Differential Roles of Estrogen Receptors Alpha and Beta in the Nongenomic Activation of Endothelial Nitric Oxide Synthase by Estradiol

Abstract 111 Cardiology: Signaling Mechanisms and Cardiovascular Function Platform, Sunday, 5/2

The hormone estrogen has important vasoprotective properties that are mediated at least in part by its rapid effects on the bioavailability of nitric oxide (NO). In previous studies we have demonstrated that physiologic levels of estradiol (10^-10M) cause acute (5 min) activation of endothelial nitric oxide synthase (eNOS) in ovine endothelial cells. The maximal response obtained with estradiol is comparable to that obtained with acetylcholine (ACh), and the threshold concentration for estradiol is 100-fold less than for ACh. Although most known effects of estradiol involve the modulation of gene expression, the acute stimulation of eNOS is not altered by the inhibition of transcription with actinomycin D. However, it is fully reversed by rapid, concomitant estrogen receptor (ER) antagonism with tamoxifen or ICI 182,780, suggesting that it is mediated by acute ER activation. The purpose of the present study was to use an overexpression strategy to compare the roles of the two known ER subtypes, ERα and ERβ, in the rapid activation of eNOS. Ovine fetal pulmonary artery endothelial cells were transfected with either ERα or ERβ cDNA to increase ER subtype expression. ERα or ERβ mRNA overexpression were confirmed by RT-PCR, and enhanced functional receptors were evident in studies of ER-mediated gene transcription using a specific estrogen-responsive reporter system. The increase in ER-mediated transcription was greater in ERβ-transfected cells compared to ERα-transfected cells (6-fold versus 3.3-fold greater than sham, respectively). ERα or ERβ overexpression had no effect on basal NOS activity in intact cells, which was assessed by determining ³H-L-arginine to ³H-L-citrulline conversion, and eNOS protein expression was also unchanged. However, the acute response to estradiol was increased 4.1-fold following ERα overexpression, and this augmented response was fully inhibited by ICI 182,780. In contrast, ERβ overexpression resulted in a modest 23% increase in the acute response to estradiol. Overexpression of either ER subtype had no effect on NOS activation by ACh, revealing that the enhanced response to estradiol is unique to that agonist. In nontransfected cells, constitutive ERα expression was readily detectable by RT-PCR, whereas ERβ expression was not evident. These findings reveal differential roles for ERα and ERβ in the acute activation of eNOS in endothelial cells, indicating that the response is primarily mediated by ERα. Further studies of the domains of ERα that are unique to that isoform will help reveal the mechanisms by which ER activation leads to rapid intracellular signaling.