Effects of the Catechol and Methoxy Metabolites of 17ß-Estradiol on Nitric Oxide Production by Ovine Uterine Artery Endothelial Cells.

Nitric oxide (NO) production is essential to facilitate rises in uterine blood flow (UBF) during pregnancy. It has been proposed that the metabolites of E2ß, 2-hydroxyestradiol (2-OHE2), 4-hydroxyestradiol (4-OHE2), 2-methoxyestradiol (2-ME2), and 4-methoxyestradiol (4-ME2) play a role in mediating vasodilation and rises in UBF during pregnancy. We previously showed that the E2ß metabolites stimulate prostacyclin production in pregnancy-derived ovine uterine artery endothelial cells (P-UAECs); however, it is unknown whether the E2ß metabolites also induce NO production. Herein, UAECs derived from nonpregnant and pregnant ewes were used to test the hypothesis that E2ß metabolites stimulate NO production in a pregnancy-specific manner. Specific estrogen receptor (ER) and adrenergic receptor (AR) antagonists were used to determine the roles of ERs or ARs in E2ß metabolite-induced NO production. E2ß and its metabolites increased total nitric oxide metabolites (NOx) levels (NO2 + NO3) in P-UAECs, but not in NP-UAECs. Pretreatment with combined 1 µmol/L 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP; ER-α antagonist) and 1 µmol/L 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP; ER-ß antagonist) inhibited the rises in NOx levels stimulated by E2ß and 2-ME2, but had no effect on 2-OHE2-, 4-OHE2-, or 4-ME2-stimulated rises in NOx levels. Pretreatment with yohimbine (α2-AR antagonist) and propranolol (ß2,3-AR antagonist) inhibited the rises in NOx levels stimulated by 2-OHE2, but not by E2ß, 4-OHE2, 2-ME2, or 4-ME2. These data demonstrate that E2ß metabolites stimulate NO synthesis via ERs or ARs in UAECs in a pregnancy-specific manner, suggesting that these metabolites contribute to rises in vasodilation and UBF during pregnancy.

Structural analysis of estrogen receptors: interaction between estrogen receptors and cav-1 within the caveolae†.

Pregnancy is a physiologic state of substantially elevated estrogen biosynthesis that maintains vasodilator production by uterine artery endothelial cells (P-UAECs) and thus uterine perfusion. Estrogen receptors (ER-α and ER-ß; ESR1 and ESR2) stimulate nongenomic rapid vasodilatory responses partly through activation of endothelial nitric oxide synthase (eNOS). Rapid estrogenic responses are initiated by the ∼4% ESRs localized to the plasmalemma of endothelial cells. Caveolin-1 (Cav-1) interactions within the caveolae are theorized to influence estrogenic effects mediated by both ESRs. Hypothesis: Both ESR1 and ESR2 display similar spatial partitioning between the plasmalemma and nucleus of UAECs and have similar interactions with Cav-1 at the plasmalemma. Using transmission electron microscopy, we observed numerous caveolae structures in UAECs, while immunogold labeling and subcellular fractionations identified ESR1 and ESR2 in three subcellular locations: membrane, cytosol, and nucleus. Bioinformatics approaches to analyze ESR1 and ESR2 transmembrane domains identified no regions that facilitate ESR interaction with plasmalemma. However, sucrose density centrifugation and Cav-1 immunoisolation columns uniquely demonstrated very high protein-protein association only between ESR1, but not ESR2, with Cav-1. These data demonstrate (1) both ESRs localize to the plasmalemma, cytosol and nucleus; (2) neither ESR1 nor ESR2 contain a classic region that crosses the plasmalemma to facilitate attachment; and (3) ESR1, but not ESR2, can be detected in the caveolar subcellular domain demonstrating ESR1 is the only ESR bound in close proximity to Cav-1 and eNOS within this microdomain. Lack of protein-protein interaction between Cav-1 and ESR2 demonstrates a novel independent association of these proteins at the plasmalemma.

Identification of Differential ER-Alpha Versus ER-Beta Mediated Activation of eNOS in Ovine Uterine Artery Endothelial Cells.

