Regulation of Multidrug Resistance-Associated Protein 2 by Calcium Signaling in Mouse Liver

Abstract

Multidrug resistance associated protein 2 (Mrp2) is a canalicular transporter responsible for organic anion secretion into bile. Mrp2 activity is regulated by insertion into the plasma membrane; however, the factors that control this are not understood. Calcium (Ca²⁺) signaling regulates exocytosis of vesicles in most cell types, and the type II inositol 1,4,5-triphosphate receptor (InsP₃R2) regulates Ca²⁺ release in the canalicular region of hepatocytes. However, the role of InsP₃R2 and of Ca²⁺ signals in canalicular insertion and function of Mrp2 is not known. The aim of this study was to determine the role of InsP₃R2-mediated Ca²⁺ signals in targeting Mrp2 to the canalicular membrane. Livers, isolated hepatocytes, and hepatocytes in collagen sandwich culture from wild-type (WT) and InsP₃R2 knockout (KO) mice were used for western blots, confocal immunofluorescence, and time-lapse imaging of Ca²⁺ signals and of secretion of a fluorescent organic anion. Plasma membrane insertion of green fluorescent protein (GFP)-Mrp2 expressed in HepG2 cells was monitored by total internal reflection microscopy. InsP₃R2 was concentrated in the canalicular region of WT mice but absent in InsP₃R2 KO livers, whereas expression and localization of InsP₃R1 was preserved, and InsP₃R3 was absent from both WT and KO livers. Ca²⁺ signals induced by either adenosine triphosphate (ATP) or vasopressin were impaired in hepatocytes lacking InsP₃R2. Canalicular secretion of the organic anion 5-chloromethylfluorescein diacetate (CMFDA) was reduced in KO hepatocytes, as well as in WT hepatocytes treated with 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Moreover, the choleretic effect of tauroursodeoxycholic acid (TUDCA) was impaired in InsP₃R2 KO mice. Finally, ATP increased GFP-Mrp2 fluorescence in the plasma membrane of HepG2 cells, and this also was reduced by BAPTA.

Conclusion

InsP₃R2-mediated Ca²⁺ signals enhance organic anion secretion into bile by targeting Mrp2 to the canalicular membrane.

Cytosolic Ca²⁺ is a ubiquitous intracellular messenger.¹ In the liver, Ca²⁺ signals have been linked to the regulation of such diverse functions as gene expression,^2,3 cell proliferation,⁴ apoptosis,^5-7 paracellular permeability,⁸ glucose release,^9,10 and bile flow.¹¹ Ca²⁺ signaling in hepatocytes is mainly controlled by the inositol 1,4,5-trisphosphate receptor (InsP₃R), an InsP₃-gated Ca²⁺ channel in the endoplasmic reticulum. InsP₃Rs type I (InsP₃R1) and type II (InsP₃R2)^12,13 are the two InsP₃R isoforms expressed in hepatocytes. InsP₃R2 is the most highly expressed of these isoforms and is concentrated in the region of the endoplasmic reticulum near the canalicular membrane. Thus it is in close proximity to canalicular transporters,¹⁴ which are either inserted into the canalicular plasma membrane or localized to subplasmalemmal vesicles.¹⁵ Activation of InsP₃Rs in the apical region of other epithelia generates a spatially restricted domain with Ca²⁺ concentrations as high as approximately 10 μM,¹⁶ which is sufficient to induce vesicular exocytosis.¹⁷

Secretion of a wide variety of amphiphilic organic anions into bile, including bilirubin, glutathione S-conjugates, and oxidized glutathione, occurs through multidrug resistance-associated protein 2 (Mrp2), a member of the ATP binding cassette family of transporters.¹⁸ Mrp2 activity is partially regulated by its dynamic trafficking between the canalicular plasma membrane and a nearby endosomal compartment. Cholestatic agents such as estradiol,¹⁹ lipopolysaccha-ride,²⁰ lithocholic acid,²¹ and phalloidin²² act in part by decreasing insertion of Mrp2 into the plasma membrane. Fusion of vesicles with the plasma membrane involves Ca²⁺-dependent proteins in nearly every cell type,²³ and Ca²⁺ signals induced by tauroursodeoxycholic acid (TUDCA) have been linked to exocytosis in hepatocytes^24,25; yet the role of Ca²⁺ in regulating organic anion secretion is not known. Therefore, we investigated the role of InsP₃R2-mediated Ca²⁺ release in the localization and activity of Mrp2.

