Journal of Hepatology
Volume 42, Issue 4 , Pages 592-603, April 2005

Regulated vesicle trafficking of membrane transporters in hepatic epithelia

  • Raúl A. Marinelli

      Affiliations

    • Instituto de Fisiología Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 570, 2000 Rosario, Santa Fe, Argentina
    • Corresponding Author InformationCorresponding author. Tel.: +54 341 430 5799; fax: +54 341 439 9473.
    • ,
  • Pamela S. Tietz

      Affiliations

    • Center for Basic Research in Digestive Diseases, Departments of Internal Medicine and Biochemistry and Molecular Biology, Mayo Medical School, Clinic, and Foundation, Rochester, MN 55905, USA
    • ,
  • Nicholas F. LaRusso

      Affiliations

    • Center for Basic Research in Digestive Diseases, Departments of Internal Medicine and Biochemistry and Molecular Biology, Mayo Medical School, Clinic, and Foundation, Rochester, MN 55905, USA

published online 25 January 2005.

Article Outline

Abbreviations:: Ntcp, Na+/bile salt co-transporter, Bsep, bile salt export pump, Mrp2, multi-drug resistance-related protein 2, Mdr1, multi-drug resistance P-glycoprotein 1, Mdr2, multi-drug resistance P-glycoprotein 2, AQP-8, aquaporin-8 water channels, ASBT, Na+/bile salt co-transporter, CFTR, Cystic fibrosis transmembrane regulator Cl-channels, AE2, Cl-/HC03- anion exchanger 2, AQP-1, aquaporin-1 water channels

 

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1. Introduction 

The vectorial movement of solutes, ions and water molecules within hepatic epithelia (i.e. hepatocytes and cholangiocytes) is achieved by specialized transport systems located in the basolateral and apical plasma membrane domains. In recent years, growing evidence has accumulated that epithelial cells, including hepatic epithelia, contain populations of membrane transporters in specific intracellular membrane vesicles. In such cells, an important mechanism of transport regulation involves the targeted trafficking to and membrane fusion of these vesicles with the apical or basolateral plasma membrane in response to appropriate stimuli (Fig. 1). Thus, cellular specific transport activities can be regulated by the resulting insertion of additional transport proteins into the plasma membrane. When the stimulus is withdrawn, or when a different stimulus is applied, the transporters are removed by retrieval endocytosis and remain for a period of time in vesicles (i.e. early endosomes) that are capable of re-fusion after re-stimulation. Eventually, the transporters may move to a non-recycling compartment (i.e. late endosomes/multi-vesicular body) where they would be finally degraded by lysosomes. Some proteins may be targeted for proteolysis through the ubiquitin-proteasome system. Ubiquitin, a 76-amino acid peptide, serves as a tag for the recognition of proteins by the multi-subunit proteolytic particle known as the proteasome. This system degrades misfolded proteins and misassembled oligomeric protein complexes at the level of the endoplasmic reticulum. Proteasomes have also been implicated in the degradation of membrane transporters from the cell surface [1], [2]. Recent evidence suggests that ubiquitin plays a role in regulating the plasma membrane expression of integral proteins. Ubiquitination would serve to trigger endocytic internalization and degradation of membrane proteins by proteasome and/or lysosomal proteases [3].

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  • Fig. 1. 

    Hepatic epithelia contain membrane transporters in specialized intracellular vesicles. Specific transport activities can be regulated by trafficking to and insertion of these transporters into the plasma membrane in response to appropriate stimuli. When the stimulus is withdrawn, the transport proteins are retrieved by endocytosis and remain for a period of time in vesicles (i.e. early endosomes). The transporters may undergo re-fusion after re-stimulation or move to a non-recycling compartment (i.e. late endosomes) where they would be finally degraded by lysosomes. Some transporters might be targeted for proteolysis through the ubiquitin-proteasome pathway. Thus, the abundance of a transporter in the plasma membrane at any given time would result from the net balance between the rate of exocytic insertion and endocytic retrieval.

Thus, the abundance of a transporter in the plasma membrane at any given time would result from the net balance between the rate of exocytic insertion and endocytic retrieval (Fig. 1). Well known examples of this recycling regulatory mechanism in non-hepatic cells are: (i) the insulin-induced insertion of the glucose transporter, GLUT-4 (SLC2A4), into the plasma membrane of adipose and muscle cells; (ii) the antidiuretic hormone-regulated insertion of aquaporin-2 water channels in the cortical collecting duct; and (iii) the secretagogue-regulated insertion of H+/K+-ATPase in gastric parietal cells (for reviews, see Refs. [4], [5], [6]). In liver, a number of transporters responsible for key physiological functions have been proposed to undergo regulated vesicle trafficking.

This review summarizes current findings on vesicle trafficking of membrane transporters in hepatic epithelia, its modulation by specific stimuli, and its implications for bile secretory physiology and pathophysiology. In addition, we also review the methodologies that have been employed.

