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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

The canalicular plasma membrane is constantly exposed to bile acids acting as detergents. Bile acids are essential to mediate release of biliary lipids from the canalicular membrane. Membrane microdomains (previously called lipid rafts) are biochemically defined by their resistance to detergent solubilization at cold temperature. We aimed to investigate the canalicular plasma membrane for the presence of microdomains, which could protect this membrane against the detergent action of bile acids. Highly purified rat liver canalicular plasma membrane vesicles were extracted with 1% Triton X-100 or 1% Lubrol WX at 4°C and subjected to flotation through sucrose step gradients. Both detergents yielded detergent-resistant membranes containing the microdomain markers alkaline phosphatase and sphingomyelin. However, cholesterol was resistant to Lubrol WX solubilization, whereas it was only marginally resistant to solubilization by Triton X-100. The microdomain marker caveolin-1 was localized to the canalicular plasma membrane domain and was resistant to Lubrol WX, but to a large extent solubilized by Triton X-100. The two additional microdomain markers, reggie-1 and reggie-2, were localized to the basolateral and canalicular plasma membrane and were partially resistant to Lubrol WX but resistant to Triton X-100. The canalicular transporters bile salt export pump, multidrug resistance protein 2, multidrug resistance-associated protein 2, and Abcg5 were largely resistant to Lubrol WX but were solubilized by Triton X-100. Conclusion: These results indicate the presence of two different types of microdomains in the canalicular plasma membrane: “Lubrol-microdomains” and “Triton-microdomains”. “Lubrol-microdomains” contain the machinery for canalicular bile formation and may be the starting place for canalicular lipid secretion. (HEPATOLOGY 2009.)

Bile formation involves vectorial secretion of bile acids and other cholephilic compounds across hepatocytes from the sinusoidal blood plasma into bile canaliculi.1, 2 The main bile constituents are bile salts, organic anions, phospholipids, and cholesterol, which aggregate in bile into mixed micelles.3 Hence, bile fluid is exquisitely suited for the excretion of water-insoluble substances. The most important transporters involved in canalicular secretion of bile salts, organic anions and phosphatidylcholine, are the bile salt export pump (Bsep) (Abcb11) (BSEP in humans),4, 5 the multidrug resistance-associated protein 2 (Mrp2) (Abcc2) (MRP2 in humans),6 and the phosphatidylcholine translocator Mdr2 (Abcb4) (MDR3 in humans),7 respectively. Export of cholesterol from the canalicular plasma membrane (cLPM) is mediated by the heterodimeric protein Abcg5/Abcg8 (ABCG5/ABCG8 in humans).8 All these transporters are members of the adenosine triphosphate (ATP) binding cassette (ABC) protein superfamily and utilize ATP hydrolysis for transport function.

Canalicular phospholipid secretion has been extensively studied both in inherited human liver disease and in animal models. The results indicate that, once secreted into the bile canaliculi, bile salts extract phosphatidylcholine from the outer leaflet of the cLPM and solubilize it within mixed micelles.3 This principle has been worked out in various animal models that demonstrated that in the absence of bile salt secretion canalicular phospholipid secretion ceases. However, in mice with disrupted Mdr2 function, phospholipid secretion cannot be stimulated, even at high rates of bile salt output.9 This highlights the importance of functional Mdr2 for canalicular phospholipid secretion. Furthermore, inherited forms of human liver diseases with defective expression and/or function of BSEP (e.g., progressive familial intrahepatic cholestasis type 2, PFIC 2) or of MDR3 (PFIC 3) provide strong evidence that the same mechanism of biliary phospholipid secretion is also valid in human liver.10 Furthermore, the finding that animals without biliary phospholipid secretion display no biliary cholesterol secretion9 demonstrates that the presence of mixed bile salt/phospholipid micelles is required in bile in order to maintain normal cholesterol secretion. In addition, the heterodimeric ABC transporter Abcg5/Abcg8 is involved in canalicular cholesterol secretion because (1) mice with disrupted Abcg5 or Abcg8 display a marked reduction in biliary cholesterol secretion,11 and (2) patients with mutations in the ABCG5/ABCG8 gene display hypercholesterolemia and β-sitosterolemia.8 Hence, at least the three ABC-transporters Bsep, Mdr2, and Abcg5/Abcg8, are needed for maintenance of normal biliary cholesterol secretion.3 The exact role of an ATP-independent phosphatidylcholine translocator that is also expressed at the cLPM is not understood at present.12, 13

