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...

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Autores principales:	Naoki Dohi, Momoka Yamaguchi, Reina Hase, Ryosuke Suzuki, Yumeto Wakabayashi, Ryota Nishiyama, Shin-Ya Saito, Tomohisa Ishikawa
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/253e4b840554416e9c0b451e52aa0110
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spelling	oai:doaj.org-article:253e4b840554416e9c0b451e52aa01102021-12-02T20:15:17ZQuantitative real-time measurement of endothelin-1-induced contraction in single non-activated hepatic stellate cells.1932-620310.1371/journal.pone.0255656https://doaj.org/article/253e4b840554416e9c0b451e52aa01102021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0255656https://doaj.org/toc/1932-6203Although 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.Naoki DohiMomoka YamaguchiReina HaseRyosuke SuzukiYumeto WakabayashiRyota NishiyamaShin-Ya SaitoTomohisa IshikawaPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 8, p e0255656 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Naoki Dohi Momoka Yamaguchi Reina Hase Ryosuke Suzuki Yumeto Wakabayashi Ryota Nishiyama Shin-Ya Saito Tomohisa Ishikawa Quantitative real-time measurement of endothelin-1-induced contraction in single non-activated hepatic stellate cells.
description	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.
format	article
author	Naoki Dohi Momoka Yamaguchi Reina Hase Ryosuke Suzuki Yumeto Wakabayashi Ryota Nishiyama Shin-Ya Saito Tomohisa Ishikawa
author_facet	Naoki Dohi Momoka Yamaguchi Reina Hase Ryosuke Suzuki Yumeto Wakabayashi Ryota Nishiyama Shin-Ya Saito Tomohisa Ishikawa
author_sort	Naoki Dohi
title	Quantitative real-time measurement of endothelin-1-induced contraction in single non-activated hepatic stellate cells.
title_short	Quantitative real-time measurement of endothelin-1-induced contraction in single non-activated hepatic stellate cells.
title_full	Quantitative real-time measurement of endothelin-1-induced contraction in single non-activated hepatic stellate cells.
title_fullStr	Quantitative real-time measurement of endothelin-1-induced contraction in single non-activated hepatic stellate cells.
title_full_unstemmed	Quantitative real-time measurement of endothelin-1-induced contraction in single non-activated hepatic stellate cells.
title_sort	quantitative real-time measurement of endothelin-1-induced contraction in single non-activated hepatic stellate cells.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/253e4b840554416e9c0b451e52aa0110
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Quantitative real-time measurement of endothelin-1-induced contraction in single non-activated hepatic stellate cells.

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