The steroid hormone estrogen and its classical estrogen receptors (ERs), ER-α and ER-β, 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.

SUMMARY 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 sub-cellular eNOS partitioning between plasma membrane caveolar domains and non-caveolar compartments.eNOS translocation from the plasma membrane to intracellular compartments is important for eNOS activation and subsequent NO biosynthesis. We present data reviewing and interpreting information: 1) the coupling of endothelial plasma membrane receptor systems in the caveolar structure relative to eNOS trafficking; 2) how eNOS trafficking relates to specific protein-protein interaction for inactivation and activation of eNOS; and 3) how these complex mechanisms confer specific subcellular location relative to eNOS multi-site phosphorylation and signaling.Dysfunction in regulation of eNOS activation may contribute to several disease states; in particular gestational endothelial abnormalities (preeclampsia, gestational diabetes, etc) that have life-long deleterious health consequences that predispose the offspring to develop hypertensive disease, type II diabetes and adiposity.1

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 Ser1177eNOS and Ser635eNOS versus inhibitory Thr495eNOS 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 Ser1177eNOS and Ser635eNOS, a decrease in Thr495eNOS, and an increase in NOx levels. In contrast, ER-beta activation only reduced Thr495eNOS without changes in Ser1177eNOS or Ser635eNOS. 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.

Both ER-α and ER-β maintain similar spatial partitioning between the plasmalemma and nucleus of UAECs and have similar interactions with Cav-1 at the plasmalemma. Using Transmission Electron Microscopy (TEM), we observed numerous caveolae structures in UAECs, while immunogold labeling and subcellular fractionations identified ER-α and ER-β in 3 subcellular locations: membrane, cytosol and nucleus. Bioinformatics' approaches to analyze ER-α and ER-β transmembrane domains identified no regions that facilitate ER interaction with the plasmalemma. However, sucrose density centrifugation and Cav-1 immunoisolation columns demonstrated a very high association only between ER-α, but not ER-β, with Cav-1. These data demonstrate: 1) both ERs localize to the plasmalemma, cytosol and nucleus; 2) neither ER-α nor ER-β contain a classic region that crosses the plasmalemma to facilitate attachment; and 3) ER-α, but not ER-β, can be detected in the caveolar subcellular domain demonstrating ER-α is the only ER in close proximity to Cav-1 and eNOS within this microdomain. Lack of protein-protein interaction between Cav-1 and ER-β demonstrates a novel independent association of these proteins at the plasmalemma.