Endotheline-1 induces contraction in quiescent hepatic stellate cells mediated by myosin light chain kinase and Rho kinase

[Speaker] Shin-ya Saito:1
[Co-author] Ryosuke Suzuki:1, Yumeto Wakabayashi:1, Ryota Nishiyama:1, Momoka Yamaguchi:1, Tomohisa Ishikawa:1
1:Department of Pharmacology, University of Shizuoka, Japan

Background: The inflammation of the liver allows hepatic stellate cells (HSCs) to transdifferentiate from quiescent to activated form, the latter play major part at collagen production in inflammation. The pathophysiological function of activated form is well studied while physiological function of quiescent form is still not clear. Although quiescent HSCs is thought to be a regulator of hepatic blood flow, there is no direct evidence whether HSCs can contract. Therefore, the development of the method measuring the contraction of quiescent HSCs is important to understand its physiological function. According to this purpose, we developed a new method to evaluate the contraction of single HSC, and apply this method to study an effect of receptor agonist on the contractile function of HSCs. Methods: Freshly isolated mouse quiescent HSCs were seeded on collagen gel. Fluorescent beads were scattered on the surface of the gel, so that the contraction of HSCs gather the beads, and relaxation disperse the beads. Based on these movements, we evaluated the contraction by measuring the distance of the beads moved. Results: 10 nM endothelin-1 (ET-1) caused slowly developing contraction, which took 25 min reaching to peak in quiescent HSCs, and 1 μM bosentan inhibited this contraction, which suggested that ET-1 stimulated endothelin receptor and caused contraction in HSCs. 1 μM ML-9, the myosin light chain kinase (MLCK) inhibitor, inhibited this contraction, and calcium free condition reduced ET-1-induced contraction but still sustained contraction was remained in the latter case. These results suggested that calcium and MLCK were involved in ET-1-induced contraction. The sustained part in calcium free condition was diminished by combination use with 1 μM H-1152, the Rho kinase inhibitor, Conclusions: Use of the method measuring single cell movement provide us, for the first time, clear results that ET-1 caused contraction in HSCs which is mediated by calcium-MLCK system and Rho kinase system.
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