Transport characteristics of three fluorescent conjugated bile acid analogs in isolated rat hepatocytes and couplets

Abstract

The transport properties of three different synthetically prepared fluorescent conjugated bile acid analogs (FBA), all with the fluorophore on the side chain, were determined using isolated rat hepatocytes and hepatocyte couplets. The compounds studied were cholylglycylamidofluorescein (CGamF), cholyl(N epsilon-nitrobenzoxadiazolyl [NBD])-lysine (C-NBD-L), and chenodeoxycholyl-(N epsilon-NBD)-lysine (CDC-NBD-L). When hepatocytes were incubated at 37 degrees C with 0.3 mumol/L of FBA and 0.15 mol/L of Na+, cell fluorescence increased linearly with time at a rate (U/min) of 7.8 +/- 0.5 for CGamF, 7.2 +/- 0.3 for C-NBD-L, and 13.7 +/- 1.0 for CDC-NBD-L (mean, +/- SE; n = 40 to 90). Uptake was concentration dependent for concentrations less than 20 mumol/L and was saturable. The Michaelis constant (Km) value (mumol/L) for CGamF was 10.8, for C-NBD-L was 3.8, and for CDC-NBD-L was 3.0. In the absence of Na+, the uptake rate was decreased by 50% for CGamF and by 38% for C-NBD-L; but uptake of CDC-NBD-L was unchanged and thus Na+ independent. Cellular uptake of all three derivatives was specific to hepatocytes and was absent in several nonhepatocyte cell lines. For CGamF and C-NBD-L, both Na(+)-dependent and Na(+)-independent uptake was inhibited by 200-fold excess concentrations of cholyltaurine, dehydrocholyltaurine, and cholate, but for CDC-NBD-L, these nonfluorescent bile acids did not inhibit initial uptake. The intracellular fluorescence of CGamF was strongly pH dependent at an excitation wavelength of 495 nm, but pH independent at 440 nm excitation. In contrast, intracellular fluorescence of C-NBD-L and CDC-NBD-L was pH independent. All three FBA were secreted into the canalicular space of approximately 50% to 60% of couplets. Cellular adenosine triphosphate (ATP) depletion with either CN- or atractyloside inhibited secretion of all three FBA. The multispecific organic anion transporter (MOAT) inhibitor, chlorodinitrobenzene, blocked secretion of fluorescent MOAT substrates at a concentration of 1 mumol/L. At this concentration it did not affect secretion of the three FBA. At higher concentrations, chlorodinitrobenzene partially inhibited the canalicular secretion of CGamF but not the other two FBA. In conclusion, all three FBA are secreted by canalicular membrane bile acid transporters, but the sinusoidal uptake characteristics of CGamF and C-NBD-L are more similar than those of CDC-NBD-L to the transport properties of cholyltaurine. Therefore, C-NBD-L appears to be the best of the three for studies of conjugated trihydroxy-bile acid transport in hepatocytes.

Efficient In Vitro vectorial transport of a fluorescent conjugated bile acid analogue by polarized hepatic hybrid WIF-B and WIF-B9 cells

“…The proportion of fluorescent BC did not change with time of incubation (65%-75%) compared with that found in isolated hepatocyte doublets(50%-60% ). 19 Moreover, CGamF transport to the BC was almost abolished at 4°C, and was partially sodium-dependent as happens in normal hepatocytes in culture. 19 Our results are in good agreement with the idea that CGamF is transcellularly transported across the basolateral membrane pole and the canalicular pole via specific active transporters.…”

“…29 Moreover, this transport was temperaturedependent, saturable, specific to hepatocytes, and absent in several nonhepatic cell lines. 19 CGamF was secreted into the canalicular spaces of approximately 50% to 60% of rat hepatocyte couplets, and cellular adenosine triphosphate depletion inhibited this secretion. 19 Because of the chole- Experiments were carried out in duplicate or triplicate in three to five different cultures and a total of at least 500 BC were counted for each condition.…”

“…19 CGamF was secreted into the canalicular spaces of approximately 50% to 60% of rat hepatocyte couplets, and cellular adenosine triphosphate depletion inhibited this secretion. 19 Because of the chole- Experiments were carried out in duplicate or triplicate in three to five different cultures and a total of at least 500 BC were counted for each condition. *P Ͻ .005 when compared with control experiments.…”

“…Cells were washed twice in a HEPES-buffered saline solution with the following composition in mmol/L: NaCl, 144; KCl, 5; NaH 2 PO 4 , 2; CaCl 2 , 1.25; MgSO 4 , 1; HEPES, 10; pH 7.4. 19 Then they were incubated in that solution containing 2-5 µmol/L CGamF (prepared as a stock solution in ethanol) or 5 µmol/L FDA. To stop the incubation, dishes containing the cells were placed on ice, rinsed five times with 2 mL of cold HEPES-buffered saline solution and the coverslips were immediately mounted with this solution.…”

“…Moreover, the fluorescent bile acid was not always distributed homogeneously in the cytoplasm, but it was rather concentrated in punctuate bright structures, as reported for hepatocyte couplets. 19,29 WIF-B and WIF-B9 cells were incubated with 2 µmol/L CGamF for 2 minutes at 37°C or at 4°C. Canalicular fluorescence was dramatically reduced when the incubation was done at 4°C (Fig.…”