Characterization of transport in isolated human hepatocytes: A study with the bile acid taurocholic acid, the uncharged ouabain and the organic cations vecuronium and rocuronium

doi:10.1016/0006-2952(94)90255-0

Biochemical Pharmacology

Volume 47, Issue 12, 15 June 1994, Pages 2193-2200

https://doi.org/10.1016/0006-2952(94)90255-0 Get rights and content

Abstract

The uptake and efflux of three categories of substrates were measured in isolated human hepatocytes and compared to those in rat hepatocytes. In addition, the extent to which the in vitro experiments quantitatively reflect liver function in vivo in both species was investigated. The anionic bile acid taurocholic acid was taken up by isolated human hepatocytes at a considerably lower rate than observed in isolated rat hepatocytes. Taurocholic acid uptake both in human hepatocytes and in liver plasma membrane vesicles showed sodium dependency. The uptake rate of taurocholic acid in isolated hepatocytes of both species was quantitatively compatible with the reported liver clearance of the bile acid in vivo. Ouabain uptake rate in isolated human hepatocytes was lower than in rat hepatocytes. This species difference was in accordance with pharmacokinetic studies in vivo on hepatic clearance of ouabain in man and rat. Uptake of vecuronium into human hepatocytes was about a factor of 10 lower than that in rat hepatocytes. Uptake into and efflux from human hepatocytes was comparable for the two short acting muscle relaxants vecuronium and rocuronium. Since distribution to the liver is considered to be a major factor in termination of action of vecuronium and rocuronium these observations were in line with the human pharmacokinetic profiles. In conclusion, the uptake rate of the studied model compounds in human hepatocytes appeared to be lower than that in rat hepatocytes. These observed transport rates reflected the relative hepatic transport rates observed in these species in the intact organism, but the absolute values in both species for some substrates may have been somewhat lower than calculated from in vivo data. It is concluded that transport studies in isolated hepatocytes are suitable for comparative drug transport studies, but are less precise in the prediction of quantitative membrane transport.

