Short-Term Regulation of Canalicular Transport

• ABSTRACT
• CANALICULAR AND SINUSOIDAL TRANSPORT SYSTEMS
• SHORT-TERM REGULATION OF CANALICULAR TRANSPORT
• Levels of Control: General Considerations
• Substrate Availability
• Covalent Modification
• Regulation by Transporter Insertion/Retrieval into/from the Canalicular Membrane
• Regulation by Cell Hydration
• Osmoregulation of Bile Acid Excretion
• Osmoregulation of Mrp2
• Regulation by LPS, Oxidative Stress, and Phalloidin
• Regulation by Bile Acids
• Short-term Regulation by Hormones
• Insulin
• Cyclic AMP
• Ca^{2 +}-mobilizing Hormones
• PERSPECTIVE
• ACKNOWLEDGMENTS
• ABBREVIATIONS
• REFERENCES

ABSTRACT

On a short term basis, canalicular secretion is under control of the hepatocellular hydration state, substrates, cytokines, toxins, and hormones. Regulation occurs at the level of substrate availability, covalent modification of transporters, and their regulated exocytic insertion into or endocytic retrieval from the membrane. A variety of signal transduction pathways involving the activation of mitogen-activated protein kinases, protein kinases A and C, participates in these processes. However, much has still to be learned about the crosstalk of different signaling systems and their molecular targets that determine the outcome for canalicular secretion.

Canalicular secretion is a complex process that involves basolateral and apical cell membrane transport. Regulation may occur at the level of gene expression, transporter degradation, and on a short-term basis at the level of substrate availability, covalent modification of transporters, and their regulated exocytic insertion into or endocytic retrieval from the membrane. From a teleologic point of view, short-term regulation of canalicular secretion is mandatory, because the load to the liver of cholephilic compounds may vary considerably within short time periods. One example is the bile acid load to the liver, which increases rapidly in response to oral feeding, gallbladder contraction, and exhibits circadian rhythmicity. Furthermore, biliary excretion of glutathione conjugates contributes to the control of the glutathione disulfide/glutathione (GSSG/GSH) ratio and thus to the defense against oxidative stress. It appears that the canalicular excretory capacity is finely tuned to many of these demands. Although considerable progress has been made in the past years in the characterization of the transport proteins at the genome and functional level and their regulation, many issues are still unsolved.

CANALICULAR AND SINUSOIDAL TRANSPORT SYSTEMS

Bile formation is an osmotic process driven by the vectorial transport of biliary constituents from the sinusoidal blood or the cellular interior into the bile canalicular lumen. Various transport systems in the sinusoidal (basolateral) and the canalicular (apical) membrane of the hepatocyte participate in this process and were characterized at the molecular level (for reviews, see Refs. 1-10). The Na⁺/K⁺-ATPase is located in the basolateral membrane. Its activity creates an electrochemical Na⁺ gradient, which provides the driving force for Na⁺-coupled transport systems in the plasma membrane. Among these, the Na⁺-taurocholate cotransporting polypeptide (Ntcp and NTCP in rat and human, respectively) is involved in bile formation and represents the major route for uptake of conjugated bile salts from the sinusoidal space into the hepatocyte.[11] Taurocholate transport via the Ntcp is electrogenic and exhibits an Na⁺/taurocholate stoichiometry of 2:1.[12] Unconjugated (but also conjugated) bile salts such as cholate and organic anions such as sulfobromophtalein are taken up by rather unspecific Na⁺-independent transport systems including the organic anion transporting polypeptide (Oatp1, OATP).[13] In addition, bile acids are taken up in a Na⁺-independent way by the recently identified liver specific organic anion transporter LST1 (rat counterpart, rlst1).[14] Oatp also mediates the Na⁺-independent transport of many amphipathic compounds, such as conjugates of estrogens, bile acids, leukotrienes, and xenobiotics, such as ajmalin or ouabain. Oatp acts as a 1:1 organic anion exchanger, and under physiologic conditions organic anion uptake is thought to be driven predominantly by a countertransport of intracellular glutathione (GSH).[15]

Canalicular secretion in liver is brought about by specific transport ATPases, which belong to the ATP-binding cassette (ABC) transporter superfamily with common structural and functional characteristics.[1] [7] These primary active transport systems can transport bile acids, other organic anions, conjugates, and xenobiotics against steep concentration gradients across the canalicular membrane. They include a canalicular transporter for bile salts (sister of P-glycoprotein [Spgp], bile salt export pump [Bsep]), the canalicular multidrug resistance protein MRP2 (Mrp2/cMRP/cMOAT) for transport of anionic conjugates (e.g., glutathione and glucuronide conjugates), the multidrug resistance P-glycoprotein (MDR1 and Mdr1a/b for human and rat, respectively) for excretion of chemotherapeutic agents, other amphiphilic organic cations and xenobiotic compounds, and the human MDR3 P-glycoprotein (in rat Mdr2) for phospholipid excretion (for reviews, see Refs. 4-10). These transport ATPases have been cloned and characterized at the molecular level.[16] [17] [18] [19] [20] Defective expression of MRP2, a 190-kDa integral glycoprotein in the apical membrane, underlies the human Dubin-Johnson syndrome.[21] A defective MDR3 gene is found in subgroups of patients with Byler's disease[22] and homozygous disruption of the murine Mdr2 P-glycoprotein impairs phospholipid excretion into bile and leads to liver disease.[23] Transporters with similar substrate specificity as the canalicular MRP2 have also been identified in the basolateral membrane (Mrp1 and 3) and further poorly characterized members of this transporter family (Mrp4-6).[8] [13]