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2. Methods used for investigation of regulated vesicle trafficking of hepatic transporters 

Several criteria are needed to demonstrate regulated vesicle trafficking of transporters in hepatic epithelia:

(i)presence of the transporter in the plasma membrane as well as in an intracellular pool of vesicles, under basal (non-stimulated) conditions;

(ii)reciprocal changes in the amount of the transporter between the plasma and intracellular vesicle membranes, after appropriate stimulus;

(iii)rapid response to the stimulation, i.e. within minutes; and

(iv)constant cellular amount of the transport protein, i.e. de novo synthesis should not be required in the short term.

To study the regulated vesicle trafficking of transporters in hepatic epithelial cells, investigators have made use of several experimental models such as isolated hepatocytes or cholangiocytes, polarized hepatocyte couplets, intrahepatic bile duct segments, liver cell lines, and intact animals. A variety of cell/molecular biologic methods that provide complementary information have been used on these models.

One of the methods used is subcellular fractionation using high-speed centrifugation through sucrose or Percoll density gradients, followed by the assessment of the amount of transport protein by immunoblotting [7], [8], [9]. When done appropriately, this method is considered to be quantitative. The transporter of interest should be present in intracellular membrane fractions containing transport vesicles, i.e. total microsomes or endosomes. Following the addition of the stimulus, the transporter is expected to increase in the plasma membrane-enriched fraction and simultaneously to decrease in intracellular membranes (Fig. 2). Much depends on the purity of the membrane fractions tested, e.g. a significant cross-contamination could mask the redistribution of the transporter. It is also possible that a small portion of the transporter trafficking from a large intracellular pool results in a significantly increased amount of the transporter in plasma membrane, whereas any decrease in the transporter amount in intracellular membranes remains below the detection limit [10].

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  • Fig. 2. 

    Localization of AQP-8 in hepatocytes by subcellular fractionation and immunoblotting: effect of dibutyryl cAMP (DBcAMP). Hepatocytes were incubated in the absence (controls) or presence of 100μM DBcAMP for 10min at 37°C, and subcellular fractionation was performed. (A) anti-AQP8 immunoblot of plasma and intracellular microsomal membranes (20μg total protein/lane). (B) Densitometric analysis of three separate experiments in each group (control and DBcAMP). Data are expressed in percent values as mean±S.E. *, P<0.05 for DBcAMP effect (Student's t test).

Immunofluorescence followed by laser scanning confocal microscopy has proven to be a powerful tool to localize transporters in intracellular vesicle structures and to assess their redistribution after a stimulus [8], [11], [12], [13], [14], [15] (Fig. 3). This method should be considered semi-quantitative. An additional related method is immunogold electron microscopy where the location of the protein and its carrier vesicle can be unequivocally determined. Assuming a proper accessibility of the antibody to the ligand, this method allows quantification of the redistribution of the transporter through the counting of gold particles on intracellular vesicles and on cell surfaces [16], [17], [18] (Fig. 4).

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  • Fig. 3. 

    Effect of glucagon on the localization of AQP8 in hepatocytes by confocal immunofluorescence. Hepatocytes (A and B, singlets; C and D, couplets) in short term cultured were incubated at 37°C for 10min in the absence (controls) or presence of 1μM glucagon, fixed, permeabilized, and labeled with anti-QP8. Fluorescence localization was viewed by laser scanning confocal microscopy. (A and C), controls; (B and D), glucagon-treated. Magnification, ×600.

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  • Fig. 4. 

    Polarized confluent mouse cholangiocytes were exposed to media alone (basal) (A and B) or media with dibutyryl cAMP (agonist) (A1 and B1). Agonist stimulation caused a redistribution of dual labeled immunogold and demonstrates a consistent and significant movement of vesicles containing colocalizated AQP1 with CFTR and AE2 with CFTR to the apical plasma membrane (arrowheads). Quantitation of the apical membrane-associated labeling (C and D) revealed a significant 2-fold or greater increase in the apical membrane-associated redistribution. Bar=200nm at a magnification of ×30,000. *P<0.05 (Student's t-test).

A complementary approach is to immunoisolate the transporter-containing vesicles, using anti-transporter antibodies [18], in order to characterize the specialized array of proteins involved in signaling or docking/fusion events, as well as to explore for possible co-localization of different transporters on the same vesicle [18], (Fig. 5).

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  • Fig. 5. 

    (A) Immunoblotting for AQP1, CFTR, and AE2 on subcellular fractions prepared from microsomes (starting material), vesicles specifically associated with the magnetic beads (bound), and vesicles that did not bind to the beads (unbound) using AQP1 antibody. (B) Reverse vesicle immunoisolation using CFTR antibody. (C) Reverse vesicle immunoisolation using AE2 antibody. A 10% polyacrylamide gel was loaded with fractions for AQP1 (50μg of total protein/lane), CFTR (100μg of total protein/lane), and AE2 (50μg of total protein/lane). Data are expressed in arbitrary densitometry units as means±S.E. *P<0.05 (Student's t-test).

