Identification of Differential ER-Alpha Versus ER-Beta Mediated Activation of eNOS in Ovine Uterine Artery Endothelial Cells1

Abstract

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.

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

“…The former data are consistent with several others that observed the caveolae structures localize at the plasma membrane of numerous endothelial cells types and that these structures assume the classic -shaped invaginations [47,50,65,66]. We theorized that respective location of ESR1 and ESR2 would be important for understanding the rapid NO-mediated uterine vasodilatory effects on the endothelium by estrogen that we previously reported in vivo [2,5,13,29,67,68] and in vitro [24,39,41,69,70]. In the present study, ESR1 was detected in the plasma membrane as well as several intracellular locations similar to other reports that studied the cardiovascular system especially the endothelium and numerous endothelial cell types [40,47,65,71].…”

“…However, as compared to the extensive data for ESR1, less is known regarding ESR2 subcellular location to the plasma membrane caveolar structures, its interaction with Cav-1, or its mechanism to increase NO bioavailability. This is important based on our recent observations that both ESR1 and ESR2 very rapidly stimulate P-UAEC NO biosynthesis in culture [41]. Consistent with our recent data, Corcoran et al [24] also showed that in ex vivo pre-contracted human myometrial arteries agonists to both ESR1 (PPT) and ESR2 (DNP) induce partial relaxation in an endothelial and NO-dependent manner.…”

“…Our previous studies, using the pregnant uterine artery endothelial cells (P-UAECs) model that maintains a pregnancy-specific signaling and vasodilator phenotype, revealed the existence of membrane ESRs that increase NO with the membrane-impermeable estradiol-17β conjugate [39]. More recently, we demonstrated that ESR1 and ESR2 are both responsible for these rapid NO effects in UAECs independently of each other [41]. Membrane-bound ESR1 is known to co-localize to specialized, dynamic plasma membrane domains called caveolae.…”

“…In endothelial cells, ESRs are known to localize to different intracellular locations [28,[36][37][38], including potentially a small population to the plasma membrane which, in turn, serves to direct the rapid estrogenic responses, such as biosynthetic vasodilator responses, i.e. NO and prostacyclin (PGI 2 ) production [10,[39][40][41]. Moreover, studies by Acconcia et al [42] clearly identified that the ESR1 requires palmitoylation as a post-translational modification to target the receptor to the plasma membrane of HeLa cells.…”

Influence of Estrogens on Uterine Vascular Adaptation in Normal and Preeclamptic Pregnancies

“…Besides this indirect mechanism, estrogens can also induce a rapid synthesis of NO by the activation of plasma membrane-associated ERs. Studies in UA shown that estradiol-17β binds to ERs localized on the EC plasmalemma and rapidly (within 2 min) increases NO production secondary to ERK 1/2 and PI3K/PKB activation, leading to eNOS phosphorylation ( Figure 2) [69,[106][107][108]. eNOS phosphorylation occurs via ERα and ERβ, independently from each other.…”