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Abstract

Bile secretion by liver parenchymal cells is the result of vectorial transcellular transport of solutes and involves the coordinated action of transport proteins at the basolateral (sinusoidal) and apical (canalicular) membranes of the hepato-cyte. A complex network of signals controls uptake and efflux transporters on a long- and a short-term timescale, including regulation at the level of gene transcription, protein translation and maturation, covalent modification, and dynamic localization of transporter proteins, as well as substrate availability. Evidence has shown that the hepatocellular hydration state exerts powerful control on the transcellular transport of solutes, such as conjugated bile acids and glucuronide and glutathione conjugates. This is of physiological significance because liver cell hydration is a dynamic parameter, which changes within minutes under the influence of hormones, nutrients, and oxidative stress. Thus, osmoregulation of bile formation is of physiological and pathophysiological interest.

Clinic of Gastroenterology, Hepatology and Infectiology, Heinrich-Heine-University Düsseldorf,Düsseldorf, Germany

Methods in Enzymology, Volume 428 © 2007 Elsevier Inc.

ISSN 0076-6879, DOI: 10.1016/S0076-6879(07)28018-8 All rights reserved.

1. Networks of Transport Systems

Bile formation is the result of transport activity of many transport systems. According to their substrate specificities, groups of transporters form functional networks. For example, bile acid transport is mediated by the Na+-coupled uptake transporter Na+-taurocholate cotransporting polypeptide (Ntcp/Slc10a1) (Hagenbuch and Meier, 1994; Stieger et al., 1994). Ntcp/ NTCP (for the protein in animals and humans, respectively) represents the major uptake system for conjugated bile salts and is functionally complemented by members of the organic anion transporting polypeptide (Oatp/OATP) family (Hagenbuch and Meier, 2003), such as Oatp1/Oatp1a1, Oatp2/ Oatp1a4, and Oatp4/Oatp1b2 in rats or OATP2/OATP1B1 and OATP8/ OATPB3 in humans (Fig. 18.1). Net bile salt uptake is further determined by efflux transporters at the sinusoidal membrane of hepatocytes, such as the ATP-binding cassette (ABC) transporter multidrug resistance associated protein 4 (Mrp4/MRP4) and Mrp3/MRP3, which transport bile salts back into the blood at the expense of ATP (Rius et al., 2003, 2006). Bile acid secretion into the canaliculus is driven by the bile salt export pump Bsep, which belongs

Figure 18.1 Hepatobiliary transporter of liver parenchymal cells. Uptake and efflux transporters are localized in the sinusoidal membrane of liver parenchymal, while the canalicular membrane harbors efflux transporters, which transport their substrates at the expense of ATP. (Left) Transporters involved in bile salt transport (among other substrates). (Right) Transporters of other substrates than bile salts. NTCP, sodium tauro-cholate cotransporting polypeptide; OATP, organic anion transporting polypeptide; OAT, organic anion transporter; OCT, organic cation transporter; BSEP, bile salt export pump; ABCG5/G8, ABC transporter subfamily G members 5 and 8; MDR, multidrug resistance 3; FIC1, familial intrahepatic cholestasis 1; MRP, multidrug resistance associated protein; OST, organic solute transporter.

Figure 18.1 Hepatobiliary transporter of liver parenchymal cells. Uptake and efflux transporters are localized in the sinusoidal membrane of liver parenchymal, while the canalicular membrane harbors efflux transporters, which transport their substrates at the expense of ATP. (Left) Transporters involved in bile salt transport (among other substrates). (Right) Transporters of other substrates than bile salts. NTCP, sodium tauro-cholate cotransporting polypeptide; OATP, organic anion transporting polypeptide; OAT, organic anion transporter; OCT, organic cation transporter; BSEP, bile salt export pump; ABCG5/G8, ABC transporter subfamily G members 5 and 8; MDR, multidrug resistance 3; FIC1, familial intrahepatic cholestasis 1; MRP, multidrug resistance associated protein; OST, organic solute transporter.

