The endothelin-1 receptor-mediated pathway is not involved in the endothelin-1-induced defenestration of liver sinusoidal endothelial cells.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Yokomori
Hiroaki
H
Department of Internal Medicine, Kitasato Medical Center Hospital, Saitama 364-8501, Japan. yokomori-hr@kitasato.or.jp
Yoshimura
Kazunori
K
Ohshima
Susumu
S
Nagai
Toshihiro
T
Fujimaki
Kayo
K
Nomura
Masahiko
M
Oda
Masaya
M
Hibi
Toshifumi
T
D000199:Actins / Q000032:analysis; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects* / Q000648:ultrastructure; D019332:Endothelin-1 / Q000494:pharmacology*; D008099:Liver / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D008297:Male; D008855:Microscopy, Electron, Scanning; D051381:Rats; D017208:Rats, Wistar; D044022:Receptor, Endothelin A / Q000502:physiology*; D020742:rhoA GTP-Binding Protein / Q000032:analysis
D000199:Actins; D019332:Endothelin-1; D044022:Receptor, Endothelin A; D020742:rhoA GTP-Binding Protein; D002118:Calcium