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Parallel decrease of Na(+)-taurocholate cotransport and its encoding mRNA in primary cultures of rat hepatocytes
D Liang1, B Hagenbuch, B Stieger
1Department of Medicine, University Hospital, Zurich, Switzerland.
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.
Insights
Primary cultured rat hepatocytes lose bile salt uptake due to reduced Na(+)-taurocholate-cotransporting polypeptide mRNA levels. This indicates a shift towards a fetal gene expression pattern in vitro.
Area of Science:
- Hepatology
- Molecular Biology
- Cell Biology
Background:
- Primary cultured rat hepatocytes exhibit a progressive loss of Na(+)-dependent bile salt uptake over time.
- Understanding the molecular mechanisms behind this loss is crucial for liver research.
Purpose of the Study:
- To investigate the molecular basis for the decline in Na(+)-dependent bile salt uptake in primary rat hepatocyte cultures.
- To determine the role of Na(+)-taurocholate-cotransporting polypeptide (NTCP) mRNA levels in this process.
Main Methods:
- Primary rat hepatocytes were cultured on collagen with insulin, dexamethasone, and fetal calf serum for up to 72 hours.
- Na(+)-dependent taurocholate uptake kinetics were measured.
- mRNA levels for NTCP and housekeeping genes (Na+K(+)-ATPase, beta-actin, glycerol-3-phosphate dehydrogenase) were quantified using cDNA probes and Northern hybridization.
Main Results:
- While the dissociation constant for taurocholate uptake remained stable, the maximum velocity decreased significantly over 72 hours.
- Concomitantly, NTCP mRNA levels dropped from 100% at 3 hours to 4% at 72 hours.
- mRNA levels for housekeeping genes increased, indicating a dedifferentiation process.
Conclusions:
- The loss of Na(+)-dependent bile salt uptake in cultured rat hepatocytes is directly caused by decreased levels of specific NTCP mRNA.
- Cultured hepatocytes, without specific interventions, revert to a more fetal gene expression pattern, losing their differentiated liver-specific phenotype.
