Regulation of plasma membrane localization of the Na+-taurocholate cotransporting polypeptide (Ntcp) by hyperosmolarity and tauroursodeoxycholate

Abstract

In perfused rat liver, hepatocyte shrinkage induces a Fyn-dependent retrieval of the bile salt export pump (Bsep) and multidrug resistance-associated protein 2 (Mrp2) from the canalicular membrane (Cantore, M., Reinehr, R., Sommerfeld, A., Becker, M., and Häussinger, D. (2011) J. Biol. Chem. 286, 45014-45029) leading to cholestasis. However little is known about the effects of hyperosmolarity on short term regulation of the Na(+)-taurocholate cotransporting polypeptide (Ntcp), the major bile salt uptake system at the sinusoidal membrane of hepatocytes. The aim of this study was to analyze hyperosmotic Ntcp regulation and the underlying signaling events. Hyperosmolarity induced a significant retrieval of Ntcp from the basolateral membrane, which was accompanied by an activating phosphorylation of the Src kinases Fyn and Yes but not of c-Src. Hyperosmotic internalization of Ntcp was sensitive to SU6656 and PP-2, suggesting that Fyn mediates Ntcp retrieval from the basolateral membrane. Hyperosmotic internalization of Ntcp was also found in livers from wild-type mice but not in p47(phox) knock-out mice. Tauroursodeoxycholate (TUDC) and cAMP reversed hyperosmolarity-induced Fyn activation and triggered re-insertion of the hyperosmotically retrieved Ntcp into the membrane. This was associated with dephosphorylation of the Ntcp on serine residues. Insertion of Ntcp by TUDC was sensitive to the integrin inhibitory hexapeptide GRGDSP and inhibition of protein kinase A. TUDC also reversed the hyperosmolarity-induced retrieval of bile salt export pump from the canalicular membrane. These findings suggest a coordinated and oxidative stress- and Fyn-dependent retrieval of sinusoidal and canalicular bile salt transport systems from the corresponding membranes. Ntcp insertion was also identified as a novel target of β1-integrin-dependent TUDC action, which is frequently used in the treatment of cholestatic liver disease.

Keywords: Fyn; Ntcp; Src; bile acid; cyclic AMP (cAMP); integrin; protein kinase C (PKC); tauroursodeoxycholate; transporter retrieval.

FIGURE 1.

Quantification of Ntcp and Na⁺/K⁺-ATPase distribution by fluorescence densitometry. Cryosections from perfused rat liver were immunostained for Ntcp and Na⁺/K⁺-ATPase, and fluorescence pictures were recorded by confocal LSM. The profile of the fluorescence intensity was measured over a thick line (8 μm) perpendicular to the plasma membrane of adjacent hepatocytes. The mean fluorescence intensity of each pixel along this line was calculated by Image-Pro Plus, and the obtained data (pixel positions with the associated pixel intensities, red and green channel) were transferred to an MS-Excel data sheet and plotted. The ordinate shows the normalized intensity of Na⁺/K⁺-ATPase staining depending on the distance (micrometers) from the center of the basolateral membrane (set as 0) and the normalized intensity of Ntcp-bound fluorescence. The means ± S.E. of 30 measurements in each of three individual experiments are shown.

FIGURE 2.

Regulation of Ntcp in hyperosmotically perfused rat liver. A, rat livers were perfused with normo-osmotic (305 mosmol/liter) or hyperosmotic (385 mosmol/liter) Krebs-Henseleit buffer for up to 60 min and immunostained for Ntcp and Na⁺/K⁺-ATPase. Densitometric analysis of fluorescence profiles and intensity of Ntcp and Na⁺/K⁺-ATPase distribution (visualization by confocal LSM) is shown. Under control conditions (green (0 min) and black (60 min)), Ntcp-bound fluorescence was largely localized in the center of the basolateral membrane. Hyperosmotic perfusion (red (30 min) and blue (60 min)) resulted in a significant lateralization of the Ntcp-bound fluorescence, but no significant changes of Na⁺/K⁺-ATPase fluorescence profiles were observed. The fluorescence profiles depicted are statistically significantly (p < 0.05) different from each other with respect to variance and peak height. Means ± S.E. of 30 measurements in each of at least three individual experiments for each condition are shown. B, rat livers were perfused with normo-osmotic (305 mosmol/liter) or hyperosmotic (385 mosmol/liter) Krebs-Henseleit medium for up to 60 min, immunostained for Ntcp and Na⁺/K⁺-ATPase, and visualized by SR-SIM. Under normo-osmotic conditions, Ntcp was largely localized in the membrane, and after hyperosmotic exposure Ntcp, but not the Na⁺/K⁺-ATPase, was internalized. Representative pictures of at least three independent experiments are depicted. The scale bars correspond to 10 μm in the overviews (upper row) and 1 μm in the four bottom rows with higher magnification of the plasma membrane. C, primary rat hepatocytes were cultured for 24 h and thereafter stimulated with normo-osmotic (305 mosmol/liter) or hyperosmotic (405 mosmol/liter) medium for up to 24 h. RNA was extracted, and Ntcp mRNA expression levels were analyzed by real time PCR. Ntcp mRNA expression levels are given relative to the unstimulated control (0 min). Data represent the mean ± S.E. of four independent experiments *, p < 0.05 statistical significance compared with the unstimulated control. #, p < 0.05 statistical significance between normo-osmotic and hyperosmotic stimulations.