SHORT-TERM REGULATION OF CANALICULAR TRANSPORT

Levels of Control: General Considerations

Regulation of canalicular secretion may occur on a long-term basis at the level of transporter gene expression and on a short-term basis at the levels of substrate availability, allosteric and covalent modification of canalicular transporter proteins, and their regulated exocytic insertion into or endocytic retrieval from the membrane (Fig. [1]). These processes underlie the control by multiple intracellular signaling pathways comprising of different second messenger and protein kinase/phosphatase systems. They allow rapid adaptations of canalicular secretion to environmental challenges. Further determinants of canalicular secretion or its efficacy for bile flow, such as tight junctional permeability or modification of primary bile composition by bile duct epithelia, are not considered here.

Substrate Availability

Substrate availability for canalicular transport ATPases is primarily determined by substrate delivery via the portal vein, the rate of uptake from the sinusoidal space, and the biotransformation/conjugation reactions that may yield high-affinity substrates for canalicular transport ATPases. Also, intrahepatocytic synthesis of bile acids has to be considered, which may not only produce physiologic bile acids, but also, in case of inborn errors of bile acid synthesis, toxic bile acids with inhibitory effects on canalicular secretion.[24] [25] Further, competition of different substrates for one transporter or the cooperative action of different substrates on the transport ATPases may influence transport. In general, the transport capacity of sinusoidal transporters exceeds that of canalicular transport systems. This led to the widely accepted view that under physiologic conditions the canalicular excretion step is rate limiting for overall transcellular transport of most cholephilic compounds.[26] [27] However, the control strength theory[28] has not yet been applied to transcellular transport. Thus, the possibility is not ruled out that significant control on bile formation may be exerted also at the step of uptake across the sinusoidal membrane. This may be relevant, especially at physiologically low bile acid concentrations or under pathophysiologic conditions, such as sepsis or estrogen treatment, in which the expression of Ntcp and other transporters is downregulated.[29] [30] [31] The view that sinusoidal bile acid uptake may exert significant control on canalicular excretion is further suggested by the finding that taurocholate transport capacity via Ntcp underlies short term control by cAMP, Ca^{2 +}/calmodulin, and activity of okadaic acid-sensitive protein phosphatases.[32] [33] [34] [35] Here, cAMP enhances the V_max of the transporter within minutes by a microfilament-dependent translocation of intracellularly stored Ntcp molecules to the plasma membrane.[34] [36] Thus, modulation of Ntcp transport activity by transporter recruitment to the sinusoidal membrane or by changes in the driving force of the transporter (i.e., the electrochemical Na⁺ potential of the plasma membrane) is expected to exert indirect control on canalicular bile acid secretion by the canalicular bile salt export pump (Bsep).

Theoretically, substrate availability for canalicular transporters may also be compromized by activation of alternative export routes across the basolateral membrane. Whereas Mrp2 is exclusively localized in the canalicular membrane, other Mrp isoforms (i.e., Mrp1,3,6) are basolateral[8] [13] and may, albeit normally expressed at low levels, compete with Mrp2 for substrates. This may occur for glutathione-S-conjugate excretion in hepatocytes entering the cell cycle, which exhibit a switch in expression from the apical Mrp2 to the basolateral Mrp1 transporting protein.[37] Whether such a switch can also occur on a short term time scale is yet unclear; however, hyperosmotic exposure of perfused rat liver leads within minutes to an increased release of 2,4-dinitrophenyl-S-glutathione into the perfusate at the expense of excretion into bile.[38]

Covalent Modification

Putative phosphorylation and glycosylation sites have been identified in most canalicular transport ATPases; however, the role of such covalent modifications for transport function is poorly understood. Glycosylation of the MDR1 P-glycoprotein seems not to be required for functional activity but may be essential for determining the sites of cellular localization.[7] [39] The transporter has phosphorylation sites accessible for protein kinase C (PKC); however, controversy exists with regard to the functional consequences (for review, see Ref. 7). Evidence has been presented that P-glycoprotein (Mdr1) can function as a volume-regulated Cl^- channel and PKC-dependent phosphorylation of MDR1 affects cell-swelling activated Cl^- currents.[40] There is also good evidence that canalicular secretion of Bsep and Mrp2 substrates is controlled by PKC, protein kinase A (PKA), and mitogen activated protein (MAP) kinases; however, it remains to be investigated to what extent the transport proteins themselves (in addition to other proteins that are indirectly involved in transport control) are the immediate target substrates for these protein kinases. The Ntcp is a serine/threonine phosphoprotein.[33] Phosphorylation is increased by okadaic acid and is decreased by cAMP. It was proposed that cAMP-induced dephosphorylation of Ntcp triggers an increased retention of the transporter in the plasma membrane.[35]