Materials and Methods

Animals and Materials

Male Swiss black wild-type (WT) mice obtained from Taconic Farms Inc (Hudson, NY) and InsP₃R2 knockout (KO) mice described previously²⁶ were used for experiments. Animals were maintained on a standard diet and housed under a 12-hour light/dark cycle. All animal procedures were approved by the Yale University Institutional Animal Care and Use Committee. Vasopressin, ATP, bovine serum albumin, penicillin-streptomycin, and protease inhibitor cocktails were purchased from Sigma Chemical Co (St. Louis, MO). Dulbecco's modified Eagle medium, Liebowitz 15, Fluo-4/acetoxymethyl ester (AM), Cell tracker green 5-chloromethylfluorescein diacetate (CMFDA), the nuclear stain TO-PRO-3, rhodamine-conjugated phalloidin, and Alexa-488 secondary antibodies were obtained from Invitrogen (Eugene, OR). InsP₃R1 antibodies from affinity-purified specific rabbit polyclonal antiserum directed against the 19 C-terminal residues of the mouse InsP₃R1 were from Affinity Bioreagents (Golden, CO). InsP₃R2 antibodies from affinity-purified specific rabbit polyclonal antiserum directed against the 18 C-terminal residues of the rat InsP₃R2 were provided by Richard Wojcikiewicz (SUNY Syracuse, NY). Monoclonal antibodies directed against the N-terminal of InsP₃R3 were obtained from Becton Dickinson (Lexington, KY). Bilirubin total reagent was obtained from ClinicQA (San Marcos, CA). Mammalian protein extraction reagent cell lysis buffer was obtained from Pierce (Rockford, IL). Rat GFP-MRP2 expression vector was provided by Dietrich Keppler (German Cancer Research Center, Heidelberg, Germany).²⁷ HepG2 cells were purchased from ATCC (Manassas, VA). All other chemicals were of the highest quality commercially available.

Isolation and Culture of Hepatocytes and Bile Flow Studies

Isolated mouse hepatocyte couplets were used for single-cell imaging. Cells were isolated in the Cell Isolation Core of the Yale Liver Center, as described.^28,29 Briefly, mouse livers were perfused with Hanks' A and then Hanks' B medium containing 0.05% collagenase (Roche Applied Science, Indianapolis, IN) and 0.8 units of trypsin inhibitor (Sigma) per unit of tryptic activity. Livers were minced and passed through serial nylon mesh filters, and the resultant cells were washed. Isolated hepatocytes were resuspended in Liebowitz 15 medium with 50 units penicillin and 50 mg streptomycin. Cells were then seeded onto collagen-I-coated coverslips and incubated at 37°C for 2 to 4 hours before use in isolated hepatocyte experiments. For hepatocytes in collagen sandwich culture, cells were incubated for 2 hours at 37°C before being coated with a second layer of collagen-I and used 3 to 5 days after plating.³⁰ For bile flow studies, bile was collected using Intramedic Polyethylene Tubing (Becton Dickinson, Franklin Lakes, NJ) inserted into the gallbladder. Bile was collected in pre-tared tubes, and then flow measurements were normalized by the liver weight and expressed as microliters per gram of liver per minute (μL/g liver/minute). During the entire experiment (1 hour), animals were under anesthesia by inhalation of a mixture of oxygen (0.5 L/minute) and isoflurane (2.5%-3.0%) and their temperature monitored.

Immunoblotting

Immunoblots were performed as described previously.¹³ Briefly, cells were lysed at 4°C with mammalian protein extraction reagent lysis buffer; the lysate underwent centrifugation, and the protein concentration of the supernatant was determined spectrophotometrically. Sixty micrograms total cellular protein was separated by sodium dodecyl sul-fate polyacrylamide gel electrophoresis, using a 7.5% polyacrylamide gel. Membranes were blocked with nonfat milk and then incubated overnight at 4°C with InsP₃R1 or InsP₃R2, Mrp2, and alpha tubulin-specific antibodies. A rabbit anti-InsP₃R1 was used at a dilution of 1:3000, an affinity-purified rabbit antibody against the C-terminus of InsP₃R2 isoform was used at 1:100, and a mouse antibody against the N-terminus of the InsP₃R3 was used at a dilution of 1:3000. For Mrp2 immunoblotting, an affinity-purified rabbit antibody³¹ was used at dilution of 1:2000; the positive control (alpha tubulin) was used at a dilution of 1:5000. Membranes were washed and incubated with peroxidase-conjugated secondary antibodies at a dilution of 1:5000. The protein-antibody conjugates were then detected by enhanced chemiluminescence (Millipore, Billerica, MA).

