Essential role of rho kinase in the Ca2+ sensitization of prostaglandin F2α-induced contraction of rabbit aortae

Inhibition of dephosphorylation of the 20 kDa myosin light chain (MLC20) is an important mechanism for the Ca2+-induced sensitization of vascular smooth muscle contraction. We investigated whether this mechanism operates in prostaglandin F-2alpha (PGF(2alpha))-induced contraction of rabbit aortic smooth muscle and, if so, whether protein kinase C (PKC) or rho-associated kinase (rho kinase) contribute to the inhibition of dephosphorylation. In normal medium, PGF(2alpha) (10 muM) increased the phosphorylation of MLC20 and developed tension. The rho-kinase inhibitors fasudil and hydroxyfasudil inhibited these changes, despite having no effect on a phorbol-ester-induced MLC20 phosphorylation. After treatment with verapamil or chelation of external Ca2+ with EGTA, PGF(2alpha) increased the MLC20 phosphorylation and tension without an increase in [Ca2+](i), all of which were sensitive to fasudil and hydroxyfasudil. ML-9, a MLC kinase inhibitor, quickly reversed the KCl-induced MLC20 phosphorylation and contraction to the resting level. However, fractions of PGF(2alpha)-induced contraction and MLC20 phosphorylation were resistant to ML-9 but were sensitive to fasudil. Ro31-8220 (10 muM), a PKC inhibitor, did not affect the phosphorylation of MLC20 and the tension caused by PGF(2alpha), thus excluding the possibility of the involvement of PKC in the PGF(2alpha)-induced MLC20 phosphorylation. PGF(2alpha) increased phosphorylation at Thr654 of the myosin binding subunit (MBS) of myosin phosphatase, which is a target of rho kinase, and fasudil decreased the phosphorylation. These data suggest that the PGF(2alpha)-induced contraction is accompanied by the inhibition of MLC20 dephosphorylation through rho kinase-induced MBS phosphorylation, leading to Ca2+ sensitization of contraction. An actin-associated mechanism may also be involved in the PGF(2alpha)-induced sensitization.