In vitro, bile salts preferentially extract phosphatidylcholine from the cLPM, albeit phosphatidylcholine represents only 35% of total canalicular phospholipids14, 15 and the cLPM has an exceptionally high sphingomyelin content.15 This unique property of the cLPM could be due to the presence of lipid microdomains such as, for example, detergent-sensitive membrane regions enriched in phospholipids and detergent-resistant sphingomyelin/cholesterol clusters, which are also called lipid rafts.16, 17 In such microdomains, sphingomyelin and cholesterol are arranged in a tightly packed, liquid-order state16 forming highly dynamic structures, which exist in short length and time scales.18 Recently, evidence has been provided for the presence of coexisting raft and nonraft microdomains in both the basolateral plasma membrane domain (blLPM) and the cLPM domain of rat hepatocytes.19, 20 In these studies the Triton X-100-resistant lipid microdomains were enriched in alkaline phosphatase (AP), caveolin-1, cholesterol, sphingomyelin, the ganglioside GM1, and the aquaporins 8 (canalicular) and 9 (basolateral).20 Other studies have indicated that the hepatocyte plasma membrane might contain at least two different pools of cholesterol and caveolin-1-enriched microdomains, the major one (≈90%) being soluble and the minor one (≈10%) being insoluble in Triton X-100 (1%).21 Furthermore, the coexistence of different cholesterol-enriched lipid microdomains has been proposed in the apical membrane of Madin-Darby canine kidney (MDCK) cells based on its relative solubility in the nonionic detergents Triton X-100 (“Triton microdomains”) and Lubrol WX (“Lubrol microdomains”).22 And finally, in neurons and astrocytes, caveolin-1-negative lipid microdomains have been identified that are associated with the marker proteins reggie-1/flotillin-2 and reggie-2/flotillin-1.23–26 Reggie proteins are widely expressed, form oligomers at the cytoplasmic face of the plasma membrane,27 and scaffolds of plasma membrane microdomains, which are clearly distinct from caveolae.28 Reggie microdomains represent platforms for multiprotein complex formation and signal transduction in a cell-type and situation-specific manner.29, 30 They communicate, for instance, with Rho- GTPases and regulate actin cytoskeleton dynamics.31 Hence, heterogeneous lipid microdomains might coexist at the two polar plasma membrane domains of epithelial cells and differentially influence vectorial transport processes such as hepatocellular bile formation.

In this study we investigated the hypothesis that distinct lipid microdomains with different protein and lipid compositions and different sensitivities toward nonionic detergents (Lubrol WX, Triton X-100) might coexist at the cLPM of rat hepatocytes. Furthermore, we wondered whether the canalicular ABC transporters are partially or even completely compartmentalized into distinct lipid microdomains.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Antibodies and the methods describing the extraction and analysis of lipids, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, immunofluorescence, and immunoelectron microscopy are in the online supporting material.

Animals.

Male Sprague-Dawley rats (180-200 g) were obtained from Harlan (Horst, The Netherlands). They received humane care in accordance with local and federal guidelines and regulations were kept under standard conditions.

Chemicals.

Lubrol WX and Triton X-100 were purchased from Serva Feinbiochemica (Heidelberg, Germany) and MP Biomedicals (Irvine, CA), respectively. Lipid standards were from Sigma-Aldrich (St. Louis, MO). All other chemicals were of the highest grade and readily available from commercial sources.

