Differential expression of bile salt and organic anion transporters in developing rat liver
Introduction
The vectorial transport of cholephilic compounds from sinusoidal blood plasma into bile is governed by distinct transport systems at the sinusoidal (basolateral) and canalicular plasma membrane domains of hepatocytes [1]. In rat liver, basolateral uptake systems include the Na+-taurocholate cotransport protein Ntcp (gene symbol: Slc10a1) and the organic anion transporting polypeptides Oatp1a1 (Slco1a1; previously called Oatp or Oatp1/Slc21a1), Oatp1a4 (Slco1a4; previously called Oatp2/Slc21a5) and Oatp1b2 (Slco1b2; previously called Oatp4 or Lst-1/Slc21a10) [1], [2]. While Ntcp represents the major hepatocellular uptake system for conjugated bile salts, the Oatps mediate sodium-independent uptake of a large variety of amphipathic organic compounds including numerous drugs [2]. Despite this polyspecificity some Oatps exhibit unique transport properties such as Oatp1a4, which does not transport sulfobromophthalein (BSP), but exhibits high affinity transport of the cardiac glycoside digoxin [3]. At the canalicular plasma membrane, biliary secretion of bile salts and organic anions is driven by distinct members of the ABC (ATP-binding-cassette)-transporter superfamily such as the bile salt export pump Bsep/BSEP (ABCb11/ABCB11) for monovalent bile salts [4], [5] and the multidrug resistance-associated protein Mrp2/MRP2 (ABCc2/ABCC2) for a wide range of non-bile salt organic anions including bilirubindiglucuronide and glutathione [6], [7].
While expression of transport polarity is a characteristic feature of differentiated adult hepatocytes, the ontogenesis of polarized transporter expression in developing rat liver has been investigated only partially. Thus, significant quantities of Mrp2 were observed already at day 16 of gestation [8], [9] while Ntcp and Bsep became detectable on the mRNA and protein levels shortly before birth and reached adult levels by 1 week of age [10], [11], [12]. In contrast, although low level Slco1a1 gene transcription was observed around birth [13], significant expression of Oatps occurred after birth and reached adult levels by postnatal day 30 [14], [15], [16]. Although these studies provide evidence for differential ontogenic expression of the various hepatocellular bile salt and organic anion transporter genes, the development of the fully polarized (i.e. basolateral and canalicular) bile salt and organic anion transporter phenotype of hepatocytes has not been investigated in the same livers. Therefore, we performed a detailed analysis of mRNA-levels and protein immunolocalization of Ntcp, Oatp1a1, Oatp1a4, Oatp1b2, Bsep, Mrp2 and Mrp6 (ABCc6) at various pre- and postnatal stages of developing rat liver.
Section snippets
Animals
Pregnant female and adult male (200–250 g, ∼8 weeks old) Sprague–Dawley rats (SUT:SDT) were obtained from RCC Ltd. (Füllinsdorf, Switzerland). They were kept under standard conditions in accordance with the regulations of the local animal care committee. The first day of detection of sperm plugs was identified as embryonic day 1. The sex of prenatal embryos was not determined, but all postnatal studies were performed only in male newborn or adult rats.
RNA isolation and cDNA synthesis
Total liver RNA was extracted by the Trizol®
Ntcp
Immunoreactive Ntcp was first detected at E20 (Fig. 1) when mRNA levels had reached 13% of adult levels, corresponding ∼1.88×106 copies/μg RNA (Table 2). The pattern of immunoreactivity was consistent with basolateral localization of Ntcp, although intracellular immunoreactivity was also present in some hepatocytes. By 3 h after birth, mRNA levels and the number of hepatocytes expressing Ntcp at their basolateral plasma membrane had increased dramatically (Table 2, Fig. 1, P0). Predominant
Discussion
This study demonstrates that ontogenic maturation of rat hepatocytes to a fully polarized transporter phenotype requires several weeks. It starts with the expression of the multidrug resistance associated proteins Mrp2 and Mrp6 around E16 (Fig. 6, Fig. 7, Fig. 8) and is followed by the basolateral and canalicular bile salt transporters Ntcp and Bsep, respectively, shortly before birth (Fig. 1, Fig. 5, Fig. 8). On the contrast, immunologically detectable surface expression of basolateral Oatps
Acknowledgements
This work was supported by grant #31-64140.00 from the Swiss National Science Foundation. The authors thank Dr Mathias Höchli in Central Laboratory for Electron Microscopy, University of Zurich, for his help with the confocal microscopy.
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