FIGURE 3.

Determination of Ntcp and Na⁺/K⁺-ATPase distribution in hyperosmotically perfused rat livers. Rat livers were perfused with either inhibitor-containing Krebs-Henseleit buffer (i.e. apocynin (20 μmol/liter) or NAC (10 mmol/liter) or SU6656 (1 μmol/liter) or PP-2 (250 nmol/liter), black) or hyperosmotic (385 mosmol/liter) Krebs-Henseleit buffer in the absence (blue) or presence of the respective inhibitors (green). B, Ntcp and Na⁺/K⁺-ATPase were visualized after perfusion with either NAC, hyperosmotic Krebs-Henseleit buffer, or NAC plus Krebs-Henseleit buffer at t = 30 min (see perfusion plan (A)) by SR-SIM. Representative pictures of three independent experiments are depicted. The scale bar corresponds to 1 μm. C and D, densitometric analysis of fluorescence profiles and intensity of Ntcp and Na⁺/K⁺-ATPase staining at t = 30 min (see perfusion plan (A)) is shown. The means ± S.E. of 30 measurements in each of three individual experiments for each condition are shown. NAC (C), apocynin, SU6656, and PP-2 (D) all significantly inhibited hyperosmolarity-induced retrieval of Ntcp from the membrane (p < 0.05), whereas the inhibitors by themselves did not significantly affect Ntcp localization.

FIGURE 4.

Distribution of Ntcp in hyperosmotically perfused wild-type and p47^phox knock-out mouse liver. Mouse livers were perfused for 30 min with either normo-osmotic (305 mosmol/liter, black) or hyperosmotic Krebs-Henseleit buffer (385 mosmol/liter, green) and stained for Ntcp and Na⁺/K⁺-ATPase. Densitometric analysis of fluorescence profiles and intensity of Ntcp and Na⁺/K⁺-ATPase staining is shown. The means ± S.E. of 30 measurements in each of three individual experiments for each condition are shown. As shown by colocalization with Na⁺/K⁺-ATPase, Ntcp is largely localized in the membrane under normo-osmotic conditions. After hyperosmotic perfusion, Ntcp is no longer colocalized (p < 0.05) with Na⁺/K⁺-ATPase and appears inside the cells in wild-type animals but remained unchanged in p47^phox knock-out mice.

FIGURE 5.

TUDC-induced re-insertion of Ntcp into the membrane is integrin- and PKA-dependent. A, rat livers were perfused as given in the perfusion plan of the respective experiments, stained for Ntcp and Na⁺/K⁺-ATPase, and visualized by SR-SIM. Representative pictures of at least three independent experiments are depicted. Under normo-osmotic (305 mosmol/liter) conditions (t = 0), Ntcp was largely localized in the membrane, whereas after hyperosmotic (385 mosmol/liter) perfusion, Ntcp appears inside the cells and colocalizes no longer with Na⁺/K⁺-ATPase (t = 30 min). TUDC (20 μmol/liter) induced the re-insertion of Ntcp in the membrane (t = 60 min). GRGDSP (10 μmol/liter) inhibited the TUDC induced re-insertion in the membrane (p < 0.05), whereas GRADSP (10 μmol/liter) had no effect on Ntcp distribution (t = 60 min). The scale bar corresponds to 1 μm. C, rat livers were perfused as given in the perfusion plan (B), stained for Ntcp and Na⁺/K⁺-ATPase, and analyzed densitometrically. Densitometric analysis of fluorescence profiles and intensity of Ntcp and Na⁺/K⁺-ATPase at t = 60 min staining is shown. The means ± S.E. of 10 measurements in each of three individual experiments for each condition are shown. Bt₂cAMP (Db-cAMP) (50 μmol/liter) and TUDC (20 μmol/liter) significantly inhibited hyperosmolarity-induced retrieval of Ntcp from the membrane (p < 0.05). GRGDSP (10 μmol/liter) and H89 (2 μmol/liter) inhibited the TUDC-induced re-insertion in the membrane (p < 0.05), whereas GRADSP (10 μmol/liter) had no effect on Ntcp distribution. Furthermore, H89 (2 μmol/liter) inhibited the Bt₂cAMP-induced re-insertion of Ntcp (p < 0.05).