Demonstration of exocytic insertion of a transporter in response to a stimulus should be supplemented by measurement of transport activity (e.g. solute or ion membrane transport or membrane water permeability) to show that changes in transporter activity correlate well with fusion events. The relationship between the two events can be strengthened by studying the effects of maneuvers that are known to inhibit vesicle translocation, such as pretreatment with cytoskeleton inhibitors or exposure to low temperature [7], [8], [13], [19], [20].

The use of multiple experimental approaches (biochemical, morphological, and functional) provides the strongest evidence for regulated trafficking of hepatic transporters. Study at the molecular level also requires complementary experimental approaches, including the isolation of specific populations of carrier vesicles, their biochemical characterization, the reconstitution of the various steps in cell-free systems, the use of model liver cell lines after transfection, the use of transgenic animals, and more recent approaches to gene silencing (e.g. small interfering RNAs).

In the next section, we review the individual liver transporters proposed to undergo regulated vesicle trafficking. These transporters as well as the specific stimuli involved and their regulatory effects are summarized in Table 1.

Table 1. Liver transporters proposed to undergo regulated vesicle trafficking
Hepatocyte transportersStimulusRegulatory effectReferences
Solutes
NtcpcAMP; glucagon (?); osmolaritySinusoidal bile salt uptake[9], [26]
BsepcAMP; taurocholate; tauroursodeoxycholate; osmolarityCanalicular bile salt excretion[19], [33], [34], [41]
Mrp2cAMP; taurocholate; osmolarityCanalicular non-bile acid organic anion excretion[12], [13], [14], [35]
Mdr1cAMP; taurocholateCanalicular organic cation excretion[45]
Mdr2cAMP; taurocholateCanalicular phospholipid excretion[45]

Ions

AE2cAMP; pH; glucagon (?)Canalicular excretion[49], [50]
Cl−/K+ channelsInsulinUnknown[52]

H20

AQP-8cAMP; glucagonsCanalicular water permeability[8], [11], [15]

Cholangiocyte transporters


Solutes

ASBTSecretinApical bile salt uptake[61]

Ions

AE2Secretin; campApical excretion[18]
CFTRSecretin; campApical Cl− excretion[18]

H20

AQP-1Secretin; campApical water permeability[7], [67]

Ntcp, Na+/bile salt co-transporter; Bsep, bile salt export pump; Mrp2, multi-drug resistance-related protein 2; Mdr1, multi-drug resistance P-glycoprotein 1; Mdr2 multi-drug resistance P-glycoprotein 2; AQP-8, aquaporin-8 water channels; ASBT, Na+/bile salt co-transporter; CFTR, Cystic Fibrosis Transmembrane Regulator Cl− channels; AE2, Cl−/ anion exchanger 2; AQP-1, aquaporin-1 water channels.

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3. Hepatocyte membrane transporters with regulated vesicle trafficking 

3.1. Solute transporters 

3.1.1. Na+/bile salt co-transporter (Ntcp) 

Hepatocyte uptake of conjugated bile salts such as taurocholate is mediated predominantly via the basolaterally located Ntcp (for Na+ taurocholate co-transport polypeptide), (SLC10A1) [21]. The cAMP analog, dibutyryl cAMP, stimulates hepatocyte Na+/taurocholate co-transport by increasing the maximal transport rate. The effect of cAMP is mediated via protein kinase A; is potentiated, but not mediated, by Ca2+/calmodulin-dependent processes; and is downregulated by protein kinase C [22]. The cAMP-induced increase of the transport maximum of Na+/taurocholate co-transport is caused, at least in part, by insertional exocytosis of Ntcp transporters from intracellular vesicles to the basolateral plasma membrane [9]. The phosphatidylinositol 3-kinase signaling pathway has also been involved in the regulated translocation of Ntcp [23]. Two kinases, protein kinase B (also known as Akt) and atypical PKC ζ seem to act downstream of phosphatidylinositol 3-kinase in the translocation of Ntcp [23], [24], [25]. Interestingly, other maneuvers known to stimulate in hepatocytes the same signaling pathway (i.e. cell swelling) can also stimulate taurocholate uptake by translocating Ntcp to the plasma membrane [24], [26]. Glucagon, a hormone whose effects in hepatocytes are mainly mediated by cAMP, can also stimulate Na+/taurocholate co-transport. Whether glucagon is also able to induce Ntcp translocation has not yet been explored.

Ntcp is a serine/threonine phosphoprotein, and dephosphorylated Ntcp is located primarily in the plasma membrane. Treatment of hepatocytes with cAMP results in Ntcp dephosphorylation by a mechanism that involves the activation of Ca 2+/calmodulin-dependent serine-threonine PP2B (also known as calcineurin) [27]. Thus, both insertional exocytosis and dephosphorylation of Ntcp seem to be involved in the regulation of hepatic Na+/taurocholate co-transport.

