*3.5. Activation of TGR5 in HSCs Attenuates ET-1-Mediated Contractility*

Activated HSCs are able to contract, leading to an increase in portal pressure due to their perivascular location in the space of Disse [40]. ET-1 has been identified as one of the key mediators regulating HSC contractility [24,41]. Since we observed an increase in portal pressure solely in TGR5 KO mice after LCA feeding, we hypothesized that activation and contraction of HSCs may contribute to this phenotype. To determine whether ET-1-dependent HSC contraction is modulated by TGR5, HSC contraction in response to ET-1 was analyzed. Rat HSCs were cultivated on collagen lattices for 7 days [40]. After this interval, DMSO, a TGR5 agonist (10 μM), or forskolin (10 μM) were added to the culture medium 30 min prior to stimulation with ET-1 (1 nM). Measurement of the collagen lattice surface area 2 h and 24 h after addition of ET-1 revealed a significantly smaller reduction in relative surface area in TGR5 agonist- or forskolin-treated cells, indicating an attenuation of HSC contractility (Figure 6A); 2 h: DMSO/ET-1 vs. TGR5 Ago/ET-1 1.0 vs. 1.64 <sup>±</sup> 0.11 pixels2; DMSO/ET-1 vs. forskolin/ET-1 1.0 vs. 2.85 <sup>±</sup> 0.36 pixels2, *n* = 12, *p* < 0.01; 24 h: DMSO/ET-1 vs. TGR5 Ago/ET-1 1.0 vs. 1.89 <sup>±</sup> 0.27 pixels2; DMSO/ET-1 vs. forskolin/ET-1 1.0 vs. 3.96 <sup>±</sup> 0.51, *n* = 12, *p* < 0.01. It was previously shown that cAMP may desensitize the ET-1 receptor-A (ETAR) towards its ligand on activated HSCs through internalization. Thus, the ETAR's responsiveness is being shifted from picomolar to nanomolar concentrations [42]. Since TGR5 is coupled to a stimulatory G protein [6,8], we hypothesized that ligand binding to TGR5 could result in increased intracellular cAMP levels and internalization of the ETAR. Immunofluorescence staining localized the ETAR within the plasma membrane of HSCs, as demonstrated by colocalization with the plasma membrane marker wheat germ agglutinin (WGA) (Figure 6B). Treatment with the TGR5 agonist reduced the amount of ETAR within the plasma membrane, indicating internalization of the receptor (Figure 6C) (DMSO vs. TGR5 Ago (*n* = 8–9) 0.37 ± 0.06 Pearson's correlation coefficient vs. 0.11 ± 0.04, 3.4-fold decrease, *p* < 0.01).
