Activation of estrogen receptor β is a prerequisite for estrogen-dependent upregulation of nitric oxide synthases in neonatal rat cardiac myocytes

Abstract

Physiological effects of estrogen on myocardium are mediated by two intracellular estrogen receptors, ERα and ERβ, that regulate transcription of target genes through binding to specific DNA target sequences. To define the role of ERβ in the transcriptional activation of both endothelial (eNOS) and inducible nitric oxide synthase (iNOS) in cardiac myocytes, we used the complete ERβ-specific antagonist R, R-tetrahydrochrysene ( R, R-THC). R, R-THC inhibited activation of iNOS/eNOS promoter-luciferase reporter constructs (iNOS/eNOS-Luc) in a dose-dependent fashion in COS7 cells selectively transfected with ERβ, but failed to influence ERα-mediated increase of iNOS/eNOS-Luc. In neonatal rat cardiomyocytes transfected with eNOS-Luc or iNOS-Luc, incubation with 17β-estradiol (E2, 10 −8 M) for 24 h stimulated expression of eNOS and iNOS. R, R-THC (10 −5 M) completely inhibited this effect. Furthermore, eNOS and iNOS protein expression in cardiac myocytes induced by E2 was completely blocked by R, R-THC as shown by immunoblot analysis. Taken together, these results show that ERβ mediates transcriptional activation of eNOS and iNOS by E2.