The role of microfilaments and microtubules in the short-term regulation of Ntcp translocation has been studied using GFP-tagged Ntcp expressed in the HepG2 cell line. The authors reported that the targeting of Ntcp to the plasma membrane consists of two steps: the delivery of Ntcp to the region of the plasma membrane via microtubules; and the insertion of Ntcp into the plasma membrane, in a microfilament- and cAMP-sensitive fashion [20]. The requirement of intact actin filaments in regulated translocation of Ntcp was also observed in isolated rat hepatocytes [23]. It has been recently demonstrated that Ntcp is present in vesicles from the early (recycling) endosomal compartment [28]. These Ntcp-containing vesicles were also shown to express the microtubule based motors, dynein and kinesin, and the actin-based motor myosin IIa [28], supporting the role of cytoskeleton in Ntcp hepatocyte trafficking.

Studies on human Ntcp suggest that Cysteine residues 96 and 98 in the second transmembrane domain and Cys 266 in the extracellular loop of the Ntcp molecule are critical for its proper intracellular transport. In addition, since mutation of the Cys residues at 266 to alanine results in an increase in maximal transport rate, it has been suggested that Cys266 is involved in the intracellular retention of Ntcp [29]. Finally, expression studies of GFP-fused human Ntcp in non-hepatic cell lines indicate that the polarized basolateral localization of Ntcp is cell specific and mediated by a novel-sorting pathway that is brefeldin A sensitive and monensin and low-temperature shift insensitive [30].

3.1.2. Bile salt export pump (Bsep) 

The hepatocyte canalicular membrane contains several primary active transporters that couple ATP hydrolysis to the transport of specific substrates into the bile canaliculus [31]. These transporters are members of the family of ATP binding cassette (ABC) membrane transporters and are involved in secretion of bile acids (e.g. Bsep), non-bile acid organic anions (e.g. Mrp2), organic cations (e.g. Mdr1), and phospholipids (e.g. Mdr2).

Rat liver Bsep (ABCB11) is an ATP-dependent protein of about 160kDa. Bsep is responsible for the biliary excretion of bile acids and therefore is key to the elaboration of canalicular bile [31]. Immunogold electron microscopy detection of Bsep revealed that the distribution of Bsep in the rat hepatocyte is not restricted only to the canalicular membrane, but labeling of Bsep was also detected in vesicles close to the bile canaliculus [32]. Pericanalicular distribution of Bsep was also demonstrated by immunofluorescent staining of isolated rat hepatocyte couplets [12].

Experiments in intact rats as well as in isolated hepatocyte couplets showed that dibutyryl cAMP rapidly increases the amount of Bsep in the canalicular membrane and stimulates bile acid secretion, processes that are inhibited by microtubule disrupters [19].

The administration of bile salts (i.e. taurocholate or tauroursodeoxycholate) to rats or perfused livers also stimulates the vesicle targeting of Bsep to the canalicular membrane [33], [34]. Bsep (as well as other ABC transporters) seems to cycle between the bile canaliculus and at least two intracellular pools, one of which is mobilized to the canalicular membrane by cAMP and the other, by taurocholate [33]. The vesicular translocation of Bsep to the canalicular membrane stimulated by taurocholate, not by cAMP, is inhibited by wortmannin, suggesting the involvement of phosphoinositide 3-kinase [35], [36]. In addition, lipid products of phosphoinositide 3-kinase directly regulate the ATP-dependent substrate transport activity of Bsep in the canalicular membrane [37]. The tauroursodeoxycholate-stimulated canalicular Bsep translocation was found to be dependent on the activation of the mitogen-activated protein kinase, p38(MAPK) and Ca(2+)-independent protein kinase C isoforms [34], [38].

In transfected polarized WIF-B9 hepatocytes, Bsep tagged with yellow fluorescent protein (YFP) was found to constitutively cycles between apical rab 11a-containing endosomes and the canalicular membrane. Bsep cycling requires an intact functional microtubular and microfilament systems, but is unaffected by cAMP, taurocholate, or phosphatidylinositol 3-kinase inhibitors [39].

Canalicular transport of bile salts via Bsep is dependent on the hydration state of hepatocytes. In perfused rat livers, hypososmotic solutions increase translocation of intracellular Bsep to the canalicular membrane and stimulate taurocholate excretion into bile, whereas hyperosmotic buffers cause endocytic retrieval of Bsep from the canalicular membrane and inhibition of biliary taurocholate excretion [40], [41]. The osmosignaling pathways involved in the regulation of taurocholate excretion by liver cell hydration changes have been studied. A G-protein-and tyrosine kinase-dependent but protein kinase C-independent activation of mitogen-activated protein kinases, extracellular-signal-regulated kinase (Erk)-1 and (Erk)-2, seem to be involved [42].