Isolation of Detergent-Resistant Microdomains (DRMs) from Highly Purified cLPM.

cLPM were isolated as described32 and stored in liquid nitrogen until use. DRMs were isolated by performing all steps at 4°C with minor modifications as described.33 Briefly: cLPM (1 mg protein) were thawed on ice and diluted to 1 mL in TNE (150 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH 7.4) containing antipain and leupeptin 1 μg/mL each and 1 mM phenylmethylsulfonylfluoride. The suspension was mixed with 1 mL of either 2% (wt/vol) Lubrol WX or 2% (wt/vol) Triton X-100 in TNE (yielding a detergent to protein ratio [wt/wt] of 20) and homogenized by passing it 10 times through a 25-gauge needle. After incubation for 30 minutes on ice, the suspension was brought to 40% (wt/vol) sucrose by adding 2 mL 80% (wt/vol) sucrose in TNE. This mixture was overlaid with 4 mL 35% (wt/vol) and 4 mL 5% (wt/vol) sucrose both in TNE. The DRMs were floated by centrifugation for 18 hours at 40,000 rpm (270,000gav), 4°C in a TST41.14 swinging bucket rotor from Kontron Instruments (Schlieren, Switzerland). Twelve 1-mL fractions were harvested from the top and the pellet was resuspended in 1 mL TNE by vigorous vortexing and 10 passages through a 25-gauge needle. AP activity in the presence of 0.2 mM ZnSO4 was measured immediately as described.34 For all other analyses the fractions were stored in aliquots at −80°C until used.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

In order to test whether the cLPM of rat liver contains DRMs, we exposed cLPM vesicles from rat liver to the two nonionic detergents Lubrol WX and Triton X-100. As indicated in Fig. 1, after cold extraction of cLPM with 1% (wt/vol) Lubrol WX 24% of total cLPM protein (Fig. 1A) and 88% of total canalicular AP activity (Fig. 1B) floated to the top of the 35% sucrose layer during ultracentrifugation (fractions 3-5, Fig. 1), i.e., they were not solubilized by Lubrol WX and, thus, associated with Lubrol WX-resistant membrane microdomains (so called “Lubrol-microdomains”). For 1% (wt/vol) Triton X-100, the corresponding numbers were 10% (total protein) and 85% (AP activity), respectively (Fig. 1). Hence, the vast majority of the canalicular AP activity was recovered in the DRMs after extraction with either detergent. Using 2% (wt/vol) instead of 1% (wt/vol) Triton X-100 showed no difference in the distribution of AP activity after flotation, whereas extraction with 0.5% (wt/vol) Triton X-100 resulted in a lower recovery of detergent-resistant AP activity (data not shown). Consequently, subsequent experiments were performed with 1% (wt/vol) Triton X-100. Increasing Lubrol WX concentration to 2% (wt/vol) did not alter the amount of protein floating to the DRMs, nor the distribution pattern of canalicular proteins between solubilized and DRM fractions on the gradient (data not shown). Therefore, a Lubrol WX concentration of 1% (wt/vol) was chosen for this study. These data indicate that the cLPM of rat liver contains indeed DRMs that harbor a significant proportion of membrane-associated proteins and are highly enriched in glycosylphosphatidylinositol (GPI)-anchored AP, which represents a typical marker enzyme for DRMs.35

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Figure 1. Distribution of total protein (A) and AP (B) in detergent resistant microdomains (DRMs) isolated from cLPM. cLPM were extracted with 1% (wt/vol) Lubrol WX or 1% (wt/vol) Triton X-100 and floated on discontinuous sucrose gradients as described in Materials and Methods. Twelve 1-mL fractions were collected from the top and analyzed for protein content and AP activity. Low-density fractions 3-5 contained detergent-resistant microdomains (i.e., “Lubrol-microdomains” [left panels] and “Triton-microdomains” [right panels]), high-density fractions 9-12 contain solubilized proteins. (A) The protein concentration in each fraction was determined as described in Materials and Methods. The total recovery in relation to the input was 95% for Lubrol WX and 93% for Triton X-100. (B) AP activity was measured in the various fractions as described in Materials and Methods. AP was quantitatively recovered in both “Lubrol-microdomains” and “Triton-microdomains.” The total recovery of AP relative to the total inputs was 95% for Lubrol WX and 96% for Triton X-100. One representative result out of at least two independent experiments is shown.