Regulation by Transporter Insertion/Retrieval into/from the Canalicular Membrane

Regulated recruitment of transport proteins into the plasma membrane in response to specific stimuli represents a widespread regulatory mechanism, by which the total number of active transporter molecules in the cell membrane and consequently V_max of transport can be modulated within minutes. Longer known examples are the insulin-induced insertion of the glucose transporter isoform 4 (GLUT-4) into the plasma membrane of adipocytes, the antidiuretic hormone (ADH)-regulated insertion of water channels in the cortical collecting duct, and the pH-regulated insertion of H⁺-ATPases in the proximal tubule (for reviews, see Refs. 41,42). The respective transporters are partly located in submembraneous vesicles, which can be inserted within minutes into the plasma membrane in response to appropriate stimuli but can also be retrieved again from the membrane and stored in vesicles underneath the plasma membrane. Recent data suggest that such a mechanism is also involved in the short-term regulation of canalicular secretion in liver and was shown to occur for Mrp2, Bsep, canalicular ecto-ATPase, and the canalicular Cl^-/HCO₃ ^- exchanger.[43] [44] [45] [46] [47] [48] The molecular mechanisms underlying this regulated transporter trafficking in nonhepatic cell types have been reviewed recently.[41] They involve a local depolymerization of the submembraneous F-actin filaments, which opens the physical barrier of submembraneous actin bundles to allow vesicle fusion with the plasma membrane and reversible phosphorylations of actin-binding proteins, small GTP-binding proteins, microtubules, and associated structures and a variety of docking proteins. Although most knowledge was obtained from studies in nonhepatic cells, it is likely that similar mechanisms are operative in hepatocytes.

Regulation by Cell Hydration

Liver cell hydration is dynamic and can change within minutes under the influence of hormones, oxidative stress, and substrate supply to the liver.[49] [50] Such physiologic short-term modulation of cell volume due to water shifts across the plasma membrane within a narrow range were identified as an independent and potent signal that modifies cellular metabolism and gene expression (for reviews, see Refs. 49-51). Different osmosignaling pathways have been characterized in liver that couple cell hydration changes to the functional consequences for metabolism and gene expression.[51] Also, canalicular secretion is controlled by the hepatocellular water content, (i.e., the hydration state or cell volume).

Osmoregulation of Bile Acid Excretion

Canalicular excretion is thought to represent the rate-controlling step for overall transcellular bile acid transport. In perfused rat liver, the rate of transcellular taurocholate (TC) transport from the sinusoidal space into the biliary lumen is critically dependent on the hydration state of the hepatocyte.[44] [52] [53] Cell shrinkage inhibits, whereas cell swelling stimulates TC excretion into bile, regardless of whether cell volume is modified by anisotonic exposure[52] or in response to cumulative amino acid uptake[52] or ethanol (S. vom Dahl and D. Häussinger, unpublished data, 1996). Regulation of TC excretion into bile by the hepatocellular hydration state is due to rapid changes of transport capacity: an increase of hepatocyte water content of about 10% doubles within minutes the V_max of TC excretion into bile. This increase of V_max is not explained by changes of the cellular ATP content or the membrane potential but is abolished in presence of colchicine.[44] [52] This indicates the requirement of intact microtubules for the interaction between cellular hydration and TC excretion into bile. Current evidence suggests that alterations of hepatocellular hydration induce rapid changes of the TC secretion capacity due to a microtubule-dependent insertion/retrieval of canalicular bile acid transporter molecules (Bsep) into/from the canalicular membrane. Such Bsep-containing subcanalicular vesicles have been identified by immunogold-labeling electron microscopy[20] and probably correspond to the known bile acid containing vesicles, which were seen in the past to reflect ``vesicular transcellular bile acid transport.'' The role of these vesicles, however, may reside in the transport of the bile acid transporter molecules rather than in the transport of bile acids. The osmoregulated Bsep insertion/retrieval in the intact perfused rat liver was demonstrated in a recent study from our laboratory by means of immunohistochemistry and confocal laser scanning microscopy, using the statistical methods described previously[38] [43] [48] (Fig. [2], A-E). In normoosmotic control perfusions, Bsep was localized largely in the canaliculi, but some Bsep-containing vesicles were also detectable inside the hepatocytes (Fig. [2], A, D). The hyperosmotic inhibition of taurocholate excretion into bile was accompanied by a statistically significantly increased appearence of immunoreactive Bsep in the subcanalicular area (Fig. [2], B, D), whereas hypoosmotic swelling increased Bsep localization in the canalicular membrane and also TC excretion (Fig. [2], C). However, it is not clear whether other as yet unidentified bile salt carriers and covalent modifications of the transporters will contribute to the osmoregulation of canalicular bile salt secretion.