3.1.3. Multi-drug resistance-related protein 2 (Mrp2) 

Mrp2 is an ATP-dependent protein of approximately 190kDa, responsible for the transport into bile of a variety of amphiphilic organic anions, including bilirubin glucuronides, glutathione S-conjugates and oxidized glutathione [31].

Immunogold electron microscopy detection of Mrp2 in rat liver revealed that over 50% of Mrp2 resides in intracellular vesicles close to the canalicular membrane [16]. Pericanalicular vesicular distribution of Mrp2 was also demonstrated by confocal immunofluorescence in isolated rat hepatocyte couplets [12].

Studies in isolated rat hepatocyte couplets showed that dibutyryl cAMP induces the microtubule-dependent relocation of vesicular Mrp2 to the canalicular membrane [12], [13].

Independent expression studies in the non-hepatic MCDK cell line allowed the identification of targeting signals in the C-terminal tail [43] and in the N-terminal transmembrane region of Mrp2 [44] which would be required for routing to or stable association with the apical membrane.

Confocal immunofluorescence and immunogold electron microscopy studies in isolated hepatocytes and in perfused rat liver indicate that, as observed for Bsep, the subcellular localization of Mrp2 is osmosensitive. Hypoosmolarity increases translocation of intracellular Mrp2 to the canalicular membrane, whereas hyperosmolasrity causes endocytic retrieval of Mrp2 from the canalicular membrane to redistribute in pericanalicular vesicles [14], [16].

It has been observed that rat hepatocyte Bsep and Mrp2 co-localize in a fraction of microtubule-associated vesicles [12]. Nevertheless, Bsep- and Mrp2-specific vesicles seem to participate in the short-term osmoregulation of canalicular secretion. Following hyperosmolar retrieval of Mrp2 and Bsep from the canalicular membrane, the transporters only colocalize in 15% of the vesicles, whereas 85% of the vesicles either contain Bsep (61%) or Mrp2 (24%) [41].

As mentioned for Bsep, the taurocholate-stimulated vesicular translocation of Mrp2 to the canalicular membrane is also dependent on active phosphoinositide 3-kinase [35] and its canalicular transport activity is directly regulated by lipid products of phosphoinositide 3-kinase [37].

3.1.4. Multi-drug resistance P-glycoproteins (Mdr1 and Mdr2) 

Mdr1 mediates the biliary excretion of lipophilic organic cations, while Mdr2 appears to function as a flippase, translocating phosphatidylcholine through the canalicular membrane [31].

In normal rat liver, under basal conditions, Mdr1 and Mdr2 are located mostly intracellularly. After administration of taurocholate or dibutyryl cAMP, the amounts of Mdr1 and Mdr2 increase 3-fold in the canalicular membrane. Pretreatment of rats with colchicine prevents the responses to taurocholate. These results suggest that taurocholate and dibutyryl cAMP increase the specific activity of the canalicular ATP-dependent transport proteins by stimulating an increase in the relative amounts of these proteins in the membrane [33], [45]. The effects of taurocholate and dibutyryl cAMP are additive, suggesting the presence of at least two different intrahepatic pools of ABC transporters, one of which is mobilized to the canalicular membrane by cAMP and the other by taurocholate [33]. Intracellular distribution and trafficking of Mdr1 has been studied using GFP-tagged Mdr1 in stably transfected WIF-B cells [46].

3.2. Ion transporters/channels 

3.2.1. Cl−/HCO3− anion exchanger 2 (AE2) 

AE2 (SLC4A2) has been localized to the canalicular membrane domain [47]. This ion exchanger extrudes HCO3− and functions to regulate intracellular pH when hepatocytes are exposed to an alkaline load [48]. The canalicular activity of the AE2 is increased in hepatocytes exposed to bicarbonate-containing medium or in response to stimulation with cAMP. This increased activity of the exchanger can be blocked with colchicine, suggesting the microtubule-dependent targeting of vesicles containing this exchanger to the canalicular domain [49].

Glucagon has also been reported to stimulate the canalicular Cl−/HCO3− exchange activity through a microtubule and cAMP-dependent PKA pathway [50]. The stimulation of Cl−/HCO3− exchange activity by cAMP or glucagon is inhibited by PKC agonists [49], [50].

Because biliary bicarbonate secretion may be responsible for up to 50% of bile acid-independent bile flow [51], canalicular targeting of the AE2 may be an important step in the regulation of bile production.

3.2.2. Cl− and K+ channels 

In the HTC liver cell line, insulin stimulates the recruitment of new membrane containing Cl− and K+-selective ion channels. The effect of insulin was inhibited by blockers of phosphoinositide 3-kinase and by the disruption of microtubule assembly. The molecular identification of these insulin-responsive channels has not been established; they may mediate the changes in membrane conductance that are essential for the cellular response to insulin [52]. It is possible that this process is involved in bile formation since both chloride and potassium channels have been identified previously in canalicular membrane vesicles.