Immunofluorescence

Confocal immunofluorescence to detect the subcellular distribution of InsP₃R iso-forms was performed as described previously.^13,14,32 Briefly, frozen mouse liver sections were fixed in 4% formaldehyde and permeabilized with 0.1% Triton X-100. Nonspecific binding was blocked with 1% bovine serum albumin and 5% normal goat serum. Liver sections were incubated at room temperature with primary antibody directed against specific InsP₃R iso-forms or against Mrp2 (Alexis Biochemicals, Farming-dale, NY) and then rinsed with phosphate-buffered saline. The specimens were then incubated with Alexa 488 secondary antibody and co-labeled with Rhodamine-conjugated Phalloidin and TO-PRO-3 to facilitate recognition of hepatocyte morphology and nucleus, respectively. Images were obtained using a LSM 510 confocal microscope (Zeiss, Thornwood, NY) by excitation at 488 nm and observation at 505 to 530nm to detect Alexa 488. Rhodamine phalloidin was excited at 543 nm and observed at 585 nm and TO-PRO3 was detect by using a 647 nm laser line and observed above 665 nm.

Time-Lapse Imaging

Cytosolic Ca²⁺ was monitored in isolated mouse hepatocytes and HepG2 cells. CMFDA secretion was monitored in the canalicular spaces between mouse hepatocytes in collagen sandwich culture by time-lapse confocal microscopy as described previously.^13,33 Briefly, hepatocytes were incubated with either Fluo-4/AM (6 μM) for 30 minutes at 37°C for Ca²⁺ imaging or Cell tracker Green CMFDA (1 μm) to monitor organic anion secretion. Cover slips seeded with the cells were transferred to a custom-built perfusion chamber on the stage of an LSM 510 confocal microscope (Zeiss, Thornwood, NY), and the cells were then perfused with 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid-buffered solution. Cells were excited at 488 nm and observed at 505 to 550 nm. In most experiments, 40× or 63× objectives were used to observe the cells. Increases in Ca²⁺ or organic anion secretion were expressed as percent increase in Fluo-4 or CMFDA fluorescence intensity and normalized by baseline fluorescence, respectively.

Total Internal Reflection Microscopy

HepG2 cells were seeded on 35-mm glass-bottom culture dishes at 2.0 × 10⁵ cells/dish 2 days before total internal reflection fluorescence (TIRF) imaging. Cells were transfected 24 hours later with rat GFP-Mrp2 and transferred to the stage of a custom-built TIRF microscope 1 day after transfection. Cells were kept at 37°C during the experiment. Images were acquired using an Olympus inverted microscope equipped with a 1.45 NA 60× TIRFM lens, back-illuminated electron multiplying charge-coupled device camera (16-bit; iXon887; Andor Technologies), and controlled by Andor iQ software. Cells were excited using the 488-nm line of an argon laser, with exposure times of 0.15 second and an acquisition rate of 0.5Hz. Cells were imaged for 5 minutes before the addition of ATP (100 μM) to determine the average baseline membrane fluorescence. Fluorescence changes were monitored for 20 minutes in the presence of ATP. In control experiments, cells were pretreated with the intracellular Ca²⁺ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA)/AM (50 μM). The resulting series of images were background subtracted using Image J software (National Institutes of Health). The calculated evanescent field depth was approximately 150 nm.

Statistics

All results are expressed as mean ± standard error of the mean of at least three individual experiments. Student t test or analysis of variance (ANOVA) was used for comparisons between groups. A P value less than 0.05 was used to indicate a statistically significant difference. GraphPad Prism software (San Diego, CA) was used for all statistical tests.

Results

Expression and Distribution of InsP₃R Isoforms in Mouse Liver

Immunoblotting demonstrated expression of InsP₃R1 and InsP₃R2 but not InsP₃R3 (Fig. 1A-C) in wild-type (WT) mouse liver, similar to what has been observed in rat¹³ and human liver.³⁴ In InsP₃R2 KO liver, InsP₃R1 was detected but both InsP₃R2 and InsP₃R3 were absent (Fig. 1A-C). Confocal immunofluorescence of WT mouse liver slices (Fig. 2) revealed that InsP₃R2 is highly concentrated close to the canalicular membrane, whereas InsP₃R1 is distributed throughout the hepatocyte, also similar to what is observed in rats.¹³ Immunofluorescence for InsP₃R2 revealed no specific staining in InsP₃R2 KO liver, plus no appreciable modification in the diffuse subcellular distribution of InsP₃R1 (Fig. 2). Expression of InsP₃R3 was absent from hepatocytes in both types of mice. Together these results confirm the absence of InsP₃R2 in the livers of KO animals and show that there is no significant compensatory up-regulation of InsP₃R1 in the InsP₃R2 KO mice.

