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Control (TC)

Figure 18.4 Quantification of transporter distribution by fluorescence densitometry. (A) Rat livers were perfused with 25 mmol/liter of TC (control) orTCDC. After immu-nostaining with an anti-Ntcp antibody, fluorescence pictures were recorded by confocal scanning microscopy (as shown in Fig. 18.3B). Pictures were analyzed by a semiautomatic software: single cells were identified by their border strips. Thereafter, distribution of fluorescence intensity perpendicular to all straight stretches of single cells was measured and averaged as shown on the right side. InTCDC-treated livers, fluorescence profiles of Ntcp were flattened as compared toTC (control)-treatedlivers.

immunostained liver sections. Flattening or narrowing of these fluorescence profiles is detected automatically in a multitude of hepatocytes and can be taken as a measure for transporter internalization or membrane insertion, respectively (Fig. 18.4). Here, addition of the hydrophobic bile acid taur-ochenodeoxycholate, which induces cell shrinkage (Becker et al., 2007), reduces the net uptake oftaurocholate (Fig. 18.3A), alters immunoreactivity of Ntcp in liver slices (Fig. 18.3B), and results in a flattening of the Ntcp fluorescence profile (Fig. 18.4) across the plasma membrane, suggestive of an internalization of Ntcp in response to this bile acid. Accordingly, in Ntcp-transfected HepG2 cells, reduction of membrane-bound Ntcp can be measured by flow cytometry (Fig. 18.3C). Such a regulation may protect hepatocytes from bile acid overload, which would otherwise result in apopto-tic hepatocyte injury.

Several independent techniques can be used in order to demonstrate transporter insertion or retrieval in hepatocytes in response to osmotic challenges, all of them supporting the concept that bile formation is controlled at the short-term timescale by a regulated insertion/retrieval of transporter molecules. Future developments may aim toward the visualization of such transporter movements in the living cell and the disclosure of protein—protein interactions involved in this dynamic process; fluorescence resonance energy

6. Conceding Remarks

Hypoosmolarity/ cell swelling

Extracellular matrix

Choleresis

Bsep insertion

Choleresis

Bsep insertion

Figure 18.5 Cell swelling-induced signaling pathway. Cell swelling triggers integrin attachment to the extracellular matrix (ECM) and integrin activation. Focal adhesion kinases (FAK) and src kinases are thereby activated. Further signal transduction involves the dual activation of Erk-type and p38-type MAP kinases. Eventually, transporter bearing vesicles are inserted into the target membrane in a microtubule (MT)-depen-dent manner, along with an increase of transporter activity. A similar mechanism is activated nonosmotically by tauroursodeoxycholate and triggers choleresis.

transfer techniques may have here some potential. Furthermore, much has to be learned about the osmosensing and osmosignaling pathways and their nonosmotic activation, which will give new insights into the development of choleresis and cholestasis in health and disease (Fig. 18.5).

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