The osmosignaling pathways that trigger the increase of TC excretion in response to hepatocyte swelling have been identified in part. In hepatocytes and H4IIE hepatoma cells, hypoosmotic cell swelling results within a few minutes in a pertussis-toxin and genistein-sensitive but PKC-independent activation of extracellular signal regulated protein kinases Erk-1 and Erk-2,[51] [54] [55] which are members of the MAP kinase family.[56] [57] These findings suggest that cell swelling leads to a G-protein-mediated activation of a yet unidentified tyrosine kinase, which triggers the activation of Erks. Ca^{2 +} transients are probably not involved in the hypoosmotic MAP kinase activation, because hypoosmotic exposure of hepatocytes did not affect the intracellular Ca^{2 +} concentration.[58] The functional significance of this swelling-activated signaling pathway toward Erk activation for the stimulation of TC excretion was shown in inhibitor studies.[55] [59] Cholera and pertussis toxin, genistein, erbstatin, and PD098059 (i.e., inhibitors of the osmosignaling events upstream of Erks) completely abolish both, the swelling-induced Erk activation and the swelling-induced stimulation of TC excretion. The latter is also sensitive to inhibition by colchicine, but this does not hold true for the hypoosmotic Erk activation.[60] This suggests that microtubules are involved downstream of Erks in the osmosignaling cascade toward TC excretion. Recent data also indicate a swelling-induced activation of the p38^MAPK, which mediates the inhibition of autophagic proteolysis in response to cell swelling.[61] Like hypoosmotic Erk activation, also p38^MAPK activation is colchicine insensitive.[62] The upstream events of the swelling-induced p38^MAPK activation are not yet known; however, specific inhibition of p38^MAPK by SB203580 largely inhibits the hypoosmotic stimulation of TC excretion (S. vom Dahl, M. Wettstein, and D. Häussinger, unpublished data, 1999). Because SB203580 does not affect the hypoosmotic Erk activation,[61] simultaneous activation of both Erks and p38^MAPK is apparently required for transmitting the choleretic effect of cell swelling. However, the signaling events downstream of MAP kinases, which may trigger the insertion of Bsep vesicles into the canalicular membrane, are largely unclear.

MAP kinases, which exhibit a modular organization,[57] have multiple protein substrates, which may be relevant for this process. These include the microtubule-associated proteins MAP-2 and Tau and other protein kinases, such as S6 kinase and the MAP-kinase signal integrating protein kinase (MNK-1),[57] whose activation requires signal input from both Erks and p38^MAPK. Evidence has been given in several cell types that p38^MAPK and its downstream protein kinase substrate MAPKAP-2 is involved in the regulation of actin filament dynamics by phosphorylation of heat shock protein 27.[63] MAP kinases also affect microtubules, and this may relate to the stabilization of microtubules in response to cell swelling.[64] Interestingly, the osmosignaling pathway toward Erks also mediates an alkalinization of endocytotic vesicles,[58] [65] but it is not known whether alkalinization also occurs in the Bsep-bearing subcanalicular vesicles and whether this has relevance for the Bsep targeting to the canalicular membrane. However, given the important role of vacuolar acidification for receptor-ligand sorting, exocytosis, and protein targeting,[66] one is tempted to speculate that cellular hydration may also interfere with these processes. Figure [3]A depicts a current model for the osmosignaling that links cell swelling toward stimulation of TC excretion.

TC was shown to induce hepatocyte swelling,[52] [67] which by itself is expected to trigger an increase in the TC excretory capacity due to Bsep insertion into the canalicular membrane. This may represent a feed-forward control mechanism, which couples, via cell swelling, an increased TC load to the liver to an increased capacity of canalicular bile acid excretion.[55]

Interestingly, hypoosomotic liver cell swelling also increases Bsep gene expression, whereas hyperosmotic cell shrinkage downregulates expression of the transporter.[68] Thus, cell hydration controls canalicular bile acid excretion at the levels of both short- and long-term regulatory mechanisms. It should be noted that hypoosmotic cell swelling also exerts a hepatoprotective effect against a variety of noxes,[69] [70] and considerably higher concentrations of bile acids are required to induce the known cholestatic effect of high concentrations of taurocholate in swollen than in shrunken liver cells.[52]

Osmoregulation of Mrp2

Liver cell hydration also affects the function of the canalicular multispecific organic anion transporter Mrp2. In line with this, hepatocyte swelling in response to hypoosmolarity or addition of glutamine can increase within minutes the biliary excretion of cysteinyl-leukotrienes[71] and of dinitrophenol-S-glutathione (DNP-SG)[38] in lipopolysaccharide (LPS)-treated ratlivers, whereas hyperosmotic cell shrinkage inhibits DNP-SG excretion into bile. Again, canalicular secretion by the MRP2 export pump is regulated by rapid osmodependent carrier insertion and retrieval into or from the canalicular membrane, as shown by confocal laser scanning microscopy.[43] Here, hyperosmotic cell shrinkage induces the retrieval of the carrier from the canalicular membrane and its storage in intracellular vesicles beneath the canalicular membrane, whereas subsequent hypoosmotic cell swelling reverses the process by a rapid reinsertion of the transporter into the canalicular membrane[43] (Fig. [2], F-H). These findings were confirmed by immunogold labeling electron microscopy studies.[72] Hyperosmotic retrieval of Mrp2 is not accompanied by significant morphologic alterations of the canaliculi at the electron microscopic level.[72] It also exhibits selectivity in that dipeptidylpeptidase IV, another canalicular marker protein, is not simultaneously retrieved from the canalicular membrane.[72] In addition to short-term regulation of Mrp2 function by rapid carrier insertion/retrieval, osmolarity also affects Mrp2 expression. Hypoosmolarity increases, whereas hyperosmolarity decreases mRNA and protein levels of MRP2 in isolated rat hepatocytes.[73] Thus, as it is observed for Bsep,[68] also Mrp2 regulation by cell hydration occurs at the level of both short-term regulation and gene expression.