Fig. 1.

Expression of InsP₃R isoforms in WT and InsP₃R2 KO hepatocytes. (A) InsP₃R1 is not up-regulated in the liver of InsP₃R2 KO animals. Each lane is loaded with 60 μg total cell lysate prepared from freshly isolated hepatocytes. Blots were probed with an anti-InsP₃R1 antibody and show that InsP₃R1 expression levels are similar between WT and InsP₃R2 KO livers. Cerebellum was used as positive control for InsP₃R1, and alpha-tubulin was used as a loading control. (B) Blot for InsP₃R2 confirms that this isoform is absent in InsP₃R2 KO animals (lanes 1-3). (C) InsP₃R3 is absent in both WT and InsP₃R2 KO hepatocytes. Mz-ChA-1 cell extracts were used as positive controls for detection of InsP₃R3.

Fig. 2.

Cellular localization of InsP₃R isoforms in WT and InsP₃R2 KO liver. Confocal immunofluorescence microscopy was used to determine the subcellular localization of each InsP₃R isoform. (A) InsP₃R1 is diffusely distributed in the cytosol of both types of hepatocytes. Top row shows the diffuse distribution of InsP₃R1 (green) in WT liver. Subcortical f-actin is shown in red to reveal the outline of individual hepatocytes. Nuclei (blue) were stained with TO-PRO3. The rightmost panel shows the merged image. Note that the expression level and subcellular distribution of InsP₃R1 is similar in InsP₃R2 KO liver (bottom row). (B) InsP₃R2 is concentrated near the canalicular membrane of WT (top left panel) hepatocytes but is absent in InsP₃R2 KO liver (bottom left panel). Expression of InsP₃R3 is absent from hepatocytes in livers from both types of mice (data not shown). Scale bar = 50 μm.

Effects of InsP₃R2 on Ca²⁺ Signaling in Mouse Hepatocytes

To determine the effects of loss of InsP₃R2 on Ca²⁺ signaling, hepatocytes isolated from WT and InsP₃R2 KO mice were stimulated with ATP (100 μM) or concentrations ranging from 0.1 to 100 nM of arg⁸-vasopressin (AVP) to induce InsP₃-mediated cytosolic Ca²⁺ release.^12-14,35 Either stimulus increased Ca²⁺ in both WT and InsP₃R2 KO hepatocytes, although differences in the nature of the Ca²⁺ signal could be detected between the two types of cells (Fig. 3). The two kinetic parameters used to characterize these differences were the amplitude of the Ca²⁺ signal and the rise time, which is the time required for the signal to increase from 25% to 75% of the maximum response (Fig. 3A). The amplitude of the Ca²⁺ signal was significantly reduced in InsP₃R2 KO cells stimulated with either AVP (Fig. 3B) or ATP (Fig. 3C). The rise time for both agonists was significantly prolonged in InsP₃R2 KO hepatocytes compared with WT cells (Fig. 3D, E). These differences demonstrate impaired InsP₃-mediated Ca²⁺ signaling in InsP₃R2 KO hepatocytes, similar to what has been reported in rat hepatocytes in which InsP₃R2 expression was reduced by antisense.¹²

Fig. 3.

Ca²⁺ signaling is impaired in InsP₃R2 KO hepatocytes. (A) Representative tracing of Fluo4 fluorescence changes over time in WT and InsP₃R2 KO hepatocytes stimulated with ATP (100 μM). Hepatocytes were stimulated with either ATP or AVP and examined by time-lapse confocal microscopy. Using these individual tracings, the amplitude and the rise time of the Ca²⁺ signal from individual hepatocytes was determined. (B) The increase in Ca²⁺ induced by AVP (10 nM) was greater in WT as compared with InsP₃R2 KO hepatocytes (165 ± 3%, n = 309 cells versus 148 ± 2%, n = 423 cells; P < 0.0001). (C) ATP (100 μM)-induced Ca²⁺ release also was greater in WT as compared with InsP₃R2 KO cells (152.9 ± 1.16%, n = 149 versus 146.2 ± 1.18%, n = 176; P < 0.001). (D) Rise time of Ca²⁺ signal was faster in WT hepatocytes than in InsP₃R2 KO cells perfused with AVP 10 nM (11.43 ± 0.71 seconds, n = 251 versus 29.96 ± 1.30 seconds, n = 256; P < 0.0001). (E) Rise time also was shorter in WT than in InsP₃R2 KO cells when ATP 100 μM was used to induced Ca²⁺ release (1.89 ± 0.08 seconds, n = 149 cells versus 2.81 ± 0.17 seconds, n = 176 cells; P < 0.0001), reflecting slower Ca²⁺ release in cells lacking InsP₃R2.