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To further characterize the canalicular “Lubrol-microdomains” and “Triton microdomains,” we analyzed the low-density membrane material recovered from fractions 3-5 with respect to additional DRM marker proteins such as caveolin-1 and reggie-1 and reggie-2. In addition, the cytoskeletal protein actin was probed to investigate a possible association of cytoskeletal elements with the two canalicular DRMs. As shown in Fig. 2, caveolin-1 was mostly resistant to Lubrol WX (fractions 3-5), but almost completely solubilized by Triton X-100 (fractions 8-12). In contrast, reggie-1 and reggie-2 were completely resistant to Triton X-100 in this system (Fig. 2, fractions 3-5). And finally, actin was found to be associated with detergent-soluble and detergent-resistant cLPM subfractions, indicating no preferential interaction with canalicular DRMs. These data support the presence of distinct DRMs at the cLPM of rat liver. They demonstrate that solubilization of cLPM with Lubrol WX or Triton X-100 results in cLPM microdomains of different marker protein composition. “Lubrol-microdomains” contain quantitatively caveolin-1, and “Triton-microdomains” are associated with reggie-1 and reggie-2.

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Figure 2. Distribution of the DRM markers caveolin-1, reggie-1, and reggie-2 between canalicular “Lubrol-microdomains” and “Triton-microdomains.” cLPM were extracted with 1% (wt/vol) Lubrol WX (left panel) or 1% (wt/vol) Triton X-100 (right panel) and floated on discontinuous sucrose gradients as described in Materials and Methods. Fractions and the resuspended pellets (P) were subjected to Western blot analysis for marker proteins and actin. Untreated cLPM served as controls (C). Apparent molecular weights are given on the right. Caveolin-1 (Cav-1) was found to be associated predominantly with “Lubrol-microdomains,” whereas reggie-1 and reggie-2 were found to be distinct marker proteins for “Triton-microdomains.” The cytoskeletal protein actin is only minimally associated with both types of DRMs. One representative result out of at least two independent experiments is shown.

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The strong association of caveolin-1 with detergent-insoluble (Lubrol WX) and detergent-soluble (Triton X-100) cLPM subfractions (Fig. 2) was surprising, because the liver has been reported to express only low levels of caveolin-136 and the subcellular distribution of caveolin-1 in rat liver has remained controversial.37–39 Therefore, we investigated the exact hepatocellular surface distribution of caveolin-1 in more detail and compared it with established basolateral and canalicular marker proteins as well as with the surface distribution of reggie-1 and reggie-2. As illustrated in Fig. 3A, in Western blot analysis of isolated blLPM and cLPM, caveolin-1 exhibited an exclusive canalicular localization as especially evidenced by its colocalization with the canalicular marker enzyme aminopeptidase N (APN). In contrast, the expression of reggie-1 and reggie-2 was not domain-specific, but evenly distributed between blLPM and cLPM of rat hepatocytes. The antigen 1/18 has been previously shown to represent a valid basolateral marker protein in rat hepatocytes.40

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Figure 3. Immunological localization of caveolin-1 at the cLPM domain in isolated rat liver plasma membrane vesicles and in intact rat liver. (A) blLPM and cLPM rat liver plasma membrane vesicles were isolated as described in Materials and Methods and probed with specific antibodies (Western blotting) against the indicated proteins. APN and 1/18 are established marker proteins for the cLPM and blLPM domains of rat liver, respectively. (B-E) Rat liver was fixed and processed for immunofluorescence using antibodies against caveolin-1, reggie-1, and the established canalicular marker Mrp2 (B) as described in Materials and Methods. (C,D) Caveolin-1 labeled the cLPM domain (C) and colocalized with Mrp2 (D). Additional caveolin-1 labeling was also seen along the sinusoidal lining of hepatocytes (C,D), most probably reflecting caveolin-1 expression in sinusoidal lining endothelial cells. Nuclei (blue) were stained with DAPI. (E) Reggie-1 labeling was seen in the blLPM and cLPM of hepatocytes.