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NTCP is predominantly expressed at the basolateral membrane in the liver and is mostly involved in Na+-dependent uptake of bile acids from the portal blood circulation. To date, the Na+-dependent uptake via hNTCP has been corroborated by various experimental systems with isolated perfused liver [45], isolated hepatocytes [20,23,24,46], isolated membrane vesicles [47] and hNTCP expressed in mammalian cultured cells [15–17] or heterologously in X. laevis oocytes [22,43]. As far as we are aware, however, there is no direct evidence for the electrogenic nature of bile acid transport via hNTCP.
The Na⁺/taurocholate cotransporting polypeptide (NTCP) plays a major role in Na⁺-dependent bile acid uptake into hepatocytes. The purpose of the present study was to establish the heterologous expression of human NTCP (hNTCP) in Xenopus laevis oocytes and to elucidate whether the transport of bile acid via hNTCP is electrogenic using electrophysiological techniques. First, we evaluated the uptake of taurocholate (TCA) by hNTCP heterologously expressed in Xenopus oocytes utilizing [³H]-labeled TCA. The uptake of 1.2 μM TCA by cRNA-injected oocytes increased more than 100-fold compared to H₂O-injected oocytes, indicating that hNTCP is robustly expressed in the oocytes. hNTCP-mediated transport of TCA is saturable with a Michaelis constant of 10.5 ± 2.9 μM. The Na⁺-activation kinetics describing the relationship between the concentration of Na⁺ and the magnitude of the TCA uptake rate by hNTCP were sigmoidal with a Hill coefficient of 2.3 ± 0.4, indicating the involvement of more than one Na⁺ in the transport process. Ntcp in primary cultured hepatocytes from rats exhibited similar Na⁺-activation kinetics of TCA uptake rate with a Hill coefficient of 1.9 ± 0.1, suggesting that hNTCP could be expressed properly in the oocytes and exhibit the electrogenic property of Na⁺-coupled TCA transport. The transport of TCA via hNTCP was subsequently determined in the oocytes by the inward currents induced via TCA uptake under voltage (−50 mV). Two hundred micromolar TCA induced significant inward currents that were entirely abolished by the substitution of Na⁺ with N-methyl-d-glucamine (NMDG) in the perfusate, indicating that the TCA-induced currents were obligatorily dependent on the presence of Na⁺. The TCA-induced currents were saturable, and the substrate concentration needed for half-maximal induction of the current was consistent with the Michaelis constant. Transportable substrates, such as rosuvastatin and fluvastatin, also induced currents. These results in the hNTCP heterologously expressed in Xenopus oocytes directly demonstrated that hNTCP is an electrogenic Na⁺-dependent transporter.
The SLC10 carrier family: Transport functions and molecular structure
2012, Current Topics in Membranes
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In the human hepatocyte cell model, the apparent kinetic parameters of affinity (Km) and capacity (Vmax) for TC uptake differed from data of rat Ntcp. A direct comparison of both species showed that in human hepatocytes, the Km was 2–3 times higher (Sandker et al., 1994) or quite similar (Azer & Stacey, 1993) but Vmax was consistently much lower than for TC transport in rat hepatocytes (e.g. Vmax 300 pmol/min/106 human cells versus 1800 pmol/min/106 rat cells), reflecting less-efficient uptake of the BS in human hepatocytes (Azer & Stacey, 1993; Sandker et al., 1994). In NTCP transfected cells such as HeLa cell, COS cells as well as in X. laevis oocytes Km for TC transport was, however, always lower (Table 1) than in human hepatocytes (Hagenbuch & Meier, 1994; Kim et al., 1999), reflecting higher affinity in transfected cell systems.
The SLC10 family represents seven genes containing 1–12 exons that encode proteins in humans with sequence lengths of 348–477 amino acids. Although termed solute carriers (SLCs), only three out of seven (i.e. SLC10A1, SLC10A2, and SLC10A6) show sodium-dependent uptake of organic substrates across the cell membrane. These include the uptake of bile salts, sulfated steroids, sulfated thyroidal hormones, and certain statin drugs by SLC10A1 (Na⁺-taurocholate cotransporting polypeptide (NTCP)), the uptake of bile salts by SLC10A2 (apical sodium-dependent bile acid transporter (ASBT)), and uptake of sulfated steroids and sulfated taurolithocholate by SLC10A6 (sodium-dependent organic anion transporter (SOAT)). The other members of the family are orphan carriers not all localized in the cell membrane. The name “bile acid transporter family” arose because the first two SLC10 members (NTCP and ASBT) are carriers for bile salts that establish their enterohepatic circulation. In recent years, information has been obtained on their 2D and 3D membrane topology, structure–transport relationships, and on the ligand and sodium-binding sites. For SLC10A2, the putative 3D morphology was deduced from the crystal structure of a bacterial SLC10A2 analog, ASBT_NM. This information was used in this chapter to calculate the putative 3D structure of NTCP. This review provides first an introduction to recent knowledge about bile acid synthesis and newly found bile acid hormonal functions, and then describes step-by-step each individual member of the family in terms of expression, localization, substrate pattern, as well as protein topology with emphasis on the three functional SLC10 carrier members.
