*3.4. Activation of TGR5 in LSECs Lowers ET-1 Expression and Secretion*

ET-1 in liver is mainly secreted by LSECs under physiological conditions [24]. We have previously demonstrated that rat and human LSECs express TGR5 [1,4]. To determine whether secretion of ET-1 from LSECs was modulated by TGR5, LSECs were isolated from WT mice. Expression and localization of TGR5 in these primary cells was confirmed by immunofluorescence staining with an antibody against TGR5 and an antibody against vascular endothelial cadherin (Ve-Cad). TGR5 was localized in the plasma membrane, as well as in intracellular vesicular structures (Figure 5A). Treatment of primary murine LSECs with vehicle (DMSO), taurolithocholic acid (TLC, 25 μM), a non-bile acid TGR5 agonist (RO5527239, 10 μM) [27], and forskolin (10 μM) for 24 h resulted in a significant decrease in ET-1 mRNA expression (DMSO vs. TLC (*n* = 10) 1.0 vs. 0.76 ± 0.06 (1.3-fold reduction, *p* < 0.01); DMSO vs. TGR5 Ago (*n* = 7) 1.0 vs. 0.73 ± 0.08 (1.4-fold reduction, *p* < 0.05); DMSO vs. forskolin

(*n* = 6) 1.0 vs. 0.58 ± 0.12 (1.7-fold reduction, *p* < 0.05)) (Figure 5B). We have previously demonstrated that stimulation of TGR5 in LSECs through coupling to a stimulatory G protein triggered an increase in intracellular cAMP [1]. Therefore, forskolin, a direct activator of adenylate cyclase, was used as TGR5-independent positive control [1]. Furthermore, detection of ET-1 in the cell culture supernatant of these cells demonstrated a reduction in ET-1 protein levels in response to TLC, the TGR5 Ago, and forskolin (Figure 5C) (DMSO vs. TLC (*n* = 10) 1.0 vs. 0.80 ± 0.05 (1.3-fold reduction, *p* < 0.01); DMSO vs. TGR5 Ago (*n* = 9) 1.0 vs. 0.67 ± 0.12 (1.5-fold reduction, *p* < 0.05); DMSO vs. forskolin (*n* = 5) 1.0 vs. 0.54 ± 0.11 (1.9-fold reduction, *p* < 0.05)). Thus, stimulation of TGR5 not only reduces ET-1 mRNA expression, but also ET-1 secretion from LSECs.

**Figure 5.** Stimulation of TGR5 in isolated liver sinusoidal endothelial cells (LSECs) reduces endothelin-1 (ET-1) mRNA expression and secretion. (**A**) Immunofluorescence staining of TGR5 in isolated LSECs (in red) demonstrates localization of the receptor within the plasma membrane, as well as in intracellular vesicular structures. An antibody against vascular endothelial cadherin (VE-cad, shown in green) was used to visualize cell junctions near the plasma membrane. Nuclei were stained with Hoechst (shown in blue). Bar = 10 μm. (**B**) ET-1 mRNA levels in isolated murine LSECs in response to a 24-h incubation with DMSO (=control), TLC (25 μM), a TGR5 agonist (10 μM), or forskolin (10 μM). The mRNA level after DMSO treatment was set to 100%. (**C**) Detection of ET-1 protein amounts by ELISA in the cell supernatant of LSECs incubated for 24 h with DMSO (=control), TLC (25 μM), a TGR5 agonist (10 μM), or forskolin (10 μM). ET-1 protein levels after stimulation with DMSO were set to 100%. Data are presented as mean ± SEM (n = 6–9). \* Statistically significant difference as compared to DMSO-treated controls (*p* < 0.05).