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The canalicular localization of caveolin-1 was further confirmed by immunofluorescence studies using Mrp2 as a canalicular marker (Fig. 3B). Immunopositive caveolin-1 was clearly associated with the cLPM (Fig. 3C) and colocalized with canalicular Mrp2 (Fig. 3D). However, green caveolin-1 immunoreactivity was also seen along the sinusoidal lining of hepatocytes (Fig. 3C,D). Although isolated blLPM were virtually devoid of immunopositive caveolin-1 (Fig. 3A), the apparent sinusoidal caveolin-1 positivity was most probably due to caveolin-1 with endothelial cells, where it is highly expressed,41 although low-level expression of caveolin-1 at the blLPM of hepatocytes cannot be definitely excluded.38 In any case, the data demonstrate that caveolin-1 is a highly expressed and intrinsic protein of the cLPM domain of rat hepatocytes and, thus, can be used as a marker protein of canalicular DRMs in rat liver. The nonpolar expression of reggie-1 in hepatocytes (Fig. 3A) was confirmed by immunofluorescence localization (Fig. 3E). We next used immunoelectron microscopy to more precisely localize Mrp2 and caveolin-1 in the cLPM. Because aldehyde fixation of liver tissue abolished reactivity of the anti-caveolin-1 antibody, methanol fixation had to be applied. This resulted in a satisfactory structural preservation of bile canaliculi in ultrathin frozen sections (Fig. 4A) and at the same time permitted simultaneous immunogold localization of caveolin-1 and Mrp2 (Fig. 4A,B). As previously reported,42 immunogold labeling for Mrp2 was intense at the microvilli of the bile canaliculi. As expected from the confocal immunofluorescence result for caveolin-1, immunogold labeling for caveolin-1 in ultrathin frozen sections of methanol-fixed liver was sparse but also detectable at microvilli and subplasma membrane regions of the bile capillaries. Inspecting the cLPM at higher magnification revealed again expression of both proteins and they were observed in proximity, and hence corroborated the finding from the immunofluorescence experiment. However, the distance between the gold particles precludes a true colocalization. This might, at least in part, be the result of the methanol fixation method, which had to be used to detect caveolin-1.

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Figure 4. Immunoelectron microscopic localization of Mrp2 and caveolin-1 in the canaliculus: Low-power micrograph showing a bile canaliculus with immunogold labeling for Mrp2 and caveolin-1 (A). At higher magnification (B), immunogold labeling for Mrp2 (small gold particles, arrowheads) and for caveolin-1 (large gold particles, arrows) of the microvilli can be seen. Caveolin-1 immunogold labeling is also observed beneath the bile capillary plasma membrane. Original magnifications: ×38,500 (A), ×107,000 (B).

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In addition to caveolin-1, reggie-1, and reggie-2, sphingomyelin represents an additional DRM marker.16 In fact, and as illustrated in Fig. 5, sphingomyelin segregated quantitatively into both “Lubrol-microdomains” and “Triton-microdomains” (fractions 3-5). This was not the case for cholesterol and various phospholipids. Hence, whereas the caveolin-1 positive “Lubrol-microdomains” (Fig. 2) contained most canalicular phospholipids (except part of phosphatidylcholine) and all the cholesterol (Fig. 5), the reggie-1-positive and reggie-2-positive “Triton-microdomains” (Fig. 2) were devoid of any phospholipids and contained only a portion of canalicular cholesterol (Fig. 5). Whereas these data are consistent with Lubrol WX being a less selective detergent for DRMs than Triton X-100,43 they further support the presence of distinct canalicular DRMs. More specifically, the partial solubilization of cholesterol, but not sphingomyelin, by Triton X-100 indicates that part of the cLPM cholesterol is soluble independent of sphingomyelin and, thus, might represent a mobilizable cLPM cholesterol pool that can be extracted from the canalicular membrane together with phospholipids (especially phosphatidylcholine, Fig. 5) by the detergent action of intracanalicular bile salts.