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However, the Vmax for NaFluo in rat hepatocytes was approximately 13‐fold higher relative to human hepatocytes. Several fold higher Vmax values in rat versus human hepatocytes have previously been reported for TC, EG, ouabain, and the two short acting muscle relaxants vecuronium and rocuronium.29,35 The uptake of NaFluo was examined in presence and absence of sodium to determine the possible contribution of sodium‐dependent uptake by Ntcp/NTCP in rat and human hepatocytes (Figure 2a and 2c).
The aim of this study was to characterize the in vitro hepatic uptake kinetics of sodium fluorescein (NaFluo) and identify the transporters involved. NaFluo exhibited saturable uptake kinetics in suspended rat and human hepatocytes as reflected by K_m values of 22.5 and 14.1 µM, and V_max values of 98.3 and 5.8 pmol/(million cells • min), respectively. Coincubation with known inhibitors (e.g. rifampicin) of organic anion transporting polypeptide (OATP/Oatp; SLCO gene family) significantly decreased NaFluo uptake in hepatocytes. In contrast, neither inhibitors/substrates of the organic cation transporter or organic anion transporter family nor depletion of extracellular sodium resulted in significant inhibition of NaFluo uptake. To explore the contribution of individual uptake transporters, NaFluo uptake was determined in Chinese hamster ovary cells transfected with OATP1B1, OATP1B3, and OATP2B1. Transporter‐mediated uptake of NaFluo was observed in OATP1B1‐ and OATP1B3‐transfected cells (K_m = 4.2 and 10.9 µM; V_max = 30.9 and 135 [pmol/(mg protein • min)], respectively). NaFluo can be used as a probe substrate to study Oatp/OATP1B‐mediated drug interactions in fluorescence‐based in vitro transport assays of rat and human liver. Labeling of drugs or bile salts with a fluorescein moiety can be expected to result in fluorescent conjugates with substantially altered hepatic uptake characteristics as compared with the unconjugated compounds. © 2011 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:5018–5030, 2011
Determination of OATP-, NTCP- and OCT-mediated substrate uptake activities in individual and pooled batches of cryopreserved human hepatocytes
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As there are interspecies-differences in drug metabolizing enzymes and in the substrate affinity profiles of transporters (Ishizuka et al., 1999; Ye et al., 2010), hepatocytes of human origin are required to make relevant extrapolations from in vitro drug transport data towards the in vivo situation in human. A few studies have been conducted illustrating the feasibility of plated and suspended human hepatocytes to study hepatic drug transport activities (Sandker et al., 1994; Olinga et al., 1998; Shitara et al., 2003; Yamashiro et al., 2006; Bi et al., 2006). However, the chronic limitation in availability of freshly-isolated human hepatocytes may have precluded more systematic studies on transporter activities in these cells.
While the utility of cryopreserved human hepatocyte suspensions (CHHS) for in vitro drug metabolism assays has been established, less is known about the effects of cryopreservation on transporter activity in human hepatocytes. In the present study, the activities of NTCP (sodium taurocholate co-transporting polypeptide; SLC10A1), as well as of the hepatic OATP (organic anion transporting polypeptide; SLCO gene family) and OCT (organic cation transporter; SLC22A) isoforms were assessed in 14 individual and four pooled batches of CHHS. For comparative purposes, substrate accumulation rates were also measured in sandwich-cultured human hepatocytes.
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Inhibition of Na<sup>+</sup>-taurocholate co-transporting polypeptide-mediated bile acid transport by cholestatic sulfated progesterone metabolites
2010, Journal of Biological Chemistry
