Yokomori Hiroaki, Yoshimura Kazunori, Ohshima Susumu, Nagai Toshihiro, Fujimaki Kayo, Nomura Masahiko, Oda Masaya, Hibi Toshifumi
Department of Internal Medicine, Kitasato Medical Center Hospital, Saitama 364-8501, Japan.
Liver Int. 2006 Dec;26(10):1268-76. doi: 10.1111/j.1478-3231.2006.01365.x.
BACKGROUND/AIMS: We previously reported that endothelin (ET)-1 may be involved in the contraction of hepatic sinusoidal endothelial fenestrae (SEF). Rho has emerged as an important regulator of the actin cytoskeleton and consequently cell morphology. To clarify the role of ET receptors [endothelin A receptor (ETAR) and endothelin B receptor (ETBR)] in ET-1-induced defenestration, we studied the size of hepatic SEF under various experimental conditions.
Liver sinusoidal endothelial cells (LSECs) isolated from rat livers by collagenase perfusion were cultured and divided into four groups: control, ET-1 (10(-6) -10(-10) M)-treated, ET-1+selective ETAR antagonist (BQ610)-treated and ET-1+ETBR antagonist (BQ788)-treated groups. SEF morphology was observed by scanning electron microscopy. Protein expressions of ETAR and ETBR, Rho A and phosphorylated myosin light-chain kinase were analyzed by Western blotting. F-actin stress fiber formation was observed by confocal microscopy. Active Rho was measured by Ren's modification. Intracellular free Ca2+ concentration ([Ca2+]i) was measured by fluorescence digital imaging using fura-2 AM by Aqua cosmos.
ET-1 induced a reduction in the number and size of SEF. ETAR antagonist pretreatment inhibited defenestration induced by low ET-1 concentrations (10(-8) -10(-10) M), whereas ETBR antagonist pretreatment did not block defenestration at low to high ET-1 concentrations (10(-6) -10(-10) M). F-actin stress fibers, Rho A levels and phosphorylated myosin light-chain kinase levels remained the same in various treatments. Active Rho was not detected in control and various treatments. ET-1 did not increase [Ca2+]i. Western blot showed prominent ETBR but scarce ETAR protein expression in LSECs.
The present findings demonstrated that ETBR- and ETAR-induced contractile mechanisms are not involved in ET-1-induced defenestration, and that Rho is also not activated. Therefore, ET-1 induces hepatic defenestration by mechanisms other than receptor-mediated contraction.
背景/目的：我们之前报道过内皮素（ET）-1可能参与肝血窦内皮窗孔（SEF）的收缩。Rho已成为肌动蛋白细胞骨架及随之而来的细胞形态的重要调节因子。为阐明ET受体[内皮素A受体（ETAR）和内皮素B受体（ETBR）]在ET-1诱导的窗孔消失中的作用，我们研究了在各种实验条件下肝SEF的大小。
通过胶原酶灌注从大鼠肝脏分离的肝血窦内皮细胞（LSEC）进行培养，并分为四组：对照组、ET-1（10⁻⁶ - 10⁻¹⁰ M）处理组、ET-1 + 选择性ETAR拮抗剂（BQ610）处理组和ET-1 + ETBR拮抗剂（BQ788）处理组。通过扫描电子显微镜观察SEF形态。通过蛋白质印迹分析ETAR和ETBR、Rho A和磷酸化肌球蛋白轻链激酶的蛋白表达。通过共聚焦显微镜观察F-肌动蛋白应力纤维形成。活性Rho通过任氏改良法测量。使用Aqua cosmos的fura-2 AM通过荧光数字成像测量细胞内游离Ca²⁺浓度（[Ca²⁺]i）。
ET-1导致SEF数量和大小减少。ETAR拮抗剂预处理抑制低浓度ET-1（10⁻⁸ - 10⁻¹⁰ M）诱导的窗孔消失，而ETBR拮抗剂预处理在低至高浓度ET-1（10⁻⁶ - 10⁻¹⁰ M）时不阻断窗孔消失。在各种处理中，F-肌动蛋白应力纤维、Rho A水平和磷酸化肌球蛋白轻链激酶水平保持不变。在对照组和各种处理中均未检测到活性Rho。ET-1未增加[Ca²⁺]i。蛋白质印迹显示LSEC中ETBR蛋白表达突出但ETAR蛋白表达稀少。
本研究结果表明，ETBR和ETAR诱导的收缩机制不参与ET-1诱导的窗孔消失，并且Rho也未被激活。因此，ET-1通过受体介导的收缩以外的机制诱导肝窗孔消失。
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