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Figure 5. Lipid composition of canalicular DRMs: cLPM were treated with 1% (wt/vol) Lubrol WX or 1% (wt/vol) Triton X-100 and floated on discontinuous sucrose gradients as described in Materials and Methods. Lipids from the recovered 12 fractions, the resuspended pellets (P), and untreated cLPM (C) were extracted and analyzed as described in Materials and Methods. Individual lipid species were identified by comigration with the purified lipids sphingomyelin (SM), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylethanolamine (PE), and cholesterol (Ch). Whereas “Lubrol-microdomains” (fractions 3-5) contained almost quantitatively all canalicular lipid species, “Triton-domains” (fractions 3-5) were selectively enriched in sphingomyelin, contained a minor portion of cholesterol, and were completely devoid of phospholipids. A representative result of two independent experiments is shown.

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Finally, we wondered about the segregation of some typical canalicular marker enzymes and canalicular ABC transporters within and outside “Lubrol-microdomains” and “Triton-microdomain.” As illustrated in Fig. 6, the canalicular marker enzymes APN, ectoATPase, and dipeptidylpeptidase IV (DPPIV) were almost equally sensitive to solubilization by Lubrol WX and Triton X-100. Whereas APN and DPPIV minimally partitioned into both types of DRMs, ectoATPase could not be detected in DRMs at all (Fig. 6). In contrast, the ABC transporters Abcg5, Bsep, Mrp2, Mdr2, and Mdr1 were significantly associated with “Lubrol-microdomains” (Fig. 7), whereas “Triton-microdomains” contained only some minor portions of Mdr1 and even less so of Mrp2 (Fig. 7). These data, while further supporting the existence of different types of canalicular DRMs, strongly indicate that canalicular ABC transporters function within phospholipids and cholesterol-containing cLPM membrane microdomains (the caveolin-1-positive “Lubrol-microdomains”) in rat hepatocytes. In contrast, reggie-1-positive and reggie-2-positive “Triton-microdomains” might represent transporter-free and sphingomyelin-enriched, more rigid membrane microdomains that are important for the maintenance of the overall structural integrity and functional compartmentalization of the cLPM.

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Figure 6. Distribution of canalicular marker enzymes within and outside “Lubrol-microdomains” and “Triton-microdomains.” cLPM were extracted with 1% (wt/vol) Lubrol WX or 1% (wt/vol) Triton X-100 and floated on discontinuous sucrose gradients as described in Materials and Methods. The recovered 12 fractions and the resuspended pellets (P) were subjected to immunoblot analyses using antibodies against APN, ectoATPase, and DPPIV as indicated. Untreated cLPMs were used as positive control (C). Apparent molecular weights are given on the right. The marker enzymes studied were only minimally (APN, DPPIV) or not at all (ectoATPase) associated with canalicular DRMs. One representative result out of at least two independent experiments is shown.

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Figure 7. Distribution of canalicular ABC transporters within and outside “Lubrol-microdomains” and “Triton-microdomains.” cLPMs were extracted with 1% (wt/vol) Lubrol WX or 1% (wt/vol) Triton X-100 and floated on discontinuous sucrose gradients as described in Materials and Methods. The recovered 12 fractions and the resuspended pellets (P) were subjected to Western blot analysis using antibodies against the ABC transporters indicated on the left side. Untreated cLPMs were used as positive control (C). Apparent molecular weights are given on the right. Canalicular ABC transporters were found to reside in part in “Lubrol-microdomains” and to be virtually absent from “Triton-microdomains.” Only Mdr1 and Mrp2 remained associated to some degree with “Triton-microdomains.” One representative result out of at least two independent experiments is shown.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

