Significance was defined as 0.05. Results E2 stimulates protein S-NO Anxa5 in endothelial cells To determine the effects of E2 on protein S-NO in endothelial cells, endothelial cells were pre-starved in phenol red-free medium without serum; thus the cells were presumptively deprived of estrogens before E2 treatment. Pathway analysis and statistics Ingenuity pathway analysis (Ingenuity Systems, Redwood City, CA) tool was used to perform pathway analysis of the recognized endothelial value) more than 1.30 ( 0.05). Statistics were performed using SigmaStat version 3.5 (Systat Software, Inc., San Jose, CA). For comparison of data between estrogen treatment and control, we used Students test. Significance was defined as 0.05. Results E2 stimulates protein S-NO in endothelial cells To determine the effects of E2 on protein S-NO in endothelial cells, endothelial cells were pre-starved in phenol red-free medium without serum; thus the cells were presumptively deprived of estrogens before E2 treatment. We first measured total levels of nitrosoproteins in HUVEC treated with or without E2 or a NO donor GSNO. S-NO of various proteins was readily detectable in untreated control HUVEC. Treatment with 10 nm E2 or 1 mg/ml GSNO for 30 min stimulated S-NO of various proteins in HUVEC. All bands were lost in the presence of HgCl2, a reagent selectively displaces NO from S-NO bonds (26), implicating specificity of the assay. Similarly, E2 increased the levels of nitroso-proteins in UAEC (Fig. 1?1). Open in a separate window Physique 1 Total nitrosoprotein profiles in E2 and GSNO-treated HUVEC and ovine UAEC on SDS-PAGE. HUVEC and UAEC were treated with E2 (10 nm), an NO donor (GSNO, 1 mg/ml), or vehicle control (Ctl) for 30 min. Whole-cell lysates were prepared and subjected to biotin (R)-(+)-Corypalmine switch reaction with or without 0.2% HgCl2. The biotin-labeled nitrosoproteins were analyzed by 10% SDS-PAGE and detected by Western blot analysis with an antibiotin antibody. -Actin was measured for monitoring protein loading. Images shown depict a typical experiment of comparable results of three impartial experiments using HUVEC from different placentas and an experiment of UAEC from three different pregnant ewes. point to visible bands representing S-nitrosylated proteins. In HUVEC, (R)-(+)-Corypalmine total levels of nitrosoproteins began to increase by 10 nm E2 at 2 min, maximized around 10C30 min, and returned to baseline at 60 min (Fig. 2A?2A).). When treated with 0.1 nm to 1 1 m E2 for 30 min, only 10 nm E2 significantly stimulated total levels of nitrosoproteins in HUVEC. However, several individual proteins were clearly S-nitrosylated by 1 nm to 1 1 m E2 (Fig. 2B?2B).). These data suggest that E2 stimulates protein S-NO in a time and concentration-dependent manner. Open in a separate window Physique 2 Time courses and dose responses of the effects of E2 treatment on protein S-NO in HUVEC. Subconfluent HUVEC were treated with or without 10 nm E2 for (R)-(+)-Corypalmine the indicated occasions (up to 1 1 h) or with increasing concentration of E2 (0.1 nm to 1 1 m) for 30 min. Total protein extracts were harvested for determining the total nitrosoproteins. Representative blots of nitrosoproteins and -actin of one common experiment are shown. summarize data (mean sem, n = 3) from three impartial experiments using HUVEC from different placentas. *, 0.05 control. E2 activation of protein S-NO is usually mediated by specific ERs The biological function of estrogens in endothelial cells is usually mediated by specific ERs, including ER and ER (27). We detected immunoreactive ER and ER proteins HUVEC, confirming a previous statement (28) and showing that HUVEC (R)-(+)-Corypalmine are direct estrogen target cells. To test whether estrogens activation of protein S-NO in HUVEC is usually mediated by its specific receptors, we pretreated HUVEC with or without a real ER antagonist ICI 182,780 (1 m) for 1 h and then determined the effects of E2 and E2-BSA treatments on protein S-NO in HUVEC. As shown in Fig. 3?3,, in comparison with control cells, ICI 182,780 alone did not alter total nitrosoprotein levels. In the presence of ICI 182,780, the levels of total nitrosoproteins in E2 (10 nm, 20 min)-treated cells were significantly (R)-(+)-Corypalmine decreased. Estrogen-induced protein S-NO seemed to be atypical quick nongenomic action presumptively via receptors around the plasma membrane as this occurred within minutes (Fig. 2A?2A).). We then tested the effects of E2-BSA in the presence or absence of ICI 182,780.
