Abstract
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 ETA receptor antagonist ambrisentan. ET-1-induced contraction was also largely reduced in Ca2+-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 Ca2+ 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 Gd3+ 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 Ca2+-influx through cation channels other than SOCs and produces myosin II-mediated contraction of non-activated HSCs via ETA receptors, as well as via mechanisms involving Ca2+-calmodulin and Rho kinase.
Highlights
Hepatic stellate cells (HSCs), located in the space of Disse between sinusoidal endothelial cells and hepatocytes, constitute 5%–8% of the total number of liver-resident cells [1]
Since isolated quiescent HSCs (qHSCs) are well known to be activated by culture on plastic plates [14], the preparations used in these studies might have contained activated HSCs (aHSCs) when the measurement was performed
Isolated qHSCs are well known to be activated by culture on plastic plates [14]; the involvement of aHSCs in the response is hard to be ruled out when using this method
Summary
Hepatic stellate cells (HSCs), located in the space of Disse between sinusoidal endothelial cells and hepatocytes, constitute 5%–8% of the total number of liver-resident cells [1]. Isolated rat HSCs have been shown to display contractile activity in response to ET-1 [9]. The contraction of human HSCs has been suggested to be induced by a chemokine CXCL12 through a calcium-independent pathway [10]. In these studies, contraction was evaluated by observing the shrinkage of collagen lattices on which isolated HSCs were cultured. Since isolated qHSCs are well known to be activated by culture on plastic plates [14], the preparations used in these studies might have contained aHSCs when the measurement was performed. We have developed a novel method to quantitatively evaluate contractile properties in single isolated non-activated HSCs in real-time. The results shown here provide direct evidence for ET-1-induced contraction of non-activated HSCs
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