The present study provides evidence for the presence of two different types of DRMs in the cLPM of rat hepatocytes. Based on the detergents used for solubilization of isolated cLPMs we named the two distinct canalicular DRMs “Lubrol-microdomains” and “Triton-microdomains.” “Lubrol-microdomains” are associated quantitatively with caveolin-1 (Fig. 2), contain the majority of canalicular cholesterol and phospholipids (Fig. 5), portions of the marker enzymes APN and DDPIV (Fig. 6), and large portions of all ABC transporters tested (i.e., Abcg5, Bsep, Mrp2, Mdr2, and Mdr1) (Fig. 7). “Triton-microdomains” are associated quantitatively with AP (Fig. 1), reggie-1 and reggie-2 (Fig. 2), sphingomyelin (Fig. 5), and contain also minor fractions of canalicular cholesterol (Fig. 5), APN, and DDPIV (Fig. 6) as well as Mdr1 and Mrp2 (Fig. 7). Hence, our study supports the concept of distinct canalicular cholesterol-enriched lipid microdomains that can be distinguished by the marker proteins caveolin-1 and reggie-1 and reggie-2. Furthermore, a large part of canalicular ABC transporters reside within the phospholipid and cholesterol-enriched membrane regions (“Lubrol-microdomains”), suggesting that they require a complex membrane lipid environment for proper functioning.

By using the same two nonionic detergents (e.g., Lubrol WX and Triton X-100), two distinct cholesterol-based lipid microdomains have previously been proposed in the apical plasma membrane of MDCK cells.22 Our findings support this previous study and extend the concept of the coexistence of multiple, distinct types of raft-like assemblies of lipids and proteins to the cLPM (apical) of hepatocytes. Although we have not tested further the exact localization of the distinct DRMs along the cLPM, due to a lack of suitable antibodies for immunoelectron microscopy, it is tempting to speculate that, similar to MDCK cells, the caveolin-1-specific and lipid and transporter-enriched “Lubrol-microdomains” may correspond to the microvillar portions, and the reggie-1 and reggie-2 and sphingomyelin-specific “Triton-microdomains” to the more rigid planar portions of the cLPM. This interpretation would be compatible with several other previous reports: (1) Bsep, which is present in “Lubrol-microdomains,” but absent from “Triton-microdomains” (Fig. 7), has been shown to be preferentially localized in microvilli and to be virtually absent from planar portions of the cLPM,4 which is paralleled by scarce Mrp2 labeling observed in this study; (2) reggie-1 and reggie-2-containing membrane microdomains (“Triton-microdomains” in this study, Fig. 2) are clearly different from caveolin-1-containing membrane microdomains. They might represent stable membrane scaffolds or platforms with possible own regulatory functions that are distinct from caveolae25; and (3) other ABC transporters (e.g., MDR1, MRP1) have been found to be associated with “Lubrol-microdomains.”44, 45