3.3. Water channels 

3.3.1. Aquaporin-8 (AQP-8) 

The aquaporins (AQPs) are a family of recently identified channel-forming, integral membrane proteins that allow water to rapidly traverse cellular membranes in response to osmotic gradients.

Rat hepatocytes express the water channel, AQP-8, which is a N-glycosylated protein of about 34kDa. Subcellular fractionation and confocal immunofluorescence studies [8] as well as immunoelectron microscopy [53] showed that AQP-8 is largely localized in intracellular vesicles in hepatocytes. The cell permeable cAMP analog, dibutyryl cAMP, induces the redistribution of AQP-8 to the plasma membrane and increases hepatocyte membrane water permeability. The microtubule blocker colchicine, but not its inactive analog β-lumicolchicine, inhibits the dibutyryl cAMP effect on both AQP-8 redistribution to cell surface and hepatocyte membrane water permeability [8]. More recent studies in isolated rat hepatocytes [15], [54] show that glucagon can also induce the translocation of AQP-8 water channels to the hepatocyte plasma membrane, a process that requires protein kinase A and phosphatidylinositol 3-kinase activation.

Confocal immunofluorescence microscopy and functional studies in polarized isolated rat hepatocyte couplets indicate that dibutyryl cAMP induces the targeting of AQP-8 to the canalicular plasma membrane domain and facilitates osmotically-driven canalicular water secretion [11]. Thus, hepatocytes are able to modulate their canalicular membrane water permeability, a process that almost certainly is relevant to hepatocyte bile secretion.

Hepatocytes also express two other water channels, AQP-0 and 9. AQP-9 is localized exclusively in the sinusoidal plasma membrane [11], [55], [56] and has been described as a membrane channel for glycerol and urea (aquaglyceroporin) in hepatocytes [57]; AQP-0 is localized intracellularly, but it is not responsive to dibutyryl cAMP, i.e. it seems not to be regulated by vesicle trafficking, and its function remains to be elucidated.

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4. Cholangiocyte transporters with regulated vesicle trafficking 

4.1. Solute transporters 

4.1.1. Na+/bile salt co-transporter (ASBT) 

We and others have demonstrated the functional expression of ASBT (SLC10A2) in cholangiocytes [58], [59]. ASBT is located at the apical cholangiocyte plasma membrane domain and takes up bile salts from bile in a sodium dependent manner. Cholangiocytes also express t-ASBT, a spliced form of ASBT, located at the basolateral domain, which is thought to mediate the basolateral extrusion of bile salts [60]. Experiments in isolated cholangiocytes using cholyl-(Ne-NBD)-lysine, a fluorescent bile acid with high specificity for ASBT, suggest that secretin induces the microtubule-dependent apical ASBT endocytosis [61]. This secretin-induced internalization of ASBT would reduce the cholangiocyte uptake of bile acids in their transit to the intestine. Further studies are needed to clarify the regulated trafficking of ASBT and its relevance to biliary physiology. There is recent evidence indicating that cholangiocyte ASBT undergoes ubiquitin-proteasome degradation [62]. Degradation via the proteasome pathway may play a role in the regulation of membrane transporter expression (Fig. 1). As suggested for ASBT [62], the exposure of the extracellular domains of hepatic transporters to the detergent environment of bile might induce protein damage and potentially trigger degradation through the proteasome pathway.

4.2. Ion transporters/channels 

4.2.1. CFTR Cl− channels 

CFTR is expressed in a functional state in the apical domain of cholangiocytes [63], [64]. CFTR is a cAMP regulated chloride channel, therefore, the secretin-induced increase of cAMP activates CFTR. Efflux of chloride via CFTR-chloride channels is thought to generate HCO3− secretion into bile via activation of the AE2. The output of HCO3− is associated with movement of water in the bile duct lumen.

We recently found in cholangiocytes, by using specific immunoisolation of vesicles as well as dual labeled immunogold electron microscopy, a specialized population of intracellular vesicles containing CFTR as well as both AQP-1 water channels and the AE2. In vivo experiments in rats showed that secretin induces the microtubule-dependent targeting of these organelles and the consequent exocytic insertion of CFTR, AQP1 and AE2 in the apical plasma membrane domain of cholangiocytes, a mechanism that is expected to be key for ductal bile secretion [18].

4.2.2. Cl−/HCO3− anion exchanger 2 (AE2) 

AE2 has been localized to the apical domain of cholangiocytes [65]. As mentioned above, this exchanger seems to be co-localized along with CFTR Cl− channels and AQP-1, in a specialized population of intracellular vesicles [18]. Secretin induces its targeting and exocytic insertion into the apical plasma membrane of cholangiocytes, a mechanism that may account for hormone-regulated ductal bile secretion [18].