FIGURE 6.

TUDC triggers integrin- and PKA-dependent inhibition of hyperosmolarity-induced Fyn and Yes activation. A, rat livers were perfused as mentioned in the perfusion plans. B, liver samples were analyzed for activating phosphorylation of Src kinase family members Fyn, Yes, and c-Src. C, blots were analyzed densitometrically. t = 60 min was compared with the respective control (t = 0 min, set as 1), i.e. sample without institution of either hyperosmolarity (385 mosmol/liter) or TUDC (20 μmol/liter), or with the respective inhibitor (i.e. H89 (2 μmol/liter), GRADSP and GRGDSP (each 10 μmol/liter). Densitometric analysis (means ± S.E.) of at least three independent perfusion experiments is shown. In line with a previous study (1), hyperosmolarity induced within 30 min a significant phosphorylation of Fyn and Yes (*, p < 0.05), however no c-Src activation was observed. Phosphorylation of Fyn and Yes was inhibited by TUDC (#, p < 0.05). Inhibition of Fyn and Yes phosphorylation by TUDC was sensitive to H89 and GRGDSP ($, p < 0.05), however GRADSP had no effect.

FIGURE 7.

Bt₂cAMP triggers PKA-dependent inhibition of hyperosmolarity-induced Src kinase family activation. Rat livers were perfused as described under “Experimental Procedures” and as mentioned in the perfusion plans (A) of the respective experiments. B, liver samples were analyzed for activation of Src kinase family members Fyn, Yes, and c-Src. Blots were analyzed densitometrically. t = 60 min was compared with the respective control (t = 0 min, set as 1), i.e. sample without institution of either hyperosmolarity (385 mosmol/liter) or Bt₂cAMP (50 μmol/liter) or with H89 (2 μmol/liter). Representative blots and densitometric analysis (means ± S.E.) of at least three independent perfusion experiments are shown. In line with Fig. 5, hyperosmolarity induced within 30 min a significant phosphorylation of Fyn and Yes (*, p < 0.05), although no c-Src activation was observed. Phosphorylation of Fyn and Yes was inhibited by Bt₂cAMP (#, p < 0.05). Inhibition of Fyn and Yes phosphorylation by Bt₂cAMP was sensitive to H89 ($, p < 0.05).

FIGURE 8.

TUDC and cAMP induce serine dephosphorylation of Ntcp in HepG2-Ntcp cells. Ntcp-transfected HepG2 cells were stimulated with either TUDC (100 μmol/liter), Bt₂cAMP (10 μmol/liter) under normo-osmotic (305 mosmol/liter) conditions, or with hyperosmotic medium (405 mosmol/liter) without and with TUDC. GFP-tagged Ntcp was analyzed with regard to serine phosphorylation. Treatment with TUDC as well as treatment with Bt₂cAMP resulted in decreased serine phosphorylation, although hyperosmolarity triggered an increase in serine phosphorylation of the Ntcp. Ntcp serine phosphorylation was analyzed by Western blot (WB) and subsequent densitometric analysis. GFP served as a loading control. Ntcp serine phosphorylation under control condition was set to 100%. Data represent means ± S.E. of at least five independent experiments. IP, immunoprecipitation. *, p < 0.05 statistically significant compared with the unstimulated control. #, p < 0.05 statistical significance between hyperosmolarity and hyperosmolarity plus TUDC.

FIGURE 9.

