Estradiol-17β-Induced Temporal and Spatial Partitioning of eNOS and Estrogen Receptors in Ovine Uterine Artery Endothelial Cells from Pregnant Sheep.

Abstract

Pregnancy-induced elevations in uterine blood flow are closely correlated with concurrent increases in plasma estradiol-17β levels and the expression/activity of eNOS. Indeed during late ovine gestation a physiologic cause and effect relationship has been established in vivo in that unilateral blockade of estrogen receptors (w/ICI 182,780) or nitric oxide synthase (w/L-NAME) locally and by a similar magnitude reduces uteroplacental blood flow. Estradiol-17β binds to estrogen receptors alpha and beta (ERα/β) and binding to these receptors results in either rapid effects (non-classical, non-genomic) and/or chronic effects (classical-genomic). Interestingly, numerous proteins that are involved in the estradiol-17β-induced rapid eNOS activation and nitric oxide production are located in specialized membrane domains called the caveolae of uterine artery endothelial cells (UAECs). Furthermore, treatment with the calcium mobilizing agonist ATP induced rapid eNOS activation which is accompanied by eNOS repartitioning with alterations in its multi-site phosphorylation state. We hypothesized that estradiol-17β induces eNOS activation via the non-classical ER receptor and initiates the sequential and coordinated re-partitioning and activation of eNOS with altered multi-site phosphorylation. UAECs were isolated from uterine arteries of pregnant ewes (gestational day 120-130; term 147d). Confluent passage 4 UAECs were treated with vehicle (Control) or with estradiol-17β (10 nM) for 10 min. The caveolae were fractionated from UAECs using sucrose density gradient centrifugation, and immunoblotting was utilized to study the multi-site phosphorylation state of eNOS relative to the domain specific levels of ERs. In control UAECs, total eNOS was predominantly located in the caveolar domain, whereas it was detectable both in the caveolar and non-caveolar domains in estradiol-17β-treated cells. In control UAECs, stimulatory P635eNOS and P1177eNOS were both not detected in any of the fractions whereas inhibitory P114eNOS was detected strictly in the non-caveolar domain. In response to estradiol-17β, stimulatory P635eNOS was detected in all cellular domains whereas stimulatory P1177eNOS was detected mainly in the caveolar domain and barely visible in the non-caveolar domain. The inhibitory P114eNOS was decreased by estrogen to low levels in the non-caveolar domain and was not detected in the caveolar domain. In control UAECs, ERs were detected in a higher abundance in the non-caveolar domain compared to the caveolar domain. estradiol-17β did not alter the distribution of the ERs. Estradiol-17β produces temporal and spatial re-partitioning of eNOS from the caveolar to non-caveolar domains and alters its multi-site phosphorylation state and their distributions in UAECs. Furthermore, since estradiol-17β actions are not calcium mediated, the striking similarities of these results to the ATP-responses illustrate alternative activation pathways of calcium mobilizing agonists. These findings may be critical to understand estradiol-17β-induced gestational vascular adaptations. NIH HL49210, HD38843, HL87144, HL70562, R25GM083252. (platform)