*3.6. Activation of TGR5 Inhibits the ET-1-Mediated Increase in Portal Pressure*

Perfusion of mouse livers with buffer containing only DMSO as vehicle did not cause any significant changes in portal perfusion pressure. A challenge of these mice with increasing concentrations of ET-1 led to a dose-dependent increase in portal perfusion pressure. Addition of a TGR5 agonist to the ET-1-enriched perfusion buffer significantly reduced the ET-1-mediated rise in portal pressure as compared to vehicle containing perfusion buffer, indicating that TGR5 activation rapidly reduces ET-1-mediated portal hypertension (DMSO vs. TGR5 Ago/+1 nM ET-1 2.41 ± 0.26 cmH20 vs. 1.42 ± 0.18 cmH20 (1.7-fold decrease); DMSO vs. TGR5 Ago/+5 nM ET-1 2.9 ± 0.18 cmH20 vs. 1.89 ± 0.28 cmH20 (1.5-fold decrease), DMSO vs. TGR5 Ago/+15 nM ET-1 3.14 ± 0.15 cmH20 vs. 2.20 ± 0.3 cmH20 (1.4-fold decrease), (*n* = 5–6), *p* < 0.05) (Figure 7A).

**Figure 6.** Stimulation of TGR5 in isolated HSCs triggers internalization of the endothelin A receptor (ETAR), thus reducing the contractile response towards ET-1. (**A**) Contraction of rat HSCs on collagen lattices was measured in response to ET-1 after pre-incubation with DMSO, a TGR5 agonist (10 μM), or forskolin (10 μM), which was used as TGR5-independent positive control. Surface area of collagen lattices served as indirect measure of contractile activity 2 h and 24 h after ET-1 addition. Surface area of cells/collagen lattices treated with DMSO and ET-1 was set to 1.0. Data are shown as mean ± SEM (*n* = 12); \* statistically significant from DMSO/ET-1-treated cells (*p* < 0.01). (**B**) Immunofluorescence staining of murine HSCs using an antibody directed against the ETAR (in green) and Alexa-543 coupled wheat germ agglutinin (WGA, shown in red) to stain the cell surface. HSCs were incubated with either DMSO or a TGR5 agonist (10 μM) for 24 h prior to fixation and immunofluorescence staining. Colocalization of ETAR and WGA was determined using the coloc2 plugin for ImageJ. Colocalized pixels are shown in white in the merge images. (**C**) Colocalization was quantified using Pearson's correlation coefficient for colocalization, as determined by the coloc2 plugin for ImageJ for each selected region of interest (ROI). Data are shown as mean ± SEM; \* statistically significant from DMSO-treated cells (*n* = 8–9; 2–3 different ROIs per condition of three different cell isolations) (*p* < 0.05).

**Figure 7.** TGR5-dependent regulation of portal perfusion pressure. (**A**) Portal perfusion pressure of TGR5 transgenic mice (TGR5tg) was continuously measured in the presence of DMSO or a TGR5 agonist and increasing concentrations of ET-1. Perfusion was performed with either DMSO or a TGR5 agonist (10 μM) and increasing concentrations of endothelin-1 (ET-1, 5–15nM). Data are shown relative to controls, where the perfusion pressure in the presence of DMSO-containing perfusion buffer following an equilibrium phase of 15 min at the starting point was set to 1.0. All data are shown as mean ± SEM; \* statistically significant from DMSO-treated cells (*p* < 0.05) (*n* = 6–9). (**B**) Cartoon depicting the mechanisms of TGR5-dependent inhibition of ET-1 signaling within the sinusoid. Activation of TGR5 through cAMP inhibits transcription and subsequent secretion of ET-1 from LSECs. ET-1, produced by LSECs, is known to induce contraction of activated HSCs cells by binding to the ETAR, leading to an increase of intrahepatic vascular resistance and, hence, the development of portal hypertension. Activation of TGR5 in HSCs promotes internalization of the ETAR, reducing ligand binding at the cell surface and thus contractile activity. In conclusion, ligand binding to TGR5 activates two mechanisms that act synergistically to reduce intrahepatic vascular resistance and, thus, portal hypertension.