Regulation by LPS, Oxidative Stress, and Phalloidin

Cholestasis is frequent in sepsis, and the underlying mechanisms were studied in animal models after endotoxin application. In rat liver, in vivo administration of LPS decreases canalicular anion and bile acid transport[48] [74] and leads to a downregulation of Mrp2 and Bsep at the protein and mRNA level.[48] [68] [75] [76] These effects on gene expression become apparent about 6-12 hours after endotoxin application; however, an LPS-induced inhibition of TC and bromosulfalein (BSP) excretion by about 50% is already observed within 3 hours of LPS administration (i.e., at a time point when MRP2 mRNA levels were still unaltered).[48] Confocal laser scanning studies revealed that endotoxin causes within 3 hours a retrieval of MRP2 from the canalicular membrane,[48] which probably explains the early phase of cholestasis after LPS administration. Here, the transporter is found in putative subapical vesicles in the immediate vicinity of the canaliculi, as revealed by confocal laser scanning microscopy. At later time points (i.e., 6 and 12 hours after the LPS challenge) these vesicles are found more distant from the canaliculi (Fig. [2]J), and it was speculated that they may have entered a lysosomal compartment for degradation. During the first 3 hours of LPS treatment but not thereafter, the subapical MRP2-containing vesicles can probably be reinserted into the canalicular membrane in response to hypoosmotic hepatocyte swelling.[48] This is suggested by the finding that hypoosmotic exposure could restore BSP excretion to control values in livers from rats which received the LPS injection 3 hours before the experiment, whereas hypoosmolarity was ineffective in those which received LPS 6 or 12 hours before the perfusion experiments. In livers from NaCl-injected control animals, hypoosmolarity was without effect on BSP excretion. This was an expected finding, because MRP2 was already localized in the canalicular membrane during normoosmotic conditions. These data suggest a potential for reversibility of the LPS-induced MRP2 retrieval from the canalicular membrane at early stages of endotoxinemia but not at later time points. Like Mrp2, also Bsep is retrieved from the canalicular membrane after LPS treatment (R. Kubitz, M. Schmitt, and D. Häussinger, unpublished observation, 2000). Thus, LPS induces cholestasis in a short-term manner by transporter retrieval from the canalicular membrane and on a long-term basis by a downregulation of MRP2 expression.

Interestingly, administration of dexamethasone prevented both the LPS-induced retrieval of MRP2 from the canalicular membrane and the downregulation of MRP2 mRNA and protein.[48] MRP2 retrieval from the canalicular membrane under the influence of LPS was not accompanied by a retrieval of dipeptidyl peptidase IV (DPPIV)[48] (Fig. [2], I, J). This suggests selectivity of the MRP2 retrieval process. LPS-induced retrieval of Mrp2 from the canalicular membrane was also demonstrated by means of immunogold labeling of the transporter and electron microscopy[72]; however, in these studies also a substantial amount of Mrp2 was found inside the hepatocytes under control conditions. This is at variance to the immunohistochemistry confocal laser scanning microscopy findings. The reason for this is unknown but may relate to the higher resolution of electron microscopy compared with confocal laser scanning microscopy. The factors that determine the fate of the transporters after retrieval from the canalicular membrane are unclear, especially the control points when the retrieved transporters lose their potential for reinsertion and irreversibly enter a degradative pathway remain to be elucidated. It is also unclear whether the putative vesicles containing retrieved Mrp2 also contain other canalicular transport ATPases or whether there are distinct vesicle populations with specificity for a given transporter type. If the latter were true, one may hypothesize the existence of clusters enriched in one transporter type in the canalicular membrane and consequently the existence of organized functional transport domains in the canaliculi.

Oxidative stress is known to induce cholestasis.[38] [77] In isolated perfused rat liver almost complete cholestasis develops within a 30- to 60-min infusion period of t-butylhydroperoxide (BOOH; 0.5 mmol/l) or CDNB (0.1 mmol/l). This is accompanied by severe depletion of cellular glutathione either due to an increased GSSG formation at glutathione peroxidase under the influence of BOOH or an increased glutathione-S-conjugate formation with CDNB (DNP-SG) at glutathione transferases. As shown by confocal laser scanning microscopy, both compounds led to a rapid retrieval of Mrp2 from the canalicular membrane and its appearence in vesicular structures inside the cell. Again these putative vesicles do not contain dipeptidyl peptidase IV, and neither BOOH nor CDNB induced an internalization of DPPIV or affected ZO-1 localization.[38] This selectivity suggests that Mrp2 retrieval in response to oxidative stress is a regulated process rather than the consequence of an unspecific toxic cell damage. The mechanisms underlying the BOOH- and CDNB-induced retrieval of Mrp2 from the canalicular membrane are unknown; however, an oxidation of critical thiol groups due to glutathione depletion and hepatocyte shrinkage, which is induced by these compounds,[38] may contribute. Short-term Mrp2 retrieval from the canalicular membrane during severe oxidative stress may be a beneficial adaptation of the hepatocyte to this challenge, because it reduces the cellular loss of glutathione, albeit in the form of GSSG. Indeed, some evidence suggests that inhibition of GSSG secretion can protect hepatocytes from injury during oxidative stress.[10] [78] Here, this effect apparently outweighs the known inhibition of glutathione reductase by GSSG,[79] which impairs the cells' antioxidant machinery.