Bile Flow and Serum Bilirubin Are Not Altered in InsP₃R2 KO Mice

To further characterize the effects of InsP₃R2 expression on liver function, bile secretion and serum bilirubin concentration were examined in WT and InsP₃R2 KO mice. Age-matched and weight-matched animals were used to measured basal bile flow in both groups of mice. The average liver weight in WT mice was greater than in InsP₃R2 KO mice (1.483 g ± 0.0502, n = 9, and 1.234 g ± 0.0437, n = 6, respectively; P < 0.005). However, there was no significant difference in bile flow between the two groups (0.9486 μL/g liver/minute ± 0.0618, n = 9; and 1.116 μL/g liver/minute ± 0.030, n = 6, respectively). There also was no significant difference in the total bilirubin concentration in the serum of WT and InsP₃R2 KO animals (0.2170 mg/dL ± 0.01813, n = 10; and 0.2096 mg/dL ± 0.02917, n = 9, respectively).

Role of Type II InsP₃R in Organic Anion Secretion

Because translocation of Mrp2 to the plasma membrane is stimulated by protein kinase C alpha (PKCα) and this kinase is Ca²⁺-activated, we hypothesized that organic anion secretion might be impaired in InsP₃R2 KO animals. To test this, we first measured the secretion of cell tracker green CMFDA, a fluores-cent Mrp2 substrate,³⁶ by hepatocytes in collagen sandwich cultures using time-lapse confocal microscopy. This cell system was used because it preserves structural and functional polarity of hepatocytes.^37,38 CMFDA accumulated in the canalicular lumen of hepatocytes (Fig. 4A), and the kinetics could be quantified as a measure of Mrp2 function (Fig. 4B). Canalicular accumulation of CMFDA fluorescence was observed in both WT and InsP₃R2 KO hepatocytes, but luminal fluorescence in InsP₃R2 KO hepatocytes reached only 25.8% of what was observed in WT cells (Fig. 4C). Luminal fluorescence was reduced by a similar extent in WT cells treated with the cytosolic Ca²⁺ chelator BAPTA/AM (50 μM; Fig. 4C). To understand the significance of this finding in vivo, bile flow was measured in WT and InsP₃R2 KO livers infused with the cholestatic bile acid TLCA (5 uM) with or without co-infusion of taurolithocholic acid (TUDCA) (25 μM). This model was chosen because TUDCA stimulates biliary exocytosis in a Ca²⁺-dependent fashion^24,25 and reverses the cholestatic effect of TLCA.³⁹ As shown in Fig. 4D, infusion of TLCA alone induced a similar decrease in bile flow in both groups of animals, but this effect was reversed more effectively in WT than in InsP₃R2 KO animals by co-infusion of TUDCA (P < 0.02 by repeated-measures ANOVA). Confocal immunofluorescence studies also were performed in hepatocytes in collagen sandwich culture to determine whether the subcellular distribution of Mrp2 and InsP₃R2 is preserved in this model system. Both InsP₃R2 (Fig. 5A) and Mrp2 (Fig. 5b) were localized to the region of the canalicular membrane of these hepatocytes, similar to what was observed in liver sections. These results demonstrate that Mrp2 function in hepatocytes depends on expression of InsP₃R2, and suggest that this is attributable to the function of the receptor as an intracellular Ca²⁺ release channel.

Fig. 4.

