Endogenous hydrogen sulfide (H2S) is a potent vasodilator and proangiogenic second messenger synthesized from L-cysteine by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH). Estrogens are potent vasodilators that stimulate H2S biosynthesis in uterine arteries (UA) in vivo; however, the underlying mechanisms are unknown. We hypothesized that estrogens stimulate H2S biosynthesis in UA endothelial cells (UAEC) via specific estrogen receptor (ER)-dependent mechanisms. In cultured primary UAEC, treatment with estradiol-17β (E2β) stimulated CBS and CTH mRNAs and proteins in a time- and concentration-dependent fashion. As little as 0.1 nM E2β was effective in increasing CBS and CTH expressions and these stimulatory effects maximized with 10-100 nM E2β at 48-72 h. E2β also activated CBS and CTH promoters in UAEC, leading to CBS and CTH expression. Treatment with E2β stimulated H2S production, which was blocked by specific inhibitors of either CBS or CTH and their combination and the ER antagonist ICI 182780. Treatment with either specific agonist of ERα or ERβ stimulated both CBS and CTH mRNA and protein expressions and H2S production to levels similar to that of E2β. Specific antagonist of either ERα or ERβ blocked E2β-stimulated CBS and CTH mRNA and protein expressions and H2S production. Combinations of either ERα or ERβ agonists or their antagonists had no additive effects. Thus, E2β stimulates H2S production by upregulating CBS and CTH mRNA and protein expressions through specific ERα or ERβ-dependent CBS and CTH transcription in UAEC in vitro.

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-β; 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.

Uterine vasodilation dramatically increases during the follicular phase of the estrous cycle and pregnancy, which are estrogen-dominant physiological states. Uterine vasodilation is believed to be mainly controlled by local uterine artery (UA) production of vasodilators and angiogenic factors. The extremely potent vasodilator and proangiogenic hydrogen sulfide (H2S) is synthesized via metabolizing L-cysteine by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CTH). This study was designed to determine if UA H2S production increases with augmented expression and/or activity of CBS and/or CTH during the ovarian cycle and pregnancy in sheep. Uterine arteries from intact nonpregnant (NP) luteal and follicular phase and late (130-135 days, term ≈ 145 days) pregnant (P) ewes were collected; endothelium-enriched proteins (UAendo) and endothelium-denuded smooth muscle (UAvsm) were mechanically prepared for accessing CBS and CTH proteins by immunoblotting; their cellular localization was determined by semi-quantitative immunofluorescence microscopy. H2S production was measured by the methylene blue assay. Immunoblotting revealed that CBS but not CTH protein was greater in P > > > NP follicular > luteal UAendo and UAvsm (P < 0.001). H2S production was greater in P > > > NP UAendo and UAvsm (P < 0.01). Pregnancy-augmented UAendo and UAvsm H2S production was inhibited by the specific CBS but not CTH inhibitor. CBS and CTH proteins were localized to both endothelium and smooth muscle; however, only CBS protein was significantly greater in P vs NP UA endothelium and smooth muscle. Thus, ovine UA H2S production is significantly augmented via selectively upregulating endothelium and smooth muscle CBS during the follicular phase and pregnancy in vivo.

Covalent adduction of a NO moiety to cysteines (S-nitrosylation or SNO) is a major route for NO to directly regulate protein functions. In uterine artery endothelial cells (UAEC), estradiol-17β (E2) rapidly stimulated protein SNO that maximized within 10-30 min post-E2 exposure. E2-bovine serum albumin stimulated protein SNO similarly. Stimulation of SNO by both was blocked by ICI 182, 780, implicating mechanisms linked to specific estrogen receptors (ERs) localized on the plasma membrane. E2-induced protein SNO was attenuated by selective ERβ, but not ERα, antagonists. A specific ERβ but not ERα agonist was able to induce protein SNO. Overexpression of ERβ, but not ERα, significantly enhanced E2-induced SNO. Overexpression of both ERs increased basal SNO, but did not further enhance E2-stimulated SNO. E2-induced SNO was inhibited by N-nitro-L-arginine-methylester and specific endothelial NO synthase (eNOS) siRNA. Thus, estrogen-induced SNO is mediated by endogenous NO via eNOS and mainly ERβ in UAEC. We further analyzed the nitroso-proteomes by CyDye switch technique combined with two-dimensional (2D) fluorescence difference gel electrophoresis. Numerous nitrosoprotein (spots) were visible on the 2D gel. Sixty spots were chosen and subjected to matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Among the 54 identified, nine were novel SNO-proteins, 32 were increased, eight were decreased, and the rest were unchanged by E2. Tandom MS identified Cys139 as a specific site for SNO in GAPDH. Pathway analysis of basal and estrogen-responsive nitroso-proteomes suggested that SNO regulates diverse protein functions, directly implicating SNO as a novel mechanism for estrogen to regulate uterine endothelial function and thus uterine vasodilatation.

