1. 17β-Estradiol induces vasorelaxation in a G protein-coupled receptor 30- independent manner Young Mi Seok 1, Eun Jin Jang 2, Oliver Reiser 3, Markus Hager 3, and In Kyeom Kim 1,2,4 * 1 Cardiovascular Research Institute, 2 Department of Pharmacology, 4 Cell and Matrix Research Institute, Kyungpook National University School of Medicine, Daegu, 700-422, Republic of Korea; 3 Organic Chemistry institute, University of Regensburg, Universitätsstr.31, Regensburg, Germany *Correspondence and Proofs In Kyeom Kim, M.D., Ph.D. Department of Pharmacology Kyungpook National University School of Medicine 101 Dongin-2-Ga Daegu, 700-422, Republic of Korea Tel: +82-53-420-4833 Fax: +82-53-426-7345 E-mail: inkim@knu.ac.kr Reference Figure

2. ET (+) 6.0 5.5 5.0 4.5 4.0 (a)(b) ** * ** Reference Fig. 1 **

3. 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 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 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.

4. 1.0 3.0 10 30 100 30 nmol/L U46619 G1 (μmol/L) KCl 0 20 (mN) SNP (100 nmol/L) 30 nmol/L U46619 G1 (μmol/L) KCl 0 20 (mN) 1.0 3.0 10 30 100 SNP (100 nmol/L) (a) (b) ET (+) ET (-) Reference Fig. 2

5. Reference Fig. 2. Effect of sodium nitroprusside (SNP) on vascular relaxation induced by GPR30 agonist G1. Representative traces show relaxing responses to SNP. G1 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. The addition of G1 was followed by 100 nmol/L SNP.

6. ET (+) 6.0 5.5 5.0 4.5 4.0 * ** Reference Fig. 3 # ** #

7. 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 oxid e 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 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. One number sign ( # ) P < 0.05 vs. L-NAME alone.

8. ET (-) ET (+) 6.0 5.5 5.0 4.5 4.0 (a) (c) (b) (d) Reference Fig. 4 ** *

9. 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-estra diol (E2) or GPR30 agonist G1. E2 or G1 were added cumulatively to elicit relaxation when vascular contraction induced by U46619 (30 nmol/L) reac hed plateaus in endothelium-intact [ET (+), a and b] or -denuded [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 t he maximal contraction. Data are expressed as mean ± SEM. Data were a nalyzed by repeated measures ANOVA followed by Tukey’s test (n=4). On e asterisk (*) P < 0.05, two asterisks (**) P < 0.01 vs. vehicle.

10. 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.

11. * Reference Fig. 6 (a)(b) 6.0 5.5 5.0 4.5 4.0 * ET (+)

12. 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- kinase 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 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 vs. vehicle.

13. 0 20 (mN) 0 20 (mN) 0 20 (mN) KCl 30 nmol/L U46619 Isoproterenol (μmol/L) 30 nmol/L U46619 Isoproterenol (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 1.0 100 1.0 10 100 (a) Reference Fig. 7 ET (+)

14. 30 nmol/L U46619 Isoproterenol (μmol/L) Vehicle (DMSO) 0.01 0.1 1.0 10 100 KCl 0 20 (mN) 1.0 μmol/L H89 30 nmol/L U46619 Isoproterenol (μmol/L) 0.01 0.1 1.0 10 100 KCl 0 20 (mN) 10 μmol/L H89 30 nmol/L U46619 Isoproterenol (μmol/L) 0.01 0.1 1.0 10 100 KCl 0 20 (mN) (b) Reference Fig. 7 ET (-)

15. Reference Fig. 7. Effect of cAMP-dependent protein kinase inhibitor H89 on vascul ar relaxation induced by cAMP-dependent agent isoproterenol. Representative traces show relaxing responses. Isoproterenol was added c umulatively to elicit relaxation when vascular contraction induced by U4661 9 (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 minutes.

16. 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