Su Bun Jeon

FLAVONE INHIBITS VASCULAR CONTRACTION BY DECREASING PHOSPHORYLATION OF THE MYOSIN PHOSPHATASE TARGET SUBUNIT

1 Flavonoids modulate vascular tone through an endothelium‐dependent or ‐independent mechanism. Although a few mechanisms for endothelium‐independent relaxation have been suggested, such as interference with protein kinase C or cAMP or cGMP phosphodiesterase, the inhibition of Ca2+ release from intracellular stores or Ca2+ influx from extracellular fluids, the mode of action of flavonoids remains elusive. 2 We hypothesized that treatment with flavone inhibits vascular smooth muscle contraction by decreasing the phosphorylation of the myosin phosphatase target subunit (MYPT1). 3 Rat aortic rings were denuded of endothelium, mounted in organ baths and contracted with U46619, a thromboxane A2 analogue. 4 Flavone dose‐dependently inhibited the U46619‐induced contractile response and myosin light chain (MLC20) phosphorylation. At 10−7 mol/L, U46619 induced vascular contraction with the concomitant phosphorylation of MYPT1 at Thr855, but not at Thr697. Incubation with flavone (100 or 300 µmol/L) for 30 min attenuated the phosphorylation of MYPT1Thr855, but not MYPT1Thr697. 5 It is concluded that treatment with flavone inhibits vascular smooth muscle contraction by decreasing the phosphorylation of the MYPT1. These results suggest that flavone causes endothelium‐independent relaxation through, at least in part, the inhibition of p160 Rho‐associated coiled‐coil‐containing protein kinase (ROCK) signalling.

Flavone Attenuates Vascular Contractions by Inhibiting RhoA/Rho Kinase Pathway

Our previous study demonstrated that flavone inhibits vascular contractions by decreasing the phosphorylation levelof the myosin phosphatase target subunit (MYPT1). In the present study, we hypothesized that flavone attenuates vascular contractions through the inhibition of the RhoA/Rho kinase pathway. Rat aortic rings were denuded of endothelium, mounted in organ baths, and contracted with either 30 nM U46619 (a thromboxane A2 analogue) or 8.0 mM NaF 30 min after pretreatment with either flavone (100 or 300 microM) or vehicle. We determined the phosphorylation level of the myosin light chain (MLC(20)), the myosin phophatase targeting subunit 1 (MYPT1) and the protein kinase C-potentiated inhibitory protein for heterotrimeric myosin light chain phophatase of 17-kDa (CPI17) by means of Western blot analysis. Flavone inhibited, not only vascular contractions induced by these contractors, but also the levels of MLC(20) phosphorylation. Furthermore, flavone inhibited the activation of RhoA which had been induced by either U46619 or NaF. Incubation with flavone attenuated U46619-or NaF-induced phosphorylation of MYPT1(Thr855) and CPI17(Thr38), the downstream effectors of Rho-kinase. In regards to the Ca(2+)-free solution, flavone inhibited the phosphorylation of MYPT1(Thr855) and CPI17(Thr38), as well as vascular contractions induced by U46619. These results indicate that flavone attenuates vascular contractions, at least in part, through the inhibition of the RhoA/Rho-kinase pathway.