Lack of InsP₃R2 impairs bile flow and organic ion secretion. (A) Representative time-lapse series of images of CMFDA secretion in WT mouse hepatocytes in collagen sandwich culture. The cell-permeant fluorescent Mrp2 substrate was added to cells, and its secretion into the canalicular spaces was monitored every 2.5 seconds for 15 minutes by confocal microscopy. Regions of interest such as the one outlined in yellow (third panel) were used to measure the canalicular accumulation of fluorescence over time. Scale bar = 20 μm. (B) Canalicular accumulation of fluorescence is decreased in cells lacking InsP₃R2, as well as in cells in which cytosolic Ca²⁺ is chelated. Results reflect mean ± standard error of the mean increases over time in WT cells (77 canaliculi on seven coverslips; blue), InsP₃R2 KO cells (74 canaliculi on five coverslips; green), WT pretreated with 50 μM BAPTA/AM (82 canaliculi on six coverslips; gray) and WT incubated with dimethylsulfoxide (32 canaliculi on three coverslips; red), which was the vehicle for BAPTA/AM. Data are pooled from three separate WT and InsP₃R2 KO cell preparations. (C) Increases in CMDFA fluorescence were significantly greater in canaliculi from WT cells (186.7 ± 10.2 au) than from InsP₃R2 KO cells (48.2 ± 5.6 au; P < 0.0001), as well as in WT pretreated with the intracellular Ca²⁺ chelator BAPTA/AM (86.0 ± 8.4 au) as compared with WT pretreated with dimethylsulfoxide vehicle alone (166.5 ± 20.8 au; P < 0.0001 by one-way ANOVA with Bonferroni post-test). (D) Portal infusion of TLCA (5 μM) caused a similar reduction in bile flow in WT (blue tracing, n = 3 animals) and InsP₃R2 KO (green tracing, n = 4) mice. TLCA-induced cholestasis was reversed by co-infusion of TUDCA (25 μM) in WT (red tracing, n = 3) and to a lesser extent in InsP₃R2 KO animals (gray tracing, n = 4; P < 0.02 by repeated-measures ANOVA). Arrow indicates time of addition of bile acids.

Fig. 5.

InsP₃R2 localization is maintained in mouse hepatocytes in collagen sandwich culture. Cells were maintained in culture for 4 days and then examined by confocal immunofluorescence. (A) Immunofluorescence for InsP₃R2 (green) reveals that this intracellular Ca²⁺ release channel is distributed in the vicinity of the canalicular network that develops between hepatocytes in collagen sandwich culture, similar to the localization pattern in the liver. Labeling of f-actin by Rhodamine phalloidin (red) identifies the canalicular membrane, and TO-PRO3 is used to label cell nuclei (blue). Scale bar = 20 μm. (B) Labeling of Mrp2 shows that the canalicular distribution of this transporter is preserved in this cell system as well.

Role of InsP₃R2 in Mrp2 Expression and Localization

To determine whether reduced secretion of CMFDA in InsP₃R2 KO hepatocytes might reflect effects of InsP₃R2 on Mrp2 expression, the expression of Mrp2 in total cell lysates of WT and InsP₃R2 KO hepatocytes was analyzed. Both western blot (Fig. 6A) and densitometric analysis (Fig. 6B) demonstrated that there is no significant difference in Mrp2 protein expression in the InsP₃R2 KO animals when compared with the WT. The subcellular distribution of Mrp2 also was examined, in liver slices from both WT and InsP₃R2 KO animals. Confocal immunofluorescence demonstrated that Mrp2 is localized to the canalicular domain of hepatocytes in both InsP₃R2 KO and WT mice (Fig. 7A). Higher magnification images revealed that Mrp2 does not colocalize with submembranous f-actin in either WT or InsP₃R2 KO mice (Fig. 7B). Together, these results demonstrate that Mrp2 expression and localization is not altered in InsP₃R2 KO liver.

Fig. 6.

Mrp2 protein expression is not altered in InsP₃R2 KO cells. (A) Representative western blot comparing total Mrp2 protein expression in WT and InsP₃R2 KO hepatocytes shows equivalent expression levels in both cell types. Beta-actin was used as a control for protein loading. (B) Densitometric analysis of three separate blots shows that Mrp2 expression is unchanged in InsP₃R2 KO hepatocytes (87.9% ± 10.5% relative to WT; P > 0.3).

Fig. 7.

Mrp2 localization is not altered in InsP₃R2 KO cells. (A) Confocal immunofluorescence localization of Mrp2 (green), f-actin (red), and nuclei (blue) in WT (top row) and InsP₃R2 KO (bottom row) liver. Note that the canalicular localization of Mrp2 is preserved in InsP₃R2 KO liver. Scale bar = 50 μm. (B) Higher-magnification view of a canalicular region reveals that Mrp2 (green) is adjacent to but does not co-localize with subcortical f-actin (red) in both WT and InsP₃R2 hepatocytes. Scale bar = 5 μm.

