Arachidonic acid-induced Ca2+ sensitization of smooth muscle contraction through activation of Rho-kinase.
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 microM, increases force in alpha-toxin-permeabilized femoral artery but not in Triton X-100-skinned fibres. The content of Rho-kinase in the latter was lower than in alpha-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 subunit inhibits myosin phosphatase and increases myosin phosphorylation.
Araki
S
S
First Department of Internal Medicine, Mie University School of Medicine, Tsu, Japan.
Ito
M
M
Kureishi
Y
Y
Feng
J
J
Machida
H
H
Isaka
N
N
Amano
M
M
Kaibuchi
K
K
Hartshorne
D J
DJ
Nakano
T
T
D000577:Amides / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002463:Cell Membrane Permeability; D003902:Detergents / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005263:Femoral Artery; D005982:Glutathione Transferase / Q000235:genetics; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D009119:Muscle Contraction / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase; D017830:Octoxynol / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D010738:Type C Phospholipases / Q000494:pharmacology; D000077191:Wortmannin; D054460:rho-Associated Kinases
D000577:Amides; D000730:Androstadienes; D003902:Detergents; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D011725:Pyridines; D011993:Recombinant Fusion Proteins; C108830:Y 27632; D016718:Arachidonic Acid; D017830:Octoxynol; D005982:Glutathione Transferase; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D009219:Myosin-Light-Chain Kinase; D010749:Phosphoprotein Phosphatases; D043263:Myosin-Light-Chain Phosphatase; D010738:Type C Phospholipases; D002118:Calcium; D000077191:Wortmannin