A ROLE FOR RHO-KINASE IN Ca2+-INDEPENDENT CONTRACTIONS INDUCED BY PHORBOL-12,13-DIBUTYRATE

1 Phorbol‐12,13‐dibutyrate (PDBu) is an activator of protein kinase C (PKC) that causes contractions in both physiological salt solutions and Ca2+‐depleted solutions. In the present study, we tested the hypothesis that Rho‐kinase plays a role in Ca2+‐independent contractions induced by PDBu in vascular smooth muscles. 2 In Ca2+‐free solution, 0.1 and 1 µmol/L PDBu induced contraction and myosin light chain (MLC20) phosphorylation, both of which were approximately 40% of responses obtained in normal Krebs’ solution. Hydroxyfasudil (H1152; 1 µmol/L), an inhibitor of Rho‐kinase, but not ML7 (10 µmol/L), an inhibitor of myosin light chain kinase, inhibited Ca2+‐independent contractions induced by PDBu. 3 In Ca2+‐free solution, PDBu increased phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and CPI‐17 (PKC‐potentiated inhibitory protein for heterotrimeric myosin light chain phosphatase of 17 kDa). This action was inhibited by H1152, with the phosphorylation of CPI‐17 almost completely abolished by 1 µmol/L Ro31‐8220, an inhibitor of PKC. 4 In Ca2+‐free solution, PDBu increased the amount of GTP‐RhoA (an activated form of RhoA). This increase was blocked by the PKC inhibitor Ro31‐8220, but not by the Rho kinase inhibitor H1152. 5 In conclusion, RhoA/Rho‐kinase plays an important role in Ca2+‐independent contractions induced by PDBu in vascular smooth muscles. The results of the present study suggest that PDBu induces Ca2+‐independent contractions by inhibiting myosin light chain phospatase (MLCP) through activation of GTP‐RhoA and subsequent phosphorylation of MYPT1 and CPI‐17.

Fluoride induces vascular contraction through activation of RhoA/Rho kinase pathway in isolated rat aortas

We hypothesized that fluoride induces vascular contraction through activation of the RhoA/Rho kinase pathway in isolated rat aortas. Rat aortic rings were mounted in organ baths and contracted with sodium fluoride (NaF). We measured the amount of GTP-RhoA as well as vascular tension. We also determined the level of phosphorylation of the myosin light chain (MLC(20)), myosin phosphatase targeting subunit 1 (MYPT1) and PKC-potentiated inhibitory protein for heterotrimeric MLCP of 17kDa (CPI17). In both physiological salt solution and Ca(2+)-free solution, NaF increased vascular tension and MLC(20) phosphorylation in dose-dependent manners. NaF increased not only phosphorylation level of MYPT1(Thr855) and CPI17(Thr38), but also the amount of GTP-RhoA. Both H1152 and Y27632, inhibitors of Rho kinase, but not Ro31-8220, an inhibitor of PKC, attenuated NaF-induced contraction and phosphorylation level of MLC(20), MYPT1(Thr855) and CPI17(Thr38). In conclusion, fluoride induces vascular contraction through activation of the RhoA/Rho kinase pathway.

Vasorelaxation by Samhwangsasim-tang, an herb medicine, is associated with decreased phosphorylation of the myosin phosphatase target subunit

Samhwangsasim-tang (SST) is a widely used herbal medicine with vasodilatory actions in oriental countries. We hypothesized that SST modulates vascular contractility by decreasing phosphorylation of the myosin phosphatase target subunit. Rat aortic ring preparations were mounted in organ baths and subjected to contractions or relaxations. Phosphorylation of 20kDa myosin light chains (MLC(20)) and MYPT1, a target subunit of myosin phosphate 1, were examined with immunoblots. SST relaxed aortic ring preparations precontracted with phenylephrine whether endothelium was intact or denuded. Treatment of aortic rings with N(ω)-nitro-l-arginine methyl ester (l-NAME), an inhibitor of endothelial nitric oxide synthase or methylene blue, an inhibitor of guanylyl cyclase, did not affect the relaxing action of SST. Furthermore, SST inhibited vascular contractions induced by NaF or phenylephrine, but not by phorbol dibutyrate. SST also decreased vascular tension precontracted by 8.0mmol/L NaF or 1.0μmol/L phenylephrine, but not by 1.0μmol/L phorbol dibutyrate. In vascular strips, SST decreased the phosphorylation level of both MLC(20) and MYPT1 induced by 8.0mmol/L NaF. In conclusion, SST inhibited vascular contraction by decreasing phosphorylation of the myosin phosphatase target subunit.