Ca²⁺ Regulates Insertion of Mrp2 into the Plasma Membrane

Because Mrp2 activity is mediated by its dynamic trafficking, we investigated whether InsP₃R2-depent Ca²⁺ release was involved in insertion of Mrp2 into the plasma membrane. Rat GFP-Mrp2 was transiently expressed in the HepG2 liver cell line, and then GFP was imaged by total internal reflection fluorescence (TIRF) microscopy (Fig. 8). This microscopy technique allows for the visualization of fluorescence within approximately 150 nm of the plasma membrane,^40,41 without affecting resolution in the focal plane, and so is ideal to monitor events such as exocytic insertion into the plasma membrane of vesicles containing GFP-tagged proteins.⁴² Stimulation of HepG2 cells with ATP (100 μM) to increase cytosolic Ca²⁺ led to an increase in plasma membrane GFP fluorescence as compared with the increased fluorescence in unstimulated cells observed over the same time interval (125% ± 2% and 107% ± 1% for cells perifused with ATP versus N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid buffer; P < 0.0001). This increase was reduced significantly by pretreating the cells with the intracellular Ca²⁺ chelator BAPTA/AM (P < 0.0006). In control experiments, treatment of cells expressing GFP-Mrp2 with BAPTA for 30 minutes had no effect on GFP fluorescence (not shown). To verify that ATP induces InsP₃-mediated Ca²⁺ signals in these cells, confocal microscopy was used to measure Ca²⁺ signals in HepG2 cells stimulated with ATP (100 μM) in the presence or absence of BAPTA/AM, the phospholipase C inhibitor U-73122, or the InsP₃R blocker Xestospongin C. Buffering intracellular Ca²⁺ nearly completely abolished ATP-induced Ca²⁺ signals (Fig. 9A, B). Moreover, when formation of InsP₃ or its target channels were blocked, there was also a significant reduction in ATP-induced Ca²⁺ release (Fig. 9C, D). These results provide evidence that intracellular Ca²⁺ signals are required for insertion of Mrp2 into the plasma membrane, and in particular suggest that Ca²⁺ released by an InsP₃R-dependent mechanism is responsible for targeting of Mrp2.

Fig. 8.

Intracellular Ca²⁺ promotes insertion of Mrp2 into the plasma membrane. (A) Representative HepG2 cell expressing rat GFPMrp2 and imaged by TIRF microscopy. The same cell is shown before and 15 minutes after addition of ATP (100 μM) to the bath. Yellow arrows indicated regions of increased fluorescence. Cells are pseudo-colored according to the scale at right. (B) Serial magnified images of the region indicated by the white square in (A) reveals progressive accumulation of GFP-Mrp2 in that region over time. Images are separated by 2-minute intervals. (C) Mean fluorescence increases in control cells, cells stimulated with ATP alone, or pretreated with Bapta-AM (50 μM). Note that there is a significant increase in GFP-Mrp2 membrane fluorescence in cells treated with ATP (124.9% ± 2.2%, n = 81 regions in 19 cells) as compared with control cells (106.7% ± 0.8%, n = 83; P < 0.0001) and that this increase is attenuated by pretreatment with BAPTA (112.9% ± 2.2%, n = 42; P < 0.001 by oneway ANOVA with Bonferroni post-test). Data represent the average of four independent experiments.

Fig. 9.

ATP-induced Ca²⁺ signals are mediated by InsP₃R in HepG2 cells. (A) Representative tracings of ATP (100 μM)-induced Ca²⁺ signals in control and BAPTA/AM-treated cells. (B) Pretreatment with BAPTA/AM significantly reduces the amplitude of Ca²⁺ signals (12.57% ± 0.36% increase above baseline, n = 550 cells) as compared with control (109.5% ± 1.95% increase, n = 550). (C) Representative tracings of Ca²⁺ signals in control cells or cells pretreated with either U73122 (10 μM) or Xestospongin C (1 μM). (D) Ca²⁺ release was partially reduced by U73122 pretreatment (86.19% ± 5.87% increase above baseline fluorescence, n = 233 versus 178.3% ± 4.50% increase, n = 504 in control cells, P < 0.0001). Pre-incubation with the InsP₃R blocker Xestospongin C greatly reduced ATP-induced Ca²⁺ release (27.69% ± 2.31%, n= 184 versus 178.3% ± 4.50%, n = 504, P < 0.0001) by one-way ANOVA with Bonferroni post-test.