Whereas MRP2 retrieval from the canalicular membrane in response to LPS, BOOH, and CDNB is without effect on the canalicular localization of DPPIV, the situation is different for phalloidin-induced cholestasis. This toxin interacts with microfilaments and its application results within 30 min in a progressive loss of Mrp2 from the canalicular membrane and its appearence in intracellular membrane structures together with other canalicular membrane proteins including P-glycoproteins, ecto-ATPase, and DPPIV but not of structures involved in intercellular adhesion.[80] This suggests that phalloidin, but not LPS or CDNB, induces marked alterations of the hepatocyte canalicular membrane.

Regulation by Bile Acids

Bile acids are not only substrates for canalicular transport systems and a major osmotic driving force in bile formation but are also involved in the regulation of bile formation. This includes not only effects of bile acids on paracellular permeability,[81] canalicular structure,[82] gene expression of basolateral transporters[83] and enzymes involved in bile acid synthesis,[84] mobilization of biliary lipid from the outer leaflet of the canalicular membrane,[85] but also short-term regulation of canalicular transport. The potency of individual bile acids for these effects is variable, probably depends on their chemical structure and tauroursodeoxycholate (TUDC) and TC have been studied most extensively. Early studies have demonstrated that TC can increase the transport maximum for Mrp2 substrates[86] [87] [88] and stimulate horseradish peroxidase excretion into bile,[89] suggestive for stimulation of a transcellular transcytotic pathway.[89] Despite competition for transport across the sinusoidal and canalicular membrane, TUDC increases the V_max of TC excretion into bile in perfused rat liver within 20 min.[52] This stimulatory effect of TUDC on the TC excretory capacity was abolished by colchicine,[44] suggestive for a TUDC-induced microtubule-dependent recruitment of bile acid transporters to the canalicular domain of the hepatocyte. In line with this, TUDC stimulates exocytosis and the biliary secretion of horseradish peroxidase in perfused rat liver.[90]

Several signal transduction pathways have been identified that may mediate the effects of bile acids on vesicular exocytosis and the putative transporter insertion into the canalicular membrane. These include bile acid effects on mitogen-activated protein kinases, PKC and PKA, and intracellular Ca^{2 +}.

Low concentrations of TUDC activate within minutes MAP kinases of the Erk[59] and p38^MAPK type (A.K. Kurz, D. Graf, and D. Häussinger, unpublished data, 2000). Whereas the upstream signaling events for TUDC-induced p38^MAPK activation are unknown, those for the activation of Erks were studied in more detail.[59] [91] TUDC-induced Erk activation occurs independent of PKC and requires concentrative TUDC uptake into the hepatocyte, because it is no longer observed after downregulation of Ntcp in prolonged hepatocyte culture.[59] In contrast to hypoosmotic Erk activation,[54] [55] TUDC-stimulation of Erks is not sensitive to inhibitors of G-proteins and tyrosine kinases[59] but occurs via a phosphatidylinositol-3 kinase (PI₃-kinase) dependent Ras activation.[91] An involvement of the Raf/MEK pathway in the TUDC-induced Erk activation was shown by the inhibitory effect of PD098059 and maneuvres that increase the levels of cAMP.[59] cAMP is known to inhibit this signaling pathway via PKA at the level of Ras/Raf.[92] These studies suggest that TUDC activates a signal transduction pathway involving PI₃-kinase/Ras/Raf/MEK toward Erks (Fig. [3]B). The functional significance of this pathway for TUDC-stimulated bile acid excretion was shown in perfused rat liver. Inhibition of TUDC-induced Erk activation at the level of either PI₃ kinase by wortmannin or LY294002, Ras/Raf by cAMP, or MEK by PD098059 completely abolished the stimulation of TC excretion by TUDC.[59] [91] [93] This stimulation was also largely inhibited when the TUDC-induced activation of p38^MAPK was inhibited by SB203580 (S. vom Dahl, A.K. Kurz, and D. Häussinger, unpublished data, 1999). These findings suggest that the rapid stimulatory effect of both hypoosmotic hepatocyte swelling and TUDC on canalicular bile acid excretion is explained by an activation of both Erks and p38^MAPK, which may trigger in a microtubule-dependent way the recruitment of Bsep to the canalicular membrane. However, TUDC and osmosignaling differ in the signaling events upstream of Ras/Raf (Fig. [3]B).

