Quantitative real-time measurement of endothelin-1-induced contraction in single non-activated hepatic stellate cells

Although quiescent hepatic stellate cells (HSCs) have been suggested to regulate hepatic blood flow, there is no direct evidence that quiescent HSCs display contractile abilities. Here, we developed a new method to quantitatively measure the contraction of single isolated HSCs and evaluated whether endothelin-1 (ET-1) induced contraction of HSCs in a non-activated state. HSCs isolated from mice were seeded on collagen gel containing fluorescent beads. The beads around a single HSC were observed gravitating toward the cell upon contraction. By recording the movement of each bead by fluorescent microscopy, the real-time contraction of HSCs was quantitatively evaluated. ET-1 induced a slow contraction of non-activated HSCs, which was inhibited by the non-muscle myosin II inhibitor blebbistatin, the calmodulin inhibitor W-7, and the ET(A) receptor antagonist ambrisentan. ET-1-induced contraction was also largely reduced in Ca(2+)-free conditions, but sustained contraction still remained. The tonic contraction was further diminished by the Rho-kinase inhibitor H-1152. The mRNA expression of P/Q-type voltage-dependent Ca(2+) channels (VDCC), as well as STIM and Orai, constituents of store-operated channels (SOCs), was observed in mouse non-activated HSCs. ET-1-induced contraction was not affected by amlodipine, a VDCC blocker, whereas it was partly reduced by Gd(3+) and amiloride, non-selective cation channel blockers. However, neither YM-58483 nor SKF-96365, which inhibit SOCs, had any effects on the contraction. These results suggest that ET-1 leads to Ca(2+)-influx through cation channels other than SOCs and produces myosin II-mediated contraction of non-activated HSCs via ET(A) receptors, as well as via mechanisms involving Ca(2+)-calmodulin and Rho kinase.