Currently, no single marker is sensitive and specific enough to be considered a reliable biomarker for prenatal alcohol exposure. To identify a proteomic signature profile for maternal alcohol consumption, we carried out high-throughput proteomics on maternal endothelial caveolae exposed to moderate binge-like alcohol conditions. In these specialized lipid-ordered microdomains that contain a rich assembly of proteins, we demonstrate that moderate binge-like alcohol resulted in a distinctive maternal caveolar proteomic signature with important proteins being dramatically decreased/knocked out in the alcoholic profile. These proteins span from histones and basic structural proteins like α tubulin to proteins involved in trafficking, deubiquitination, cell signaling, and cell-cell adhesion. The profile also suggests an important role for the mother and the uteroplacental compartment in the pathogenesis of fetal alcohol spectrum disorders (FASD). These data demonstrate that the caveolar proteomic signature created by alcohol shows a promising direction for early detection of FASD.

ATP leads to endothelial NO synthase (eNOS)/NO-mediated vasodilation, a process hypothesized to depend on the endothelial caveolar eNOS partitioning and subcellular domain-specific multisite phosphorylation state. We demonstrate herein that, in both the absence and presence of ATP, the uterine artery endothelial caveolae contain specific protein machinery related to subcellular partitioning and act as specific focal "hubs" for NO- and ATP-related proteins. ATP-induced eNOS regulation showed a complex set of multisite posttranslational phosphorylation events that were closely associated with the enzyme's partitioning between caveolar and noncaveolar endothelial subcellular domains. The comprehensive model that we present demonstrates that ATP repartitioned eNOS between the caveolar and noncaveolar subcellular domains; specifically, the stimulatory (PSer635)eNOS was substantially higher in the caveolar pool with subcellular domain-independent increased levels on ATP treatment. The stimulatory (PSer1179)eNOS was not altered by ATP treatment. However, the inhibitory (PThr495)eNOS was regulated predominantly in the caveolar domain with decreased levels on ATP action. In contrast, the agonist-specific (PSer114)eNOS was localized in the noncaveolar pool with increased levels on ATP stimulation. Thus, the endothelial caveolar membrane system plays a pivotal role(s) in ATP-associated subcellular partitioning and possesses the relevant protein machinery for ATP-induced NO regulation. Furthermore, these subcellular domain-specific phosphorylation/dephosphorylation events provide evidence relating to eNOS spatio-temporal dynamics.

Normal pregnancy is associated with dramatic increases in uterine blood flow to facilitate the bidirectional maternal-fetal exchanges of respiratory gases and to provide sole nutrient support for fetal growth and survival. The mechanism(s) underlying pregnancy-associated uterine vasodilation remain incompletely understood, but this is associated with elevated estrogens, which stimulate specific estrogen receptor (ER)-dependent vasodilator production in the uterine artery (UA). The classical ERs (ERα and ERβ) and the plasma-bound G protein-coupled ER (GPR30/GPER) are expressed in UA endothelial cells and smooth muscle cells, mediating the vasodilatory effects of estrogens through genomic and/or nongenomic pathways that are likely epigenetically modified. The activation of these three ERs by estrogens enhances the endothelial production of nitric oxide (NO), which has been shown to play a key role in uterine vasodilation during pregnancy. However, the local blockade of NO biosynthesis only partially attenuates estrogen-induced and pregnancy-associated uterine vasodilation, suggesting that mechanisms other than NO exist to mediate uterine vasodilation. In this review, we summarize the literature on the role of NO in ER-mediated mechanisms controlling estrogen-induced and pregnancy-associated uterine vasodilation and our recent work on a "new" UA vasodilator hydrogen sulfide (H2S) that has dramatically changed our view of how estrogens regulate uterine vasodilation in pregnancy.

Estrogens dramatically dilate numerous vascular beds with the greatest response in the uterus. Endogenous hydrogen sulfide (H2S) is a potent vasodilator and proangiogenic second messenger, which is synthesized from L-cysteine by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). We hypothesized that estrogen replacement therapy (ERT) selectively stimulates H2S biosynthesis in uterine artery (UA) and other systemic arteries. Intact and endothelium-denuded UA, mesenteric artery (MA), and carotid artery (CA) were obtained from ovariectomized nonpregnant ewes (n = 5/group) receiving vehicle or estradiol-17β replacement therapy (ERT). Total RNA and protein were extracted for measuring CBS and CSE, and H2S production was determined by the methylene blue assay. Paraffin-embedded UA rings were used to localize CBS and CSE proteins by immunofluorescence microscopy. ERT significantly stimulated CBS mRNA and protein without altering CSE mRNA or protein in intact and denuded UA. Quantitative immunofluorescence microscopic analyses showed CBS and CSE protein localization in endothelium and smooth muscle and confirmed that ERT stimulated CBS but not CSE protein expression in UA endothelium and smooth muscle. ERT also stimulated CBS, but not CSE, mRNA and protein expression in intact and denuded MA but not CA in ovariectomized ewes. Concomitantly, ERT stimulated UA and MA but not CA H2S production. ERT-stimulated UA H2S production was completely blocked by a specific CBS but not CSE inhibitor. Thus, ERT selectively stimulates UA and MA but not CA H2S biosynthesis by specifically up-regulating CBS expression, implicating a role of H2S in estrogen-induced vasodilation and postmenopausal women's health.