Distribution of the hepatobiliary transporter Bsep. A, cryosections from perfused rat liver were immunostained for Bsep and ZO-1, and fluorescence images were recorded by confocal LSM. The profile of the fluorescence intensity was measured over a thick line (8 μm) perpendicular to the canaliculus. The mean fluorescence intensity of each pixel along the line was calculated by Image-Pro Plus, and the obtained data (pixel positions with the associated pixel intensities, red and green channel) were transferred to an MS-Excel data sheet and plotted. The ordinate shows the normalized intensity of ZO-1 staining depending on the distance (micrometers) from the center of the canaliculus (set as 0) and the normalized intensity of Bsep-bound fluorescence. The means ± S.E. of 30 measurements in each of three individual experiments are shown. Under control conditions, ZO-1 fluorescence profiles show two peaks, whereas Bsep-bound fluorescence is largely localized in the canalicular membrane between the ZO-1 fluorescence. B, rat livers were perfused as given in the perfusion plan, stained for Bsep and ZO-1, and visualized by confocal laser scanning microscopy. Representative pictures of at least three independent experiments are depicted. The scale bar corresponds to 5 μm. Under normo-osmotic conditions (t = 0), Bsep was largely localized in the canalicular membrane, whereas after hyperosmotic perfusion Bsep appears inside the cells (t = 30 min). TUDC (20 μmol/liter) induced re-insertion in the membrane (t = 60 min). Under control conditions (black, t = 0 min), Bsep-bound fluorescence was largely localized in the canalicular membrane. Hyperosmotic perfusion (green, t = 30 min) resulted in a significant lateralization of the Bsep-bound fluorescence. TUDC (20 μmol/liter) induced re-insertion in the membrane (blue, t = 60 min). The fluorescence profiles depicted are statistically significantly (p < 0.05) different from each other with respect to variance and peak height. Under control conditions (305 mosmol/liter, black), ZO-1 fluorescence profiles show two peaks. Liver perfusion experiments with hyperosmolarity (green) and hyperosmolarity plus TUDC (blue) resulted in no significant changes of ZO-1 fluorescence profiles with respect to the distance of the peaks and to the variance of fluorescence profiles. Means ± S.E. of 30 measurements in each of at least three individual experiments for each condition are shown.

FIGURE 10.

Inhibition of hyperosmolarity-induced generation of reactive oxygen species. Primary rat hepatocytes were cultured for 24 h, loaded with 5 μmol/liter CM-H₂DCFDA as described under “Experimental Procedures,” and thereafter stimulated with hyperosmotic medium (405 mosmol/liter) or hyperosmotic medium plus TUDC (100 μmol/liter) for 15 min. When indicated, PKCζ pseudosubstrate (100 μmol/liter), chelerythrine (20 μmol/liter), Gö 6976 (200 nmol/liter), or Gö 6850 (10 μmol/liter) were preincubated for 30 min. ROS levels of at least three independent experiments were expressed as the mean-fold increase over control (set as 1) ± S.E. * indicates the statistical significance compared with the control (p < 0.05); $ indicates the statistical significance of the TUDC effect (p < 0.05), and # indicates the statistical significance of the inhibitor effect (p < 0.05). n.s., not significant.

FIGURE 11.

Effect of PKC inhibitors on hyperosmotic Fyn phosphorylation and inhibition of hyperosmotic Ntcp internalization by the broad spectrum PKC inhibitor Gö 6850. A, primary rat hepatocytes were cultured for 24 h and thereafter stimulated with hyperosmotic medium (405 mosmol/liter) for 30 min. When indicated, PKCζ pseudosubstrate (100 μmol/liter), chelerythrine (20 μmol/liter), Gö 6976 (200 nmol/liter), or Gö 6850 (10 μmol/liter) were preincubated for 30 min. Fyn was immunoprecipitated and detected for phosphorylation by Western blotting. Total Fyn served as a loading control. Hyperosmolarity-induced Fyn phosphorylation was sensitive to inhibition of PKCζ (PKCζ pseudosubstrate, chelerythrine, and Gö 6850). Fyn phosphorylation under control condition was set to 1. Data represent means ± S.E. of three independent experiments. *, p < 0.05 statistically significant compared with the unstimulated control. #, p < 0.05 statistical significance between hyperosmolarity and hyperosmolarity plus PKC inhibitor. B, rat livers were perfused with either Gö 6850 (1 μmol/liter)-containing normo-osmotic (305 mosmol/liter) buffer (black) or hyperosmotic (385 mosmol/liter) buffer in the absence (blue) or presence of the inhibitor (green) (see perfusion plan in Fig. 3A) and immunostained for Ntcp and Na⁺/K⁺-ATPase. Densitometric analysis of fluorescence profiles and intensity of Ntcp and Na⁺/K⁺-ATPase staining at t = 30 min (see perfusion plan) is shown. The means ± S.E. of 10 measurements in each of three individual experiments for each condition are shown. Gö 6850 significantly inhibited hyperosmolarity-induced retrieval of Ntcp from the membrane (p < 0.05), whereas the inhibitor by itself did not significantly affect Ntcp localization.

FIGURE 12.

Short term regulation of bile salt transporters in rat liver. Schematic illustration of signaling pathways involved in the short term regulation of basolateral Ntcp and canalicular Bsep and Mrp2. Hyperosmolarity-induced and NADPH-dependent ROS formation triggers activation of the Src family kinase Fyn and induces cholestasis by endocytosis not only of canalicular Bsep and Mrp2 but also of basolateral Ntcp. TUDC induces re-insertion of Ntcp by activation of β₁-integrin signaling.