TY - JOUR
T1 - Arachidonic acid-induced Ca2+ sensitization of smooth muscle contraction through activation of Rho-kinase
AU - Araki, Shigemasa
AU - Ito, Masaaki
AU - Kureishi, Yasuko
AU - Feng, Jianhua
AU - Machida, Hirofumi
AU - Isaka, Naoki
AU - Amano, Mutsuki
AU - Kaibuchi, Kozo
AU - Hartshorne, David J.
AU - Nakano, Takeshi
N1 - Copyright:
Copyright 2007 Elsevier B.V., All rights reserved.
PY - 2001
Y1 - 2001
N2 - Arachidonic acid activates isolated Rho-kinase and contracts permeabilized smooth muscle fibres. Various assays were carried out to examine the mechanism of this activation. Native Rho-kinase was activated 5-6 times by arachidonic acid but an N-terminal, constitutively-active fragment of Rho-kinase, expressed as a glutathione-S-transferase (GST) fusion protein and including the catalytic subunit (GST-Rho-kinase-CAT), was not. GST-Rho-kinase-CAT was inhibited by a C-terminal fragment of Rho-kinase and arachidonic acid removed this inhibition. These results suggest that the C-terminal part of Rho-kinase, containing the RhoA binding site and the pleckstrin homology domain, acts as an autoinhibitor. It is suggested further that activation by arachidonic acid is due to its binding to the autoinhibitory region and subsequent release from the catalytic site. Arachidonic acid, at concentrations greater than 30 μM, increases force in α-toxin-permeabilized femoral artery but not in Triton X-100-skinned fibres. The content of Rho-kinase in the latter was lower than in α-toxin-treated or intact fibres. The arachidonic acid-induced contraction was not observed at a pCa above 8.0 and was inhibited by Y-27632 and wortmannin, inhibitors of Rho-kinase and myosin light-chain kinase (MLCK), respectively. The activation of Rho-kinase and subsequent phosphorylation of the myosin phosphatase target sub-unit inhibits myosin phosphatase and increases myosin phosphorylation.
AB - Arachidonic acid activates isolated Rho-kinase and contracts permeabilized smooth muscle fibres. Various assays were carried out to examine the mechanism of this activation. Native Rho-kinase was activated 5-6 times by arachidonic acid but an N-terminal, constitutively-active fragment of Rho-kinase, expressed as a glutathione-S-transferase (GST) fusion protein and including the catalytic subunit (GST-Rho-kinase-CAT), was not. GST-Rho-kinase-CAT was inhibited by a C-terminal fragment of Rho-kinase and arachidonic acid removed this inhibition. These results suggest that the C-terminal part of Rho-kinase, containing the RhoA binding site and the pleckstrin homology domain, acts as an autoinhibitor. It is suggested further that activation by arachidonic acid is due to its binding to the autoinhibitory region and subsequent release from the catalytic site. Arachidonic acid, at concentrations greater than 30 μM, increases force in α-toxin-permeabilized femoral artery but not in Triton X-100-skinned fibres. The content of Rho-kinase in the latter was lower than in α-toxin-treated or intact fibres. The arachidonic acid-induced contraction was not observed at a pCa above 8.0 and was inhibited by Y-27632 and wortmannin, inhibitors of Rho-kinase and myosin light-chain kinase (MLCK), respectively. The activation of Rho-kinase and subsequent phosphorylation of the myosin phosphatase target sub-unit inhibits myosin phosphatase and increases myosin phosphorylation.
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U2 - 10.1007/s004240000462
DO - 10.1007/s004240000462
M3 - Article
C2 - 11294240
AN - SCOPUS:0012322071
SN - 0031-6768
VL - 441
SP - 596
EP - 603
JO - Pflugers Archiv European Journal of Physiology
JF - Pflugers Archiv European Journal of Physiology
IS - 5
ER -