to the ABC transporter family. Likewise, the conjugate export pump Mrp2 brings about canalicular secretion of glucuronide and glutathione conjugates. Thus, vectorial bile acid transport is accomplished by the concerted action of Ntcp/Bsep, whereas other organic anions are transported by the Oatp/Mrp2 system. In general, the transport capacity of sinusoidal transporters exceeds that of canalicular transport systems. This led to the widely accepted view that the canalicular excretion step is rate limiting for overall transcellular transport of most cholephilic compounds under physiological conditions. However, the control strength theory (Groen et al., 1982) has not yet been applied to transcellular transport. Thus, the possibility is not ruled out that significant control on bile formation is also exerted at the step of uptake across the sinusoidal membrane. This may be relevant, especially at physiologically low bile acid concentrations or under pathophysiologic conditions, which are accompanied by a downregulation of Ntcp expression (Green et al., 1996; Simon et al., 1996).

2. Osmoregulation of Canalicular Secretion

Transporter insertion and retrieval into and from the canalicular membrane is a major mechanism of short-term regulation of bile formation (Haussinger et al., 2000; Kubitz et al., 1997). By this mechanism, the total number of transporter molecules in the canalicular and sinusoidal membrane and therefore transport capacity (Vmax) can change within minutes. One major regulator of this process are changes in hepatocellular hydration, which occur in response to hormones, nutrients, oxidative stress, or changes in ambient osmolarity (for review, see Haussinger, 1996; Lang et al., 1998). In perfused rat liver, cell shrinkage inhibits, whereas cell swelling stimulates taurocholate (TC) excretion into bile, regardless of whether cell volume is modified by anisotonic exposure, insulin, cumulative amino acid uptake, or ethanol (Hallbrucker et al., 1992; Haussinger et al., 1992, 2000). Regulation of TC excretion into bile by the hepatocellular hydration state is due to rapid changes of transport capacity: a 10% increase of hepatocyte water content doubles the Vmax of TC excretion into bile within minutes, whereas hyperosmotic cell shrinkage decreases Vmax. The swelling-induced increase in transport capacity is abolished in the presence of colchicine, indicating the requirement of intact microtubules (Haussinger et al., 1993). As shown by immunohistochemistry (Schmitt et al., 2001), hyperosmotic hepatocyte shrinkage triggers a rapid retrieval of Bsep from the canalicular membrane and its transfer into an intracellular vesicular compartment, whereas cell swelling triggers the insertion of intracellularly stored Bsep into the canalicular membrane. Likewise, hyperosmotic hepatocyte shrinkage triggers retrieval of the canalicular conjugate pump Mrp2 from the canalicular membrane, whereas hypoosmotic hepatocyte swelling stimulates a rapid insertion of Mrp2 into the canalicular membrane (Dombrowski et al., 2000; Kubitz et al., 1997), which is also reflected at the functional level, that is, corresponding changes in the canalicular secretion of the glutathione conjugates (Dombrowski et al., 2000; Wettstein et al., 1995). Interestingly, following hyperosmotic hepatocyte shrinkage, Bsep and Mrp2 are retrieved into different intracellular vesicular compartments. As shown by immuno-histochemistry, only 15% of the retrieved vesicles contained both Bsep and Mrp2, whereas the remainder of vesicles contained either Mrp2 or Bsep (Schmitt et al., 2001); accordingly, BSEP and MRP2 can be found in different vesicles in human livers (Fig. 18.2). This may either indicate different retrieval mechanisms for the two transporters in response to hyperosmolarity or suggest the existence of canalicular membrane domains being enriched in either Bsep or Mrp2. In support of the latter suggestion, immunocytochemistry of pseudocanaliculi formed between two adjacent HepG2 cells indeed shows canalicular membrane patches with enrichment of either Bsep or Mrp2 and only a few patches with transporter colocaliza-tion (Kubitz and Haussinger, unpublished result). Also, the expression of Bsep and Mrp2 was found to be osmoregulated. Hypoosmotic hepatocyte swelling increases Bsep and Mrp2 expression, whereas hyperosmotic cell shrinkage decreases (Kubitz et al., 1999a; Warskulat et al., 1999).

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