Enhanced Na+-dependent bile salt uptake by WIF-B cells, a rat hepatoma hybrid cell line, following growth in the presence of a physiological bile salt

Abstract

Western blots immunostained for the basolateral Na ؉ -dependent plasma membrane protein, ntcp, revealed the appropriate Ϸ50-kd band in control and TC-grown cells, and confocal immunofluorescence microscopy demonstrated staining along the basolateral plasma membrane. Northern blots hybridized with a cDNA probe directed against ntcp indicated a modest TC-induced increase in mRNA levels. Reverse-transcriptase polymerase chain reaction (RT-PCR) using RNA isolated from WIF-B cells and oligonucleotide primers specific for rat ntcp or human NTCP transcripts revealed only the presence of the rat ntcp transcript. We conclude that bile salts, at concentrations normally found in mammalian portal blood, may be capable of promoting enhanced hepatocellular bile salt uptake via an increase in basolateral Na ؉ -dependent plasma membrane transport capacity. (HEPATOLOGY 1998;27:191-199.)The mammalian liver is continually exposed to low concentrations of bile salts present in portal and peripheral blood. Hepatocytes avidly take up bile salts across the basolateral plasma membrane and rapidly secrete them into bile; less than 5% of secreted bile salts are derived from de novo synthesis within the hepatocyte. 1 In rats, the postprandial bile salt concentration in the portal blood is Ϸ60 µmol/L, with taurocholate (TC) comprising up to half of the bile salt species. 2 In humans, the bile salt load reaching the liver fluctuates considerably over a 24-hour period, with portal vein plasma concentrations ranging from Ϸ14 µmol/L in the fasting state to Ϸ43 µmol/L in the postprandial state. 3 Hepatocytes are thus exposed routinely to changes in rates of bile salt delivery, and therefore must respond with variable rates of bile salt uptake across the basolateral plasma membrane. High plasma bile salt concentrations (up to 1 mmol/L) are associated with down-regulation of basolateral bile salt uptake into hepatocytes. 4 It is unclear, however, whether plasma bile salt concentrations within physiological ranges may promote bile salt uptake into hepatocytes.Difficulties are encountered when examining the physiological role of bile salts in intact animals, because many variables change simultaneously when conducting in vivo studies. A major experimental problem also is encountered when addressing normal bile salt regulation of hepatic physiology in vitro, because of the difficulty in maintaining liver preparations with stable rates of basolateral bile salt uptake. Isolated perfused rat livers maintain physiological rates of bile salt uptake and secretion for up to 3 hours, but bile flow decreases thereafter. 5 Isolated rat hepatocytes exhibit profound decreases in rates of bile salt uptake over the first 72 hours of primary culture. [6][7][8] Bile salt transport may be virtually absent in cultured hepatocyte tumor cell lines. 9 Recently, an in vitro polarized hepatocyte cell system, WIF-B cells, has been reported by Hubbard et al. 10,11 as a refinement of an existing rat hepatoma-human fibroblast cell line developed by Cassio et al. 12 WIF-B cell...

Targeting of aminopeptidase n to bile canaliculi correlates with secretory activities of the developing canalicular domain

“…Recently, another polarized hepatic cell line was generated by fusion between rat hepatoma cells and human fibroblasts. 27,28 These cells could also form BC-like structures in vitro, and exhibit various secretory functions as shown in this report.…”

“…Recently, progress has been made in obtaining better hepatic polarity by plating primary cultured hepatocytes in sandwiched collagen gels 20 and in coculture experiments. 21,22 In another approach, we and other researchers have taken advantage of differentiated hepatoma cell lines [23][24][25][26] and cell hybrids 27,28 as models to investigate the processes of canalicular differentiation. Human hepatoma cell lines can be classified into well-and poorly differentiated types based on morphology, hepatocyteassociated enzymes, hepatitis B virus genome expression, 29,30 plasma protein secretion, 23,31 and major histocompatability complex class II molecules.…”

Endotoxin-stimulated macrophages decrease bile acid uptake in WIF-B cells, a rat hepatoma hybrid cell line

“…On day 4, the cells were treated with CM, UM, or recombinant TNF-␣ for 24 hours and harvested thereafter. 18 In summary, cells grown on glass cover slips were preincubated for 10 minutes at 37°C in Na ϩ or Choline ϩ Ringer' s solutions (Na ϩ Ringer' s: 145 mmol/L NaCl, 4. Northern Blot Analyses.…”

“…20 We have previously shown that the WIF-B hepatoma cell line expresses bile salt capacity at a stable level over 12 days in culture, and is amenable to regulation of that capacity by ambient conditions. 18 Accordingly, we first tested whether exogenous recombinant cytokines were able to diminish bile acid transport across the basolateral membrane of WIF-B cells. WIF-B cells were grown for 9 days in culture and then exposed for 24 hours to TNF-␣, IL-1␤, and IL-6.…”

“…The protein primarily responsible for bile acid uptake in rat hepatocytes, ntcp, exhibits a molecular weight of approximately 50 kD and has been localized to the basolateral membrane in rat hepatocytes 26,27 and in WIF-B cells. 18 WIF-B cells are hybrid cells derived from rat hepatoma and human fibroblast cells that express only the rat ntcp gene. 18 To determine the effects of CM on ntcp gene expression, RNA was extracted from WIF-B cells and primary rat hepatocytes according to the procedures described in the Materials and Methods section.…”