Parallel decrease of Na+ -taurocholate cotransport and its encoding mRNA in primary cultures of rat hepatocytes

Abstract

We investigated the molecular mechanism underlying the progressive loss of Na(+)-dependent bile salt uptake in primary cultured rat hepatocytes. A specific cDNA probe was used to quantitate the levels of mRNA encoding the Na(+)-taurocholate-cotransporting polypeptide at various culture times. Hepatocytes were cultured on collagen in the presence of insulin (10(-7) mol/L), dexamethasone (10(-7) mol/L) and 10% fetal calf serum for up to 72 hr. During this time period the dissociation constant of Na(+)-dependent taurocholate uptake remained stable (19 to 39 mumol/L), whereas the maximum velocity values decreased from 100% at 3 hr to 55%, 22% and 4% at 24, 48 and 72 hr, respectively. Concomitantly the levels of the Na(+)-taurocholate-cotransporting polypeptide mRNA also decreased from 100% at 3 hr to 41%, 24% and 4% at the later time points. In contrast, Northern hybridization with complementary DNA probes for three common housekeeping gene products revealed a 1.8- to 3.4-fold increase in the levels of mRNA encoding the alpha-subunit of the Na+K(+)-ATPase, beta-actin and glycerol-3-phosphate dehydrogenase. These data indicate that the loss of Na(+)-dependent bile salt uptake in primary cultures of rat hepatocytes is caused by decreased levels of its specific mRNA. Hence the studies further confirm that without specific measures (primary) cultured rat hepatocytes reverse their liver-specific phenotype to a more fetal pattern of gene expression.

Protein-protein interactions and membrane localization of the human organic solute transporter

“…Na ϩ -dependent and -independent taurocholate (TC) influx assays were performed in 12-well plates or using a Transwell filter culture system as previously described (15,18). Briefly, for the Transwell filter system (Costar, Cambridge, MA), transfected and untransfected cells were grown to confluence for 4 -5 days on 0.45-m pore size Transwell filter inserts.…”

The Hepatitis B Virus Receptor

“…Its expression is downregulated in most human liver diseases and upregulated in a few others (Bechmann et al 2013). After isolation, the expression of NTCP is rapidly downregulated in primary hepatocytes from rats (Liang et al 1993) (Rippin et al 2001) and treeshrews (Konig et al 2014, Yan et al 2012). These observations may help explain why malignant hepatoma cells do not support infection with HBV and HDV and why primary hepatocytes are susceptible to HBV only for a few days after isolation.…”

Basal efflux of bile acids contributes to drug-induced bile acid-dependent hepatocyte toxicity in rat sandwich-cultured hepatocytes

“…For example, the mRNA expression levels of BA uptake transporters (i.e., Ntcp and Oatp1a1) are dramatically diminished in both rat primary hepatocytes (Liang et al, 1993) and SCHs (Tchaparian et al, 2011). The downregulation of uptake transporters supports our rationale for employing an extremely high concentration range for total BAs (227-681 lM) compared with BA levels observed in clinical situations.…”