In isolated rat hepatocytes, TUDC at low concentrations causes a marked and sustained elevation of the intracellular Ca^{2 +} concentration,[90] [94] which results from an initial mobilization from IP₃-sensitive Ca^{2 +} stores through an IP₃-independent mechanism and a subsequent influx of extracellular Ca^{2 +} via Ni^{2 +}-sensitive Ca^{2 +} channels (for review, see Ref. 95). Sustained increases in the cytosolic Ca^{2 +} concentration stimulate exocytosis in a variety of cells (for review, see ref. 96), and evidence has been presented for a role of TUDC-induced [Ca^{2 +}]_i increases for the TUDC-induced stimulation of exocytosis and horseradish peroxidase secretion.[90] Ca^{2 +}-dependent and Ca^{2 +}-independent pathways for MAP kinase activation have been described[97]; however, it is unclear whether the TUDC-induced increase of [Ca^{2 +}]_i is involved in TUDC-induced MAP kinase activation. In isolated rat hepatocytes, TUDC-induced increases in [Ca^{2 +}]_i are accompanied by an activation and selective translocation of the α-isoform of PKC to the cell membrane.[98] [99] From these data, it was suggested that stimulation of canalicular secretion by TUDC involves a Ca^{2 +} and PKC-dependent activation of vesicular exocytosis and thereby insertion of transport proteins into the canalicular membrane.[90] [98] However, it remains to be established whether exocytosis, when assessed by horseradish peroxidase excretion, also involves vesicles carrying the Bsep. Apart from Ca^{2 +}, several mechanisms may underly the TUDC-induced PKC activation, such as increased formation of diacylglycerol,[98] direct effects of bile acids on PKC,[100] or diacylglycerol stabilization in the plasma membrane.[101] Ursodeoxycholic acid-induced activation of PKC-α was suggested to be involved in the bile acid-induced inhibition of cAMP synthesis by glucagon.[102] Such a mechanism could provide one link between PKC and MAP kinase signaling pathways and augment the stimulation of bile acid excretion by TUDC, because cAMP-via PKA-is known to inhibit TUDC-signaling toward Erks at the level of Ras/Raf[59] and to induce MAP-kinase phosphatase-1 (MKP-1), which triggers inactivation of Erks.[103] Further studies are required to get more insight into the cross-talk between the different signaling systems that are affected by bile acids and into their relevance for canalicular excretion.

Short-term Regulation by Hormones

The short-term regulation of biliary secretion by hormones has been studied in isolated perfused liver and isolated hepatocytes, and besides insulin, hormones that increase cAMP, activate PKC, or are linked to a Ca^{2 +} mobilizing device were found to be effective modulators of bile secretion.

Insulin

In isolated perfused rat liver, insulin leads to a rapid increase of V_max of TC excretion.[52] This effect may at least in part involve insulin-induced cell swelling, which was shown to be important for triggering the insulin-induced inhibition of proteolysis.[61] [104] Like TUDC or hypoosmotic hepatocyte swelling, also insulin simultaneously activates both p38^MAPK and Erks.[61] It is therefore conceivable that the choleretic effect of insulin likewise resides in an agonist-induced insertion of Bsep into the canalicular membrane, as it was shown to occur in response to hypoosmotic swelling. Interestingly, hyperosmolarity and loop diuretics inhibit the insulin-induced activation of both p38^MAPK and Erks and confer insulin resistance by blocking a swelling component within the insulin signaling pathway.[105] Whether such a mechanism contributes to cholestasis under in vivo conditions, however, remains to be demonstrated.

Cyclic AMP

Besides stimulating bile acid synthesis,[106] glucagon and cAMP were shown to stimulate sinusoidal bile acid uptake[34] [35] [36] [107] and bile secretion in isolated rat hepatocytes.[46] [47] [108] [109] [110] [111] The latter effect is microtubule dependent[108] [110] [111] and involves an increased vesicular transport of horseradish peroxidase in perfused liver.[110] More recently, evidence has been given that cAMP and its analogues stimulate targeting of vesicles containing the Cl^-/HCO₃ ^- exchanger, ecto-ATPase,[46] and Mrp2[109] to the apical membrane in isolated rat hepatocyte couplets. Further, in this experimental model, cAMP significantly increases the excretion of bile acids[47] and Mrp2 substrates[109] and the pseudocanalicular circumference[47] and transport of sphingolipids to the bile canalicular membrane.[85] These findings are consistent with an overall stimulating effect of cAMP on apically directed membrane traffic including transporter insertion into the canalicular membrane, which may underlie the choleretic effect of cAMP. This process may not only involve the delivery of apical transporters, which were endocytosed during the cell preparation procedure, but also of newly synthesized apical transport proteins to the canalicular domain and the recruitment of transporters from a regulated subapical sorting compartment. The mechanisms underlying the glucagon- or cAMP-induced apical targeting are unclear but may involve activation of PKA and a glucagon-induced increase of intracellular Ca^{2 +}. In addition to cAMP, also cGMP and nitric oxide were suggested to stimulate canalicular secretion.[112]

