al, 2009). CGamF was synthesized by conjugation of 5-aminofluorescein with the carboxylic group of the natural bile acid cholylglycine yielding a fluorescent bile salt analogue (Holzinger et al, 1998). The utility of CGamF as a convenient fluorescent probe simulating bile salt transport in various in vitro and in vivo models of different species has been demonstrated previously (Maglova et al, 1995;Bravo et al, 1998;Holzinger et al, 1998;Mita et al, 2006).…”

“…CGamF was synthesized by conjugation of 5-aminofluorescein with the carboxylic group of the natural bile acid cholylglycine yielding a fluorescent bile salt analogue (Holzinger et al, 1998). The utility of CGamF as a convenient fluorescent probe simulating bile salt transport in various in vitro and in vivo models of different species has been demonstrated previously (Maglova et al, 1995;Bravo et al, 1998;Holzinger et al, 1998;Mita et al, 2006). Mita et al (2006) showed that CGamF as well as other fluorescent bile salt analogues were transported across LLC-PK1 cells co-expressing NTCP (SLC10A1) and BSEP (ABCB11), albeit at a substantially lower rate than the natural bile salt taurocholate.…”

Síntesis de Derivados Fotoactivos de Ácidos Biliares para Aplicaciones Biológicas

“…El compuesto final que se obtuvo está descrito y es conocido como colilamidofluoresceína (CamF). [72,[79][80] Brevemente, la ruta sintética descrita para su preparación se basó en la condensación de la 4-aminofluoresceína comercial con el ácido cólico utilizando Así pues, partiendo de ácido cólico se puede preparar una familia de intermedios versátiles que permitan la conjugación con otros fluoróforos.…”