4.3. Water channels 

4.3.1. Aquaporin-1 (AQP-1) 

Rat cholangiocytes express the water channel AQP-1 [66], which is a non-glycosylated protein of 28kDa. Subcellular fractionation as well as light and immunoelectron microscopy analysis showed that AQP-1 is present mainly in the cholangiocyte apical plasma membrane domain and in an intracellular vesicular pool [7], [18], [67].

Studies in freshly isolated cholangiocytes as well as in intact rats, indicate that secretin induces the microtubule-dependent redistribution of AQP-1 from intracellular vesicles to the apical plasma membrane domain and increases cholangiocyte membrane water permeability [7], [18], [67].

Inhibitory studies in isolated rat and mouse bile duct units suggest that AQP-1 plays a key role in osmotically-driven apical water secretion during hormone-regulated ductal bile formation [68], [69]. Indeed, studies in perfused rat bile duct units in which AQP-1 was silenced in vitro using small interfering RNA technology provided unambiguous evidence for a key role for AQP-1 in ductal bile formation [70].

In contrast, studies in bile duct units isolated from AQP-1 knockout mice have been interpreted to suggest that AQP1 does not play a major role in bile secretion [71]. The reason for this discrepancy is unclear, although differences in the experimental models as well as species differences may be involved.

Thus, cholangiocytes are able to modulate their apical membrane water permeability by altering the number of AQP-1 water channels present. The relevance of this process to the formation and regulation of ductal bile secretion continues under active investigation.

AQP-1 is expressed in erythrocytes and in fluid transporting epithelia throughout the body at sites of constitutive (not regulated) water transport [5]. Liver appears to be an exception in that AQP-1 is present in cholangiocytes in a pool of AQP-1 carrying-vesicles able to traffic to the plasma membrane in a regulated fashion [7], [18], [67]. Thus, it may be that a protein that is constitutively expressed in one cell type is regulated in another. Proteins are under the control of mechanisms that include interactions between signals within the protein itself and the cellular sorting machinery. These signals can be differentially interpreted by various cell types.

Cholangiocytes also express the water channel AQP-4 [72]. AQP-4 is secretin unresponsive and it seems to be constitutively localized to the cholangiocyte basolateral membrane, mediating water transport across that plasma membrane domain. Other members of the aquaporin family of proteins were found to be expressed in cholangiocytes [73], but their possible regulated trafficking has not yet been explored.

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5. Implications for bile secretory pathophysiology 

Bile secretion by hepatocytes and cholangiocytes results from the coordinated interactions of several membrane-transport systems. As detailed in previous sections, there is increasing experimental evidence suggesting that the vesicle translocation of some transporters to hepatic epithelia plasma membranes plays an important role in the short-term regulation of bile formation. Thus, it is conceivable that a disruption of vesicle-based trafficking of transporters may lead to alterations of normal bile physiology. Bile formation can also be modulated on the long term basis by modifying the gene expression of those transporters [74].

Bile formation is an osmotic secretory process. Conceptually, the generation of bile flow is related to the number of apical transporters involved in the secretion of osmotically active solutes and to the osmotic water permeability of the apical membrane (assuming that water flows across the hepatocyte and cholangiocyte epithelial barriers by a predominantly transcellular route). Thus, an impaired balance in membrane insertion/retrieval of solute transporters and AQPs may lead to bile secretory dysfunction (i.e. cholestasis). It is interesting that the normal vesicle trafficking of solute transporters that are key to the generation of canalicular bile secretion, such as Bsep and Mrp2, is altered in experimental models of cholestasis.

Experimental maneuvers leading to cholestasis in rats, such as treatment with the microfilament disturbing drug, phalloidin [75], the endotoxin, lipopolysaccharide, [16], [76], oxidative stress inducers [77], the bile salt, taurolithocholate [17], or with the endogenous metabolite of estradiol, estradiol-17beta-d-glucuronide [78] all result in an increased number of Mrp2-containing vesicles in the pericanalicular cytoplasm of rat hepatocytes and reduced functional expression of Mrp2 in the canalicular membrane. Similar findings have also been found for Bsep in cholestasis induced by lipopolysaccharide, [40], taurolithocholate [79] or by estradiol-17beta-d-glucuronide [80]. This accumulation of transporter-carrying vesicles around the bile canaliculi is thought to be due to increased canalicular endocytic retrieval, a process that would be independent of microtubules [81]. Nevertheless, a reduced exocytic fusion to the canalicular membrane might also be involved. Although the mechanisms implicated in these processes are largely unknown, it was recently found that proper canalicular localization of Mrp2 requires radixin, a protein that crosslinks actin filaments and integral membrane proteins [82]. Consistent with this, a defective canalicular localization of Mrp2 and radixin was observed in a cholestatic liver disease, i.e. primary biliary cirrhosis [83]. Thus, a defective radixin-dependent anchoring of canalicular Mrp2 might trigger its endocytic retrieval or prevents its proper exocytic insertion.