Discussion

The current work shows that InsP₃R2 KO hepatocytes have defective Ca²⁺ signaling and organic anion secretion, and that insertion of Mrp2 into the plasma membrane is Ca²⁺ dependent. InsP₃R2 KO mice develop normally, and no gross phenotypical alteration has been described. However, double-knockout mice lacking both InsP₃R2 and InsP₃R3 have deficient secretion of saliva and pancreatic zymogens, which results in an inability to fully digest and absorb nutrients. Pancreatic acinar cells isolated from these double-knockout animals have decreased Ca²⁺ release and accumulate zymogen granules, presumably reflecting impaired exocytosis.⁴³ In the current work, Ca²⁺ signals in hepatocytes isolated from InsP₃R2 KO mice showed reduced amplitude, and there was an increase in rise time of the Ca²⁺ signals. Together these kinetic parameters of Ca²⁺ signaling indicate a significant impairment in Ca²⁺ release in the InsP₃R2 KO cells. This effect is consistent with the observation that pericanalicular InsP₃R2 are essential to create a trigger zone to initiate and propagate Ca²⁺ waves in rat hepatocytes,^12,14 much like what is observed in pancreatic acinar cells^16,44 and in cholangiocytes.^32,45

The results presented here also demonstrate decreased Mrp2 activity in sandwich cultures of InsP₃R2 KO hepatocytes. This effect is likely attributable to the action of InsP₃R2 as an intracellular Ca²⁺ release channel, because the effect was duplicated by chelation of cytosolic Ca²⁺. This apparent effect of Ca²⁺ could be attributable to regulation of transporter activity, trafficking, or expression. The absence of InsP₃R2 did not affect either expression levels or localization of Mrp2, however. Instead, TIRF measurements suggested that release of intracellular Ca²⁺ promotes insertion of rat Mrp2 into the plasma membrane. This is consistent with previous observations that Ca²⁺ and PKC act as regulators of exocytosis in the liver⁴⁶ and also with reports showing that tauroursodeoxycholic acid induces Ca²⁺ release²⁴ and Ca²⁺-dependent exocytosis²⁵ and activates conventional PKCs, leading to Mrp2 insertion into the canalicular membrane.³⁹ However, the current work cannot exclude the possibility that Ca²⁺ also may directly regulate transporter activity, perhaps through PKC-dependent phosphorylation.³⁹

Mutations that affect Mrp2 expression and trafficking are found in patients with Dubin-Johnson syndrome,⁴⁷ an inherited form of hyperbiluribinemia, as well as in the GY/TR⁻ and Eisai rat strains,^48,49 both of which exhibit a specific defect in organic anion transport. One might therefore predict that InsP₃R2 KO animals would also have increased serum bilirubin levels. However, our results show serum bilirubin levels in InsP₃R2 KO mice that are similar to what is found in WT mice. This may reflect appropriate localization of Mrp2 in basal conditions in the KO animals (Fig. 7). Together, these findings suggest that InsP₃R2-dependent Ca²⁺ release may be important for recruitment and insertion of additional Mrp2 transporters into the canalicular membrane but would not be essential for the behavior of this transporter under basal conditions. An alternative explanation is that any reduction in bile acid-independent bile flow, the fraction of bile flow that is directly regulated by Mrp2 activity,⁵⁰ is compensated for by transport events taking place downstream, at the level of the biliary tree.

Other second messengers and signaling pathways have been implicated in transporter trafficking in hepatocytes. Most notably, cyclic adenosine monophosphate (cAMP) stimulates insertion of Mrp2 into the plasma membrane in rat hepatocytes in short-term culture,³⁶ and this is partially mediated by activation of PI3K and PKCδ.⁵¹ Cyclic AMP also potentiates Ca²⁺ oscillations in isolated rat hepatocytes.^52,53 Moreover, cAMP specifically enhances InsP₃R2-dependent Ca²⁺ release independent of the activation of protein kinase A.⁵⁴ In light of our findings that InsP₃R-mediated (most likely InsP₃R2) Ca²⁺ release enhances canalicular insertion of Mrp2, these observations raise the interesting possibility that cAMP-mediated canalicular targeting of Mrp2 occurs via the effects of cAMP on InsP₃R2 and Ca²⁺ release. However, the importance of this particular cross-talk pathway between Ca²⁺ and cAMP signaling remains to be demonstrated in hepatocytes. Moreover, it remains to be determined whether InsP₃R2-dependent Ca²⁺ signals also control the trafficking and canalicular targeting of other transporters that are important for bile formation.