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

Publisher: Public Library of Science Country of Publication: United States NLM ID: 101285081 Publication Model: eCollection Cited Medium: Internet ISSN: 1932-6203 (Electronic) Linking ISSN: 19326203 NLM ISO Abbreviation: PLoS One Subsets: MEDLINE

Original Publication: San Francisco, CA : Public Library of Science

Cell Physiological Phenomena/*drug effects ; Endothelin-1/*pharmacology ; Hepatic Stellate Cells/*metabolism ; Signal Transduction/*drug effects; Animals ; Calcium/metabolism ; Calcium Channels, N-Type/genetics ; Calcium Channels, N-Type/metabolism ; Calmodulin/antagonists & inhibitors ; Calmodulin/metabolism ; Cells, Cultured ; Endothelin Receptor Antagonists/pharmacology ; Heterocyclic Compounds, 4 or More Rings/pharmacology ; Male ; Mice ; Myosin Type II/antagonists & inhibitors ; Myosin Type II/metabolism ; Phenylpropionates/pharmacology ; Pyridazines/pharmacology ; RNA, Messenger/genetics ; Receptor, Endothelin A/metabolism ; Sulfonamides/pharmacology ; rho-Associated Kinases/metabolism

Competing Interests: The authors have declared that no competing interests exist.
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.

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0 (Calcium Channels, N-Type)
0 (Calmodulin)
0 (Endothelin Receptor Antagonists)
0 (Endothelin-1)
0 (Heterocyclic Compounds, 4 or More Rings)
0 (Phenylpropionates)
0 (Pyridazines)
0 (RNA, Messenger)
0 (Receptor, Endothelin A)
0 (Sulfonamides)
0 (voltage-dependent calcium channel (P-Q type))
20WC4J7CQ6 (blebbistatin)
65595-90-6 (W 7)
EC 2.7.11.1 (rho-Associated Kinases)
EC 3.6.1.- (Myosin Type II)
HW6NV07QEC (ambrisentan)
SY7Q814VUP (Calcium)

Date Created: 20210803 Date Completed: 20211203 Latest Revision: 20211214

20250114

PMC8330899

10.1371/journal.pone.0255656

34343209