17 β -estradiol induces vasorelaxation in a g protein-coupled receptor 30-independent manner

17β-Estradiol induces vasorelaxation in a G protein-coupled receptor 30-indepen-dent manner

Young Mi Seok1, Eun Jin Jang2, Oliver Reiser3, Markus Hager3, and In Kyeom Kim1,2,4* 1Cardiovascular Research Institute, 2Department of Pharmacology, 4Cell and Matrix

Research Institute, Kyungpook National University School of Medicine, Daegu, 700-422, Republic of Korea; 3Organic Chemistry institute, University of Regensburg, Universitätsstr.31, Regensburg, Germany

*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: +82-53-426-7345E-mail: [email protected]

Reference Figure

E2 (-Log mol/L)

Rel

axat

ion

(%)

0

20

40

60

80

100

Vehicle100 mol/L L-NAME10 mol/L ICI100 mol/L MPP

G1 (-Log mol/L)R

elax

atio

n (%

)

0

20

40

60

80

100

Vehicle100 mol/L L-NAME10 mol/L ICI100 mol/L MPP

ET (+) ET (+)

6.0 5.5 5.0 4.5 4.0 6.0 5.5 5.0 4.5 4.0

(a) (b)

***

**

Reference Fig. 1

**

Reference Fig. 1. Effect of various antagonists on vascular relaxation induced by

17β-estradiol (E2) or GPR30 agonist G1. E2 (a) or G1 (b) were added cu-

mulatively to elicit relaxation when vascular contraction induced by

U46619 (30 nmol/L) reached plateaus in endothelium-intact rat aortic rings

pretreated with the nitric oxide synthase inhibitor N-nitro-l-arginine methyl

ester (L-NAME, 100 μmol/L), the ERα/ERβ antagonist ICI 182,780 (10

μmol/L), the ERα-specific antagonist methyl-piperidino-pyrazole (MPP, 100

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

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

± SEM. Data were analyzed by repeated measures ANOVA followed by

Tukey’s test (n=4 per group). One asterisk (*) P < 0.05, two asterisks (**) P

< 0.01 vs. vehicle.

1.0 3.0 10 30 100

30 nmol/L U46619G1 (μmol/L) KCl

0

20

(mN)

SNP (100 nmol/L)

30 nmol/L U46619G1 (μmol/L) KCl

020

(mN)

1.0 3.0 1030 100 SNP (100 nmol/L)

(a)

(b)

ET (+)

ET (-)

Reference Fig. 2

Reference Fig. 2. Effect of sodium nitroprusside (SNP) on vascular relaxation

induced by GPR30 agonist G1. Representative traces show relaxing re-

sponses to SNP. G1 was added cumulatively to elicit relaxation when vas-

cular contraction induced by U46619 (30 nmol/L) reached plateaus in en-

dothelium-intact (a) or –denuded (b) rat aortic rings. The addition of G1

was followed by 100 nmol/L SNP.

G1 (-Log mol/L)

Rel

axat

ion

(%)

0

20

40

60

80

100

VehicleL-NAMEL-NAME + G15G15

ET (+)

6.0 5.5 5.0 4.5 4.0

***

Reference Fig. 3

#**#

Reference Fig. 3. Effect of GPR30 antagonist G15 on vascular relaxation induced

by GPR30 agonist G1. G1 was added cumulatively to elicit relaxation

when vascular contraction induced by U46619 (30 nmol/L) reached

plateaus in endothelium-intact rat aortic rings pretreated with the nitric ox-

ide synthase inhibitor N-nitro-l-arginine methyl ester (L-NAME, 100 μmol/

L), L-NAME and G15 (100 μmol/L), G15 alone or vehicle (0.1% DMSO) for

30 minutes. Relaxation is expressed as a percentage of the maximal con-

traction. Data are expressed as mean ± SEM. Data were analyzed by re-

peated measures ANOVA followed by Tukey’s test (n=4 per group). One

asterisk (*) P < 0.05, two asterisks (**) P < 0.01 vs. vehicle. One number

sign (#) P < 0.05 vs. L-NAME alone.