Canalicular expression of Mrp2 also seems to require interaction with the PDZ domain protein PDZK1 [84]. Bsep, Mdr1, and Mdr2 do not contain obvious PDZ-interacting motifs, and canalicular expression of Mdr proteins is not affected in radixin knockout animals [82]. Thus, the targeting and trafficking of these transporters seems to be regulated by a different array of proteins. HAX-1 has been recently identified as a binding partner for canalicular Bsep, Mdr1, and Mdr2 [85]. HAX-1 is a cytoskeleton-associated protein that interacts with the F-actin-binding protein, cortactin. There is evidence that HAX-1 and cortactin participate in clathrin-mediated endocytosis of Bsep from the canalicular plasma membrane [85]. Derangement of this protein-interacting system might be present in experimental cholestasis with defective canalicular localization of Bsep.

Interestingly, there is evidence to suggest that the anticholestatic drugs ursodeoxycholate and sylimarin, are able to prevent or reverse defective canalicular insertion of transporters in cholestasis [17], [86], providing additional support to the central role that vesicle trafficking plays in normal bile secretory physiology.

It is unknown if other canalicular transporters consider to be relevant to bile formation, such as, AE2 or AQP-8, undergo membrane endocytic retrieval during cholestasis. Interestingly, obstructive cholestasis induced by bile duct ligation also results in an increased number of vesicles in the pericanalicular cytoplasm of rat hepatocytes [87]. We recently reported experimental evidence suggesting a defective translocation or membrane insertion of AQP-8 in hepatocytes with extrahepatic obstruction [88].

Progressive familial intrahepatic cholestasis type 2 and the Dubin-Johnson syndrome are two inherited disorders associated with mutations in Bsep and in Mrp2, respectively [74], [89]. When these mutations were introduced into Bsep [90], [91] or Mrp2 molecules [89], [92], [93] and expressed in cell lines, some of these mutated proteins failed to traffic to the plasma membrane and were retained intracellularly and degraded mainly by the ubiquitin/proteasome system. This suggests that an impairment in the intracellular trafficking of Bsep or Mrp2 is involved in the molecular pathogenesis of these diseases.

As mentioned in the previous section, there is clear experimental evidence showing that the disruption of vesicular trafficking of key cholangiocyte transporters (i.e. AE2, CFTR, and AQP-1) markedly inhibits secretin-induced ductal bile flow [18], [67]. Interestingly, it has been recently described [94], [95] that the cAMP-dependent HCO3− and Cl− ductal bile secretion is impaired in inflammatory cholangiopathies (disorders in which cholangiocytes are the principal target cells of immune-mediated damage). Although the molecular pathogenesis of ductal cholestasis in cholangiopathies is still unclear, an altered trafficking of cholangiocyte transporters might certainly be involved.

Microtubules are dynamic structures that interact with intracellular vesicles by means of associated proteins which include the microtubular motors, dynein and kinesin [96]. As detailed in the previous section, a body of experimental evidence suggests a major role for microtubule-based vesicle trafficking in the movement of hepatic membrane transporters. Thus, an impairment of hepatic microtubular function by direct effects on microtubules or through inhibition of microtubular motors activity, as observed in experimental cholestasis [96], has pathophysiological potential by causing defective trafficking and targeting of key transporters.

There is significant support for the proposal that separate domains with different lipid and protein compositions can exist in cellular membranes. Detergent-insoluble membranes appear to arise from sphingolipid-cholesterol rich membrane domains ‘rafts’ in a tightly packed liquid ordered state [97]. These raft domains act as platforms to recruit proteins into these domains based on their biophysical properties, thereby coordinating a variety of cellular functions including protein trafficking and signal transduction [97]. Our unpublished observations suggest that rafts are present in hepatocytes and that AQP-8 water channels are targeted to raft domains. These lipid microdomains may represent localized target areas for clustering of transport systems involved in bile formation and may provide a molecular correlate for the impaired trafficking of key transporters and the consequent loss of osmotic driving forces for bile secretion can result in the functional changes that occur in cholestasis.

In conclusion, the vesicle trafficking of membrane transporters in hepatic epithelial cells seems to be central in the short-term regulation of bile formation. The cellular and molecular basis of certain liver diseases associated with bile secretory dysfunction may ultimately be understood in terms of derangement of normal intracellular trafficking of transport proteins.

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Acknowledgements 

This work was supported by Grant PICT 05-10590 (R.A. Marinelli) from Agencia Nacional de Promoción Científica y Tecnológica, and by Grant PIP 03020 (to R.A. Marinelli) from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), and by grant DK24031 (N.F. LaRusso) from the National Institutes of Health.

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PII: S0168-8278(05)00008-5

doi:10.1016/j.jhep.2005.01.001

Journal of Hepatology
Volume 42, Issue 4 , Pages 592-603, April 2005

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