Arachidonic acid-induced Ca2+ sensitization of smooth muscle contraction through activation of Rho-kinase

Arachidonic acid activates isolated kinase and contracts permeabilized smoo th muscle fibres. Various assays were carried out to examine the mechanism of this activation. Native Rho-kinase was activated 5-6 times by arachidoni c acid but an N-terminal, constitutively-active fragment of Rho-kinase, exp ressed as a glutathione-S-transferase (GST) fusion protein and including th e catalytic subunit (GST-Rho-kinase-CAT), was not. GST-Rho-kinase-CAT was i nhibited by a C-terminal fragment of Rho-kinase and arachidonic acid remove d this inhibition. These results suggest that the C-terminal part of Rho-ki nase, containing the RhoA binding site and the pleckstrin homology domain, acts as an autoinhibitor. It is suggested further that activation by arachi donic acid is due to its binding to the autoinhibitory region and subsequen t release from the catalytic site. Arachidonic acid, at concentrations grea ter than 30 muM, increases force in a-toxin-permeabilized femoral artery bu t not in Triton X-100-skinned fibres. The content of Rho-kinase in the latt er was lower than in a-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 ph osphorylation of the myosin phosphatase target subunit inhibits myosin phos phatase and increases myosin phosphorylation;.