G1 (-Log mol/L)

Rel

axat

ion

(%)

0

20

40

60

80

100

Vehicle1.0 mol/L ODQ10 mol/L ODQ

E2 (-Log mol/L)

Rel

axat

ion

(%)

0

20

40

60

80

100


ET (-) ET (-)

E2 (-Log mol/L)

Rel

axat

ion

(%)

0

20

40

60

80

100


ET (+)

G1 (-Log mol/L)

Rel

axat

ion

(%)

0

20

40

60

80

100


ET (+)

6.0 5.5 5.0 4.5 4.0

6.0 5.5 5.0 4.5 4.0

6.0 5.5 5.0 4.5 4.0 6.0 5.5 5.0 4.5 4.0

(a)

(c)

(b)

(d)

Reference Fig. 4

**

**

***

Reference Fig. 4. Effect of soluble guanylate cyclase blocker 1H-[1,2,4]-

oxadizolo[4,3-a]quinoxalin-1-one (ODQ) on vascular relaxation induced by

17β-estradiol (E2) or GPR30 agonist G1. E2 or G1 were added cumula-

tively to elicit relaxation when vascular contraction induced by U46619 (30

nmol/L) reached plateaus in endothelium-intact [ET (+), a and b] or -de-

nuded [ET (-), c and d] rat aortic rings pretreated with ODQ (1.0, or 10

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

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

± SEM. Data were analyzed by repeated measures ANOVA followed by

Tukey’s test (n=4). One asterisk (*) P < 0.05, two asterisks (**) P < 0.01

vs. vehicle.

Reference Fig. 5

17β-Estradiol attenuates vascular contraction through inhibition of RhoA/Rho kinase pathway. Naunyn Schmiedebergs Arch Pharmacol. 2009 Jul;380(1):35-44.

*

Reference Fig. 6

E2 (-Log mol/L)

Rel

axat

ion

(%)

0

20

40

60

80

100

Vehicle10 mol/L LY29400210 mol/L LY303511

E2 (-Log mol/L)R

elax

atio

n (%

)

0

20

40

60

80

100

Vehicle10 mol/L H89

(a) (b)

6.0 5.5 5.0 4.5 4.0 6.0 5.5 5.0 4.5 4.0

*ET (+) ET (+)

Reference Fig. 6. Effect of various antagonists on vascular relaxation induced by

17β-estradiol (E2). E2 was added cumulatively to elicit relaxation when

vascular contraction induced by U46619 (30 nmol/L) reached plateaus in

endothelium-intact rat aortic rings pretreated with the phosphatidylinositol

3-kinase inhibitor LY294002 (10 μmol/L, a), the phosphatidylinositol 3-ki-

nase inert LY analogue LY303511 (10 μmol/L, a), the cAMP-dependent

protein kinase inhibitor H89 (10 μmol/L, b), or vehicle (0.1% DMSO) for 30

minutes. Relaxation is expressed as a percentage of the maximal contrac-

tion. Data are expressed as mean ± SEM. Data were analyzed by re-

peated measures ANOVA followed by Tukey’s test (n=4 per group). One

asterisk (*) P < 0.05 vs. vehicle.

0

20

(mN)

0

20

(mN)

0

20

(mN)

KCl

KCl

KCl

30 nmol/L U46619Isoproterenol (μmol/L)


30 nmol/L U46619

Isoproterenol (μmol/L)

Vehicle (DMSO)

1.0 μmol/L H89

10 μmol/L H89

0.01 0.1 1.0

0.01 0.1 10

0.01 0.1

10 100

1.0100

1.0 10 100

(a)

Reference Fig. 7

ET (+)


Vehicle (DMSO)

0.01 0.1 1.0 10 100

KCl 0

20

(mN)

1.0 μmol/L H89


0.01 0.1 1.0 10 100

KCl 0

20

(mN)

10 μmol/L H89


0.01 0.1 1.0 10 100

KCl 0

20

(mN)

(b)

Reference Fig. 7

ET (-)

Reference Fig. 7. Effect of cAMP-dependent protein kinase inhibitor H89 on vascu-

lar relaxation induced by cAMP-dependent agent isoproterenol. Representa-

tive traces show relaxing responses. Isoproterenol was added cumulatively

to elicit relaxation when vascular contraction induced by U46619 (30 nmol/

L) reached plateaus in endothelium-intact (a) or -denuded (b) rat aortic rings

pretreated with H89 (1.0 or 10 μmol/L) or vehicle (0.1% DMSO) for 30 min-

utes.

Protein kinase A-dependent and -independent effects of isoproterenol in rat isolated mesenteric artery: interactions with levcromakalim.J Pharmacol Exp Ther. 2001 Sep;298(3):917-24.

Reference Fig. 8

17 β -estradiol induces vasorelaxation in a g protein-coupled receptor 30-independent manner

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