The association of a significant portion of ABC transporters (Fig. 7) and all different lipid species (Fig. 4) with “Lubrol-microdomains” indicates that ATP-dependent canalicular bile salt, organic anion, phospholipid, and cholesterol secretion requires a complex lipid environment for proper functioning. In this regard the high cholesterol content of “Lubrol-microdomains” appears especially interesting because it has recently been shown in a heterologous expression system that the ATPase activity of the canalicular ABC-transporter ABCG2 is stimulated by cholesterol loading.46 As the methodology used to isolate DRMs has an inherent considerable quantitative variability,47 we could not quantitatively assess the enrichment of cholesterol in DRMs compared to cLPM. Nevertheless, by inserting or retrieving canalicular export systems into cholesterol-enriched microdomains (i.e., “Lubrol-microdomains”), hepatocytes could regulate the activity of the ABC transporters and thus canalicular bile formation. Such a putative regulatory mechanism could also involve actin (Fig. 2), as it was shown for insulin in rat liver plasma membrane microdomains,21, 48 as well as the cytoskeletal web and intermediate filaments underneath the cLPM.49, 50 Furthermore, stimulation of the choleretic activity of hepatocytes leads to an upregulation of aquaporin-8 in caveolin-1-enriched canalicular microdomains.20 Also, partitioning of the serotonin transporter and the sodium-phosphate cotransporter into membrane microdomains has been demonstrated as a regulatory mechanism for these transporters.51, 52 Finally, and most important, the observation that Lubrol WX was able to partially and preferentially solubilize phosphatidylcholine (Fig. 5) mirrors the finding that taurocholate preferentially releases phosphatidylcholine from cLPM in vitro.15 Hence, caveolin-1 and ABC transporter-positive “Lubrol-microdomains” might represent the canalicular microdomains from where biliary phospholipids are preferentially solubilized by intracanalicular bile salts. Although this latter conclusion remains to be experimentally verified, our data strongly suggest that “Lubrol-microdomains” are representative for cLPM microdomains that localize the entire functional machinery for maintenance of ongoing canalicular bile formation including bile salt (Bsep), organic anion (Mrp2), phospholipid (Mdr2), and cholesterol (Abcg5/8) secretion and therefore could also be called “bile salt microdomains.”

In contrast to “Lubrol-microdomains,” “Triton-microdomains” appear to represent more stable non-caveolin-1 (caveolae)-associated canalicular DRMs, which might not be directly involved in bile secretory processes. “Triton-microdomains” contain notably fewer different lipid species than “Lubrol-microdomains.” To what extent different biophysical properties of the two different DRMs and to what extent the presence of inside-out and right-side-out oriented cLPM during detergent extraction contribute to this difference remains open at this moment. In addition, Triton X-100 is more selective in DRM isolation, such that it disrupts more lipid-protein interactions than Lubrol WX.43 Whereas the association of “Triton-microdomains” with AP, sphingomyelin, and some cholesterol (Figs. 1, 5) is in agreement with previous findings,53 the observation of a specific association of reggie-1 and reggie-2 with “Triton-microdomains” is novel. Reggie-2/flotillin-1 upon its identification as a DRM component was found to be extracted together with caveolin-1 after cold extraction of 3T3-L1 adipocytes with Triton X-100.54 However, electron microscopy studies have since shown that reggie proteins demarcate microdomains (“reggie-microdomains”) distinct from caveolae in all cell types analyzed so far.23, 24, 55 Preliminary experiments showed only partial colocalization of reggie-1 and Mrp2 (data not shown), indicating a distinct expression of reggie-1 and caveolin-1 in the cLPM. In contrast, caveolin-1 and Mrp2 showed complete colocalization in the cLPM (Fig. 3D). Opposite to 3T3-L1 cells, hepatocytes are highly polarized cells. Hence, the dissociation of “Lubrol-microdomains” and “Triton-microdomains” may be cell-type-specific and/or related to the degree of cell polarity. This view is supported by the identification of distinct microdomains in the apical membrane of MDCK cells,22 which is paralleled by an expression of caveolin-1 and reggie-1 and reggie-2 in different membrane subdomains.56 Whereas the expression of reggie-2 in mouse liver has been reported,57 in the same study expression of reggie-1 could not be demonstrated. The present study reports, to our knowledge for the first time, the expression of reggie-1 in liver and the localization of reggie-1 and reggie-2 to both plasma membrane domains of hepatocytes. The lack of detection of reggie-1 in the previous study may be due to the use of tissue lysates compared to highly purified and enriched plasma membrane fractions used in this study for Western blotting.

In conclusion, by using Lubrol WX and Triton X-100, we have presented strong evidence for the presence of two different microdomains in the cLPM of rat liver: “Lubrol-microdomains” and “Triton-microdomains.” The “Lubrol-microdomains” may play an essential role in canalicular bile formation because they contain the entire machinery for the generation of canalicular bile salt-dependent and -independent bile flow.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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