The effect of 17 beta-estradiol on intracellular calcium homeostasis in human endothelial cells

Abstract

The cardiovascular effects of estrogen are mediated in part by augmenting the function of endothelial nitric oxide synthase. Endothelial nitric oxide synthase activity is dependent on many cofactors including Ca(2+). Hence, we investigated the effect of chronic 17 beta-estradiol treatment on the intracellular Ca(2+) concentration and endothelial nitric oxide synthase protein expression in the human endothelial cell line, EA.hy926, using spectrofluorometry and Western blot, respectively. Inhibiting the sarco(endo)plasmic reticulum Ca(2+) ATPase with thapsigargin caused an increase in the intracellular Ca(2+) concentration, which was higher in chronically 17 beta-estradiol-treated (1muM, 24h) cells loaded with Fura-2-acetoxymethyl ester compared to vehicle-treated cells, suggesting a higher endoplasmic reticulum Ca(2+) content in 17 beta-estradiol-treated cells. An enhanced Ca(2+) influx pathway in chronically 17 beta-estradiol-treated cells was also observed. In addition, 17 beta-estradiol-treated cells expressed higher levels of endothelial nitric oxide synthase protein in comparison to vehicle-treated cells. The chronic effect of 17 beta-estradiol on Ca(2+) homeostasis and endothelial nitric oxide synthase expression was attenuated with the nonselective estrogen receptor inhibitor, ICI 182,780 (10muM, 7alpha, 17beta-[9-[(4,4,5,5,5-Pentafluoropentyl)sulfinyl]nonyl] estra-1,3,5(10)-triene-3,17-diol). Furthermore, analysis of the thapsigargin-evoked Ca(2+) response in chronically 17 beta-estradiol-treated estrogen receptor alpha-knockdown cells showed no significant difference in Ca(2+) response compared to vehicle-treated estrogen receptor alpha-knockdown cells, indicating that the regulation of Ca(2+) homeostasis by 17 beta-estradiol is mediated through an estrogen receptor alpha-dependent pathway. These data revealed an estrogen receptor alpha-dependent modulation of Ca(2+) homeostasis accompanying the enhancement of endothelial nitric oxide synthase expression in 17 beta-estradiol-treated human endothelial cells.

Figure 1. Estrogen treatment enhances endothelial nitric oxide synthase protein expression

Western blot analysis of endothelial nitric oxide synthase (eNOS) expression in cells undergoing long-term treatments with vehicle, ICI 182,780 (ICI), 17 β-estradiol (E₂), and ICI 182,780 plus 17 β-estradiol (ICI + E₂). Significant difference from the control is indicated (*, P<0.05, n=3), analyzed using one-way ANOVA, followed by a Bonferroni post hoc test.

Figure 2. Thapsigargin-evoked Ca²⁺ response in the presence of 1.2 mM extracellular Ca²⁺

A) Representative Ca²⁺ traces from EA.hy926 in response to thapsigargin (1 μM) in the presence of 1.2 mM extracellular Ca²⁺, followed by Ni²⁺ (5 mM) treatment in vehicle-treated, 17 β-estradiol-treated (E₂), and ICI 182,780 plus E₂-treated (ICI+E₂) cells. B) The mean ± standard deviation of the area under the curve from the thapsigargin response in the presence of extracellular Ca²⁺. Significant differences among individual treatment groups are indicated (•—•, P<0.05, n=5), analyzed using a one-way ANOVA, followed by a Bonferroni post hoc test.

Figure 3. Thapsigargin-evoked Ca²⁺ response in the absence of extracellular Ca²⁺

A) Representative Ca²⁺ traces from EA.hy926 in response to thapsigargin (1 μM) in Ca²⁺-free Hank's balanced salt solution, followed by the addition of extracellular Ca²⁺ (1.2 mM) and Ni²⁺ (5 mM) in vehicle-treated, 17 β-estradiol-treated (E₂), and ICI 182,780 plus 17 β-estradiol-treated (ICI+E₂) cells. B) The mean ± standard deviation of the area under the curve from the thapsigargin-transient response in Ca²⁺-free Hank's balanced salt solution. C) The mean ± standard deviation of the area under the curve from the thapsigargin response upon the addition of extracellular Ca²⁺. Significant differences among individual treatment groups are indicated (•—•, P<0.05, n=5), as analyzed using a one-way ANOVA, followed by a Bonferroni post hoc test.

Figure 4. Analysis of estrogen receptor α and endothelial nitric oxide synthase expression in Lipofectamine-transfected cells, nonspecific short inhibiting RNA-transfected cells, and estrogen receptor α-knockdown cells

Analysis of estrogen receptor α mRNA expression with real time RT-PCR (A) and estrogen receptor α protein expression with Western blot (B) show that estrogen receptor α specific short inhibiting RNA can effectively knockdown estrogen receptor α mRNA and protein expression, respectively. In B, the protein bands represented by a, b, and c are from Lipofectamine-transfected cells, nonspecific short inhibiting RNA-transfected cells, and estrogen receptor α-knockdown cells, respectively. C) Transfections with siRNA, specific or nonspecific, did not affect endothelial nitric oxide synthase (eNOS) mRNA expression. Values are expressed as mean ± standard deviation, significance among groups are indicated (•—•, P<0.05, n=3), analyzed using one-way ANOVA, followed by a Bonferroni post hoc test.

Figure 5. Thapsigargin-evoked Ca²⁺ response in Lipofectamine-transfected cells, nonspecific short inhibiting RNA-transfected cells, and estrogen receptor α-knockdown cells

Representative tracings of the Ca²⁺ signal from cells in response to thapsigargin (1 μM) in Ca²⁺-free Hank's balanced salt solution, followed by the addition of extracellular Ca²⁺ (1.2 mM). A) Inhibition of sarco(endo)plasmic reticulum Ca²⁺ ATPase with thapsigargin induced a higher Ca²⁺ release and a higher Ca²⁺ influx in 17 β-estradiol-treated Lipofectamine-transfected cells as compared with vehicle-treated control cells. B) 17 β-estradiol-treated nonspecific short inhibiting RNA-transfected cells also showed a higher thapsigargin-evoked Ca²⁺ release and influx. C) 17 β-estradiol-treated estrogen receptor α-knockdown cells had the same Ca²⁺ profile as vehicle-treated estrogen receptor α-knockdown cells. Comparisons were made using area under the curve in thapsigargin-evoked Ca²⁺ release (D) and influx (E) due to estrogen receptor α knockdown. Significant difference among groups are indicated (•—•, P<0.05, n=3), analyzed using one-way ANOVA, followed by a Bonferroni post hoc test.

Figure 6. Thapsigargin-evoked Ca²⁺ response in 5 min 17 β-estradiol-treated cells

A) Representative Ca²⁺ traces from cells treated for 5 min with either vehicle or 17 β-estradiol in response to thapsigargin (1 μM) in Ca²⁺-free Hank's balanced salt solution. B) The mean ± standard deviation of the area under the curve from the transient thapsigargin response in Ca²⁺-free Hank's balanced salt solution and Ca²⁺ response upon the addition of extracellular Ca²⁺ (1.2 mM). There was no significant difference among individual treatment groups (P>0.05, n=5), analyzed using a t-test.