Korean J Physiol Pharmacol.  2009 Jun;13(3):201-207. 10.4196/kjpp.2009.13.3.201.

Flavone Attenuates Vascular Contractions by Inhibiting RhoA/Rho Kinase Pathway

• Baek I
• Jeon SB
• Song MJ
• Yang E
• Sohn UD
• Kim IK

• KMID: 2071672
• DOI: http://doi.org/10.4196/kjpp.2009.13.3.201

Abstract

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 micrometer) or vehicle. We determined the phosphorylation level of the myosin light chain (MLC20), 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 MLC20 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 Ca2+-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.

Keyword

Flavone; RhoA; Rho kinase; CPI-17; MYPT1; Vasorelaxation

MeSH Terms

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid
Animals
Baths
Blotting, Western
Contracts
Endothelium
Flavones
Myosin Light Chains
Myosin-Light-Chain Phosphatase
Myosins
Phosphorylation
Phosphotransferases
Protein Kinases
Rats
rho-Associated Kinases
Thromboxane A2
Vasodilation
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid
Flavones
Myosin Light Chains
Myosin-Light-Chain Phosphatase
Myosins
Phosphotransferases
Protein Kinases
Thromboxane A2
rho-Associated Kinases

Figure

• 

  Fig. 1. Inhibitory effect of flavone on contractions and the phosphorylation level of MLC20 induced by U46619 or NaF in rat aorta. U46619 (30 nM) (A) or NaF (8.0 mM) (C) was each added to elicit tension 30 min after pretreatment with flavone (100 or 300 μM) or vehicle to denuded aortic rings. Developed tension is expressed as a percentage of the maximum contractions to 50 mM KCl (4 aortic rings). When the tension reached plateaus, the phosphorylation levelof MLC20 in response to U46619 (B) or NaF (D) was measured and was expressed as a percentage of the total MLC20 (4 aortic rings). Data are expressed as means with vertical bars showing S.E.M. ##p<0.01 vs. basal. ∗p<0.05, ∗∗p<0.01 vs. U46619 or NaF alone. (A) and (C) were analyzed by repeated measures ANOVA whereas (B) and (D) by one-way ANOVA followed by the post-hoc Tukey's test.

• 

  Fig. 2. Inhibitory effect of flavone on RhoA activation induced by U46619 or NaF in rat aorta. RhoA activation was assessed by G-LISA™ Activation Assay (4 aortic rings). Thirty nM U46619 (A) or 8 mM NaF (B) was each added to elicit tension 30 min after pretreatment with flavone (100 or 300 μM) or vehicle to denuded aortic rings. Absorbance at the control (OD of about 0.4) was expressed as 1 arbitrary unit. Data were expressed as means with vertical bars showing S.E.M. ∗p<0.05, ∗∗p<0.01 vs. U46619 or NaF alone. ##p<0.01 vs. basal (one-way ANOVA followed by post-hoc Tukey's test).

• 

  Fig. 3. Inhibitory effect of flavone on the phosphorylation of MYPT1 induced by U46619 or NaF in rat aorta. U46619 (A) or NaF (B) was added to elicit tension 30 min after pretreatment with flavone (100 or 300 μM) or vehicle to denuded aortic rings. MYPT1 phosphorylation at Thr855 was assessed by Western blot analysis after treatment with 30 nM U46619 or 8 mM NaF. Upper and lower bands in representative Western blots were probed with anti-pMYPT1 and anti-MYPT1 antibodies, respectively. Densitometry shows that the ratio of density of phosphorylated MYPT1 (upper) to that of total MYPT1 (lower) for the control was expressed as 1 arbitrary unit (5 aortic rings). Data are expressed as means with vertical bars showing S.E.M. ∗p<0.05, ∗∗p<0.01 vs. U46619 or NaF alone. ##p<0.01 vs. basal (one-way ANOVA followed by post-hoc Tukey's test).

• 

  Fig. 4. Inhibitory effect of flavone on the phosphorylation of CPI17 induced by U46619 or NaF in rat aorta. U46619 (A) or NaF (B) was added to elicit tension 30 min after pretreatment with flavone (100 or 300 μM) or vehicle to denuded aortic rings. The phosphorylation of CPI17 at Thr38 was assessed by Western blots after treatment with 30 nM U46619 or 8 mM NaF. Upper and lower bands in representative Western blots were probed with anti-pCPI17 and anti-CPI17 antibodies, respectively. Densitometry shows that the ratio of density of phosphorylated CPI17 (upper) to that of total CPI17 (lower) for the control was expressed as 1 arbitrary unit (5 aortic rings). Data are expressed as means with vertical bars showing S.E.M. ∗p<0.05, ∗∗p<0.01 vs. U46619 or NaF alone. ##p<0.01 vs. basal (one-way ANOVA followed by post-hoc Tukey's test).

• 

  Fig. 5. Inhibitory effect of flavone on contractions and phosphorylation of MYPT1Thr855 and CPI17Thr38 induced by U46619 in a Ca2+-free solution. (A) Representative tracings of the development of isometric tension showed that, after initial reference contractions to KCl (50 mM) were obtained, the aortic rings were incubated in a Ca2+-free solution for 40 min, pretreated with either 100 or 300 μM flavone or vehicle for 30 min and then challenged with the addition of U46619 in a Ca2+-free solution (3 aortic rings). Phosphorylation of MYPT1 at Thr855 (B) and CPI17 at Thr38 (C) was assessed by western blots 15 min after treatment with U46619 (30 nM) or vehicle. Data are expressed as means with vertical bars showing S.E.M. ∗p<0.05, ∗∗p<0.01 vs. U46619 or NaF alone. ##p<0.01 vs. basal (one-way ANOVA followed by post-hoc Tukey's test).

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