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The IC50 and Ki values for the P4-S compounds studied are similar to the total levels of PM4-S + PM5-S observed in third trimester pregnant serum (6.2 μm; Fig. 6) and within the documented total P4-S levels in the serum of ICP patients (21), typifying the physiological importance of pregnancy levels of P4-S in women with raised serum bile acid levels. In agreement with previous reports (35), our study has revealed that Na+-dependent uptake is responsible for the majority of temperature-sensitive bile acid influx into the hepatocyte that is mediated by the NTCP transporter (Fig. 1C). We showed that PM4-S and PM5-S significantly inhibit Na+-dependent bile acid uptake (Table 1 and Fig. 3, B and C).
Sulfated progesterone metabolite (P4-S) levels are raised in normal pregnancy and elevated further in intrahepatic cholestasis of pregnancy (ICP), a bile acid-liver disorder of pregnancy. ICP can be complicated by preterm labor and intrauterine death. The impact of P4-S on bile acid uptake was studied using two experimental models of hepatic uptake of bile acids, namely cultured primary human hepatocytes (PHH) and Na⁺-taurocholate co-transporting polypeptide (NTCP)-expressing Xenopus laevis oocytes. Two P4-S compounds, allopregnanolone-sulfate (PM4-S) and epiallopregnanolone-sulfate (PM5-S), reduced [³H]taurocholate (TC) uptake in a dose-dependent manner in PHH, with both Na⁺-dependent and -independent bile acid uptake systems significantly inhibited. PM5-S-mediated inhibition of TC uptake could be reversed by increasing the TC concentration against a fixed PM5-S dose indicating competitive inhibition. Experiments using NTCP-expressing Xenopus oocytes confirmed that PM4-S/PM5-S are capable of competitively inhibiting NTCP-mediated uptake of [³H]TC. Total serum PM4-S + PM5-S levels were measured in non-pregnant and third trimester pregnant women using liquid chromatography-electrospray tandem mass spectrometry and were increased in pregnant women, at levels capable of inhibiting TC uptake. In conclusion, pregnancy levels of P4-S can inhibit Na⁺-dependent and -independent influx of taurocholate in PHH and cause competitive inhibition of NTCP-mediated uptake of taurocholate in Xenopus oocytes.
Function of Uptake Transporters for Taurocholate and Estradiol 17b-D-Glucuronide in Cryopreserved Human Hepatocytes
2003, Drug Metabolism and Pharmacokinetics
The uptake properties of taurocholate (TC) and estradiol 17β-D-glucuronide (E₂17βG) were examined in freshly isolated and cryopreserved human hepatocytes to discover if active transport is retained in cryopreserved human hepatocytes. Firstly, the uptake of TC and E₂17βG was measured before and after cryopreservation. The uptake of TC was found to be Na⁺-dependent both in fresh and cryopreserved hepatocytes. The uptake activity in cryopreserved hepatocytes was found to range from 10 to 200% of that observed in freshly isolated cells. A kinetic analysis was performed to evaluate the transport activity of TC and E₂17βG and revealed that the Michaelis constant (K_m) for these compounds in cryopreserved human hepatocytes was 2–8 and 3–18 μM, respectively. This was within the range of K_m values previously found in human Na⁺-taurocholate cotransporting polypeptides (NTCP) and organic anion transporting polypeptides (OATP) 2 and 8, respectively. The kinetic analyses also showed that the species difference between human and rat hepatocytes was more marked for the maximal uptake rate (V_max) (> 22 and > 22 times higher for TC and E₂17βG in rats than in humans, respectively) than that for K_m (2–12 and 0.7–4 times higher, respectively), compared with earlier data we obtained in primary cultured rat hepatocytes. Hence, we conclude that cryopreserved human hepatocytes, at least in part, retain their transporter functions and, therefore, can be a useful experimental system for examining the mechanism of the hepatic uptake of drugs.

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Characterization of transport in isolated human hepatocytes: A study with the bile acid taurocholic acid, the uncharged ouabain and the organic cations vecuronium and rocuronium

Abstract

Biochem Pharmacol

Biochem Pharmacol

J Biol Chem

Gastroenterology

Life Sci

Biochim Biophys Acta

Biochem Pharmacol

Br J Anaesth

Br J Anaesth

Biochem Pharmacol

Biochem Biophys Res Commun

Biochem Pharmacol

Toxicology

Biochim Biophys Acta

Biochem Pharmacol

Br J Anaesth

Transport and metabolism in the hepatobiliary system

Hepatic transport of drugs and proteins

Acinar redistribution and heterogeneity in transport of the organic cation rhodamine B in rat liver

Hepatology

A compartmental model for hepatic transport of taurocholic acid in isolated perfused rat liver

Naunyn-Schmiedebergs Arch Pharmacol

Vesicular uptake system for the cation Lucigenin in the rat hepatocyte

Mol Pharmacol

Mechanisms for the hepatic uptake of organic cations. Studies with the muscle relaxant Vecuronium in isolated rat hepatocytes

J Pharmacol Exp Ther

Transport of sodium, chloride, and taurocholate by cultured rat hepatocytes