Regulation of Plasma Membrane Localization of the Na+-taurocholate cotransporting polypeptide (Ntcp) by Hyperosmolarity and Tauroursodeoxycholate

Introduction:

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 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.

Methods:

Rat livers were perfused with either normo- or hyperosmotic Krebs-Henseleit buffer. Addition of inhibitors, Db-cAMP and tauroursodeoxycholate (TUDC) to the influent perfusate was made by dissolution into the buffer. Liver tissue was obtained for immunofluorescence Ntcp- and Bsep-staining following analyzation of transporter localization. Cell culture experiments were performed in primary rat hepatocytes and HepG2 cells. Liver tissue was also processed for immunoprecipitation and Western blot analysis of Src kinases and Ntcp. Generation of ROS was measured using H2DCFDA.

Results:

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 wildtype mice, but not in p47phox knockout mice. Paralleling the inhibition of ROS formation, the hyperosmolarity-induced Fyn phosphorylation was sensitive to inhibition of PKCζ by the specific PKCζ pseudosubstrate, and by the broad spectrum PKC inhibitors chelerythrine and Gö 6850. 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. In line with a TUDC-induced reversal of hyperosmotic Fyn activation, TUDC not only prevented the hyperosmolarity-induced Ntcp retrieval from the plasma membrane, but also the hyperosmolarity-induced Bsep retrieval from the canalicular membrane.

Discussion/Conclusion:

These findings suggest a coordinated, 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. The study provides new insights into the regulation of bile salt transport.

Corresponding author: Sommerfeld, Annika

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