3΄,4΄-Dimethoxythioflavone induces endothelium-dependent vasorelaxation through activation of EGF receptor

Eun Jin Jang1# , Young Mi Seok2#, Jae In Lee3, Hyun Min Cho1, Uy Dong Sohn4 and

In Kyeom Kim1,2,*

 1Department of Pharmacology, 2Cardiovascular Research Institute, Kyungpook

National University School of Medicine, Daegu, 700-422, Republic of Korea; 3Department of Chemistry, Duksung Women’s University, Seoul 132-714, Korea: 4College of Pharmacy,

Chung-Ang University, Seoul 156-756, Korea.

 # These authors equally contributed to this paper.

*Correspondence and Proofs In Kyeom Kim, M.D., Ph.D.Department of PharmacologyKyungpook National University School of Medicine101 Dongin-2-GaDaegu, 700-422, Republic of KoreaTel: +82-53-420-4833 Fax: +82-53-426-7345

E-mail: inkim@knu.ac.kr

Supplementary Figures

O

Supplementary Fig. 1.

Supplement Fig. 1. Chemical structure of 3’,4’-dimethoxythioflavone.

Thioflavone (-Log mol/L)

4.05.06.07.0

Re

lax

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(%

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l)

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Vehicle10 mol/L TEA 100 mol/L TEA

Thioflavone (-Log mol/L)

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Vehicle10 mol/L TEA 100 mol/L TEA

Thioflavone (-Log mol/L)

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19

)

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Vehicle1.0 mol/L Glibenclamide 10 mol/L Glibenclamide

Thioflavone (-Log mol/L)

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Vehicle1.0 mol/LGlibenclamide 10 mol/LGlibenclamide

Supplementary Fig. 2.

(a) (b)

(c) (d)

Supplement Fig. 2. Effect of ATP-sensitive K+ channels blocker glibenclamide (GB)

and nonselective K+ channel blocker tetraethylammonium (TEA) on 3΄,4΄-

dimethoxythioflavone-induced vasorelaxation. 3΄,4΄-Dimethoxythioflavone

was added cumulatively to elicit relaxation when vascular contractions

induced by KCl (50 mmol/L; a, c) or U46619 (100 nmol/L; b, d) reached

plateaus in endothelium-intact rat aortic rings. Rings were pretreated with

GB (1.0 or 10 μmol/L; a, b), TEA (10 or 100 μmol/L; c, d), or vehicle (0.1%

DMSO) for 30 min. Relaxation is expressed as a percentage of the

maximal contraction. Data are the mean ± SEM of n=4 experiments.

Thioflavone (-Log mol/L)

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619)

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Vehicle 1.0 mol/L Indomethacin 10 mol/L Indomethacin

Thioflavone (-Log mol/L)

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Vehicle

1.0 mol/L Indomethacin 10 mol/L Indomethacin

(a) (b)

Supplementary Fig. 3.

Supplement Fig. 3. Effect of cyclooxygenase blockers indomethacin on 3΄,4΄-

dimethoxythioflavone-induced vasorelaxation. 3΄,4΄-Dimethoxythioflavone

was added cumulatively to elicit relaxation when vascular contraction

induced by KCl (50 mmol/L; a), U46619 (100 nmol/L; b) (b) reached

plateaus in endothelium-intact rat aortic rings pretreated with indomethacin

(1.0 or 10 μmol/L) or vehicle (0.1% DMSO) for 30 min. Relaxation is

expressed as a percentage of the maximal contraction. Data are the mean

± SEM of n=4 experiments.

 

Thioflavone (-Log mol/L)

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Re

laxa

tio

n (

% U

466

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Vehicle1.0 mol/L Atropine 10 mol/L Atropine

Thioflavone (-Log mol/L)

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1.0 mol/L Atropine 10 mol/L Atropine

Supplementary Fig. 4.

(a) (b)

Supplement Fig. 4. Effect of muscarinic receptor antagonist atropine on 3΄,4΄-

dimethoxythioflavone-induced vasorelaxation. 3΄,4΄-Dimethoxythioflavone

was added cumulatively to elicit relaxation when vascular contractions

induced by KCl (50 mmol/L; a) or U46619 (100 nmol/L; b) reached

plateaus in endothelium-intact rat aortic rings pretreated with atropine (1.0

or 10 μmol/L) or vehicle (0.1% DMSO) for 30 min. Relaxation is expressed

as a percentage of the maximal contraction. Data are the mean ± SEM of

n=4 experiments.

Thioflavone (-Log mol/L)

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(%

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9)

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Vehicle0.1 mol/L U0126 1.0 mol/L U0126 10 mol/L U0126

Thioflavone (-Log mol/L)

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(%

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Vehicle0.1 mol/L U0126 1.0 mol/L U0126 10 mol/L U0126

ET (-) ET (-)

Thioflavone (-Log mol/L)

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(%

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Vehicle0.1 mol/L PP21.0 mol/L PP2 10 mol/L PP2

ET (-) ET (-)

(b)

(d)Thioflavone (-Log mol/L)

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Re

lax

atio

n (

% K

Cl)

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Vehicle0.1 mol/L PP2 1.0 mol/L PP2 10 mol/L PP2

(a)

ET (-)

(c)

Supplementary Fig. 5.

Supplement Fig. 5. Effect of a Src inhibitor PPT and an ERK inhibitor U0126 on

3΄,4΄-dimethoxythioflavone-induced vasorelaxation. 3΄,4΄-

Dimethoxythioflavone was added cumulatively to elicit relaxation when

vascular contractions induced by KCl (50 mmol/L; a, c) or U46619 (100

nmol/L; b, d) reached plateaus in endothelium-denuded rat aortic rings

pretreated with PPT (0.1, 1.0 or 10 μmol/L), U0126 (0.1, 1.0 or 10 μmol/L),

or vehicle (0.1% DMSO) for 30 min. Relaxation is expressed as a

percentage of the maximal contraction. Data are the mean ± SEM of n=4

experiments.

Constractor

KCl (mN) U46619 (mN) Phenylephrine (mN)

ET (+) 30.0 ± 3.9 40.0 ± 6.9 25.0 ± 5.1

ET (-) 35.8 ± 1.8 43.5 ± 4.7 36.0 ± 2.3

Table. 1. The maximum tension (mN) produced by KCl, U46619 or phenylephrine

in rat aortic rings with intact or denuded-endothelium

Data are expressed as means ± SEM of four experiments.