Endothelial nitric oxide (NO) production is partly responsible for maintenance of uterine vasodilatation during physiologic states of high circulating estrogen levels, e.g., pregnancy. Although 3%-5% of estrogen receptors (ER-alpha/beta) localize to the endothelial plasmalemma, these receptors are responsible for the nongenomic vasodilator responses. Estradiol induces endothelial NO synthase (eNOS) activation to increase NO production; however, it is unknown if eNOS regulation is dependent on both ERs. We hypothesize that ER-alpha and/or ER-beta are capable of changing eNOS phosphorylation and increasing NO production in uterine artery endothelial cells (UAECs). UAECs were 1) treated with vehicle or increasing concentrations (0.1-100 nM) or timed treatments (0-30 min) of estradiol and 2) pretreated with the inhibitors ICI 182,780 (nonspecific ER), 1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP; ER-alpha specific), or 4-[2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP; ER-beta specific) followed by estradiol to analyze the changes in eNOS stimulatory (Ser1177)eNOS and (Ser635)eNOS versus inhibitory (Thr495)eNOS via Western blot analysis. UAECs were also pretreated with MPP, PHTPP, or MPP + PHTTP followed by estradiol or treated with the agonists estradiol, 4,4',4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol, 2,3-bis(4-hydroxyphenyl)-propionitrile, or ATP to quantify total NOx levels (NO2+NO3). Estrogen and ER-alpha activation induced an increase in (Ser1177)eNOS and (Ser635)eNOS, a decrease in (Thr495)eNOS, and an increase in NOx levels. In contrast, ER-beta activation only reduced (Thr495)eNOS without changes in (Ser1177)eNOS or (Ser635)eNOS. However, ER-beta activation increased NOx levels. Lastly, the antagonism of both receptors induced a reduction in basal and stimulated NOx levels in UAECs. These data demonstrate that 1) eNOS phosphorylation changes occur via ER-alpha- and ER-beta-dependent mechanisms and 2) ER-alpha and ER-beta can both increase NO levels independently from each other.

Endothelial caveolar subcellular domain regulation of endothelial nitric oxide synthase.

Complex regulatory processes alter the activity of endothelial nitric oxide synthase (eNOS) leading to nitric oxide (NO) production by endothelial cells under various physiological states. These complex processes require specific subcellular eNOS partitioning between plasma membrane caveolar domains and non-caveolar compartments. Translocation of eNOS from the plasma membrane to intracellular compartments is important for eNOS activation and subsequent NO biosynthesis. We present data reviewing and interpreting information regarding: (i) the coupling of endothelial plasma membrane receptor systems in the caveolar structure relative to eNOS trafficking; (ii) how eNOS trafficking relates to specific protein-protein interactions for inactivation and activation of eNOS; and (iii) how these complex mechanisms confer specific subcellular location relative to eNOS multisite phosphorylation and signalling. Dysfunction in the regulation of eNOS activation may contribute to several disease states, in particular gestational endothelial abnormalities (pre-eclampsia, gestational diabetes etc.), that have life-long deleterious health consequences that predispose the offspring to develop hypertensive disease, Type 2 diabetes and adiposity.

Estrogen receptor-α and estrogen receptor-ß in the uterine vascular endothelium during pregnancy: functional implications for regulating uterine blood flow.

The steroid hormone estrogen and its classical estrogen receptors (ERs), ER-α and ER-ß, have been shown to be partly responsible for the short- and long-term uterine endothelial adaptations during pregnancy. The ER-subtype molecular and structural differences coupled with the differential effects of estrogen in target cells and tissues suggest a substantial functional heterogeneity of the ERs in estrogen signaling. In this review we discuss (1) the role of estrogen and ERs in cardiovascular adaptations during pregnancy, (2) in vivo and in vitro expression of ERs in uterine artery endothelium during the ovarian cycle and pregnancy, contrasting reproductive and nonreproductive arterial endothelia, (3) the structural basis for functional diversity of the ERs and estrogen subtype selectivity, (4) the role of estrogen and ERs on genomic responses of uterine artery endothelial cells, and (5) the role of estrogen and ERs on nongenomic responses in uterine artery endothelia. These topics integrate current knowledge of this very rapidly expanding scientific field with diverse interpretations and hypotheses regarding the estrogenic effects that are mediated by either or both ERs and their relationship with vasodilatory and angiogenic vascular adaptations required for modulating the dramatic physiological rises in uteroplacental perfusion observed during normal pregnancy.