Also in perfused rat liver, cAMP and its analogues increased TC and phospholipid excretion into bile. However, the effect on TC excretion is transient and only observed during the first 10 min,[113] [114] whereas thereafter TC excretion was no longer stimulated despite the continuing presence of cAMP.[59] Several mechanisms may contribute to the disappearence of the stimulatory cAMP effect, such as a glucagon- or cAMP-induced hepatocyte shrinkage,[104] [115] which is known to decrease TC excretion[52] and MAP kinase inhibition at the level of Ras/Raf[59] and a glucagon- or cAMP-mediated induction of MAP-kinase phosphatases.[103]

Ca^{2 +}-mobilizing Hormones

In isolated perfused rat liver, addition of vasopressin[116] [117]; extracellular ATP[118]; prostaglandin F_2α, D₂, and E₂ [119]; and perivascular nerve stimulation[120] produce a decrease in bile flow after a frequently observed initial transient increase. Inhibition of bile flow by these maneuvres is not explained by the accompanying hemodynamic changes but may be the result of complex effector-induced alterations triggered by an increase of the intracellular calcium concentration and PKC activation. This Ca^{2 +}/PKC interrelationship and the complexity of Ca^{2 +} and PKC actions may explain in part contradictory findings on bile secretion, which may also depend on the agonist concentration used. For example, high doses of endothelin-1 reduce bile flow and inhibit bile acid secretion, whereas low concentrations of this agonist enhance bile flow, bile acid, and horseradish peroxidase excretion into bile.[121] Vasopressin was reported to stimulate apical exocytosis,[122] consistent with a role of Ca^{2 +} and PKC in vesicle trafficking.[96] The Ca^{2 +}-ionophore A23187 and Ca^{2 +}-mobilizing hormones were reported to decrease with some delay hepatocellular bile acid uptake[95] [123] and to inhibit the cAMP-induced increase in TC uptake.[32] This may contribute to the decrease in bile formation in response to Ca^{2 +}-mobilizing agents. On the other hand, bile acid secretion is stimulated upon agonist-induced elevations of intracellular Ca^{2 +} (for review, see Ref. 95). This may involve not only effects on canalicular transport systems, their targeting to the canalicular membrane, but also on canalicular membrane contractility.[124] However, vasopressin was also found to induce canalicular dysfunction and impairment of tight junctional permeability.[125]

In perfused rat liver, PKC agonists decrease bile flow,[126] which may in part result from a PKC-induced increase in tight junctional permeability[127] and an inhibition of basolateral bile acid uptake.[32] On the other hand, PKC activation by TUDC was suggested to enhance biliary excretion (see above); furthermore phorbol esters and vasopressin stimulate organic anion efflux from hepatocytes[128] suggestive for a stimulation of canalicular secretion via Mrp2. This stimulatory effect was sensitive to staurosporine, in line with an involvement of PKC. The Mrp2 transporter contains putative PKC phosphorylation sites[129]; however, their role for Mrp2 function is unclear. This also holds for Mdr1 glycoprotein, which is known to be phosphorylated by PKC (for review, see Ref. 7). In perfused rat liver, activation of PKC stimulated exocytosis[122] and in MDCK cells phorbol esters stimulate apical delivery of proteins that are endocytosed from either the basolateral or the apical surface.[85] In isolated hepatocyte couplets, however, phorbol esters blocked the cAMP-induced apical targeting of the Cl^-/HCO₃ ^- exchanger[46] and inhibited in HepG2 cells apical sphingolipid transport and stimulated the disappearance of canaliculi.[130] Thus, although there is ample evidence for a regulation of canalicular secretion by PKC, no clear-cut picture has emerged yet. This may be explained not only by differences in the experimental systems used but also by the existence of different PKC isoforms with different functions and the crosstalks between PKC isoforms and other signal transduction pathways, whose input may finally determine the functional outcome.

PERSPECTIVE

The understanding of the regulation of canalicular secretion has considerably increased since the discovery of various genes involved in hepatobiliary transport and the list of such genes will continue to grow. These advances have identified the molecular basis for several inborn and acquired cholestatic syndromes during the last years. More detailed knowledge of the signal transduction events that control transporter gene expression and their short-term regulation is expected to offer new therapeutic approaches for cholestatic diseases and to provide an understanding of drugs that have been empirically used for many years. One example is ursodeoxycholate, which exerts its beneficial action on canalicular secretion by a modulation of intracellular signal transduction mechanisms. Improved knowledge of such signaling networks could allow for the design of more potent and specific drugs in the future. It is hoped that this review will stimulate research in this exciting area.

ACKNOWLEDGMENTS

Supported by Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie.

ABBREVIATIONS

aPKC protein kinase

BOOH tert-butylhydroperoxide

Bsep bile salt export pump

BSP bromosulphalein

CDNB 1-chloro-2,4-dinitrobenzene

cTC taurocholate

DNP-SG dinitrophenol-S-glutathione

GSH glutathione

GSSG glutathione disulfide

LPS lipopolysaccharide, endotoxin

MAPK mitogen-activated protein kinase

MDR multidrug resistance protein

Mrp, MRP multidrug resistance protein

Mrp2, MRP2 canalicular isoform of the

multidrug resistance protein

PKA protein kinase

Spgp sister of P-glycoprotein

TUDC tauroursodeoxycholate

ZO-1 polypeptide associated with the tight

junction (zonula occludens)

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