Oestrogen upregulates the sarcoplasmic reticulum Ca(2+) ATPase pump in coronary arteries

Abstract

The presence of circulating plasma 17β-oestradiol (E2) is beneficial in women against abnormal vascular tone development, such as coronary arterial vasospasms. Several vascular diseases have demonstrated that increased expression of the sarcoplasmic reticulum Ca(2+) -ATPase pump (SERCA2b) serves to limit the excessive accumulation of intracellular Ca(2+) . Therefore, the hypothesis of the present study was that E2 would increase SERCA2b expression in the coronary vasculature. Coronary arteries were dissected from hearts obtained from mature female pigs. Artery segments were cultured for 24 h in E2 (1 pmol/L or 1 nmol/L) and homogenized for western blot analysis. At 1 nmol/L, E2 induced an approximate 50% increase in immunoreactivity for SERCA2b. In addition, E2 increased the protein expression of the known SERCA regulatory proteins, protein kinase A (PKA) and protein kinase G (PKG). The E2-induced increase in SERCA2b was attenuated when the culture medium was supplemented with the oestrogen receptor (ER) α/β antagonist ICI 182,780 and the PKG antagonist KT5823 (10 μmol/L, 24 h for both). The PKA antagonist (KT5720; 10 μmol/L, 24 h) had no effect on SERCA2b expression. Removal of the endothelium (using a wooden toothpick) from artery segments prior to culture decreased the E2-mediated increase in SERCA2b and PKG expression by 45% and 47%, respectively. Overall, the findings suggest that one of the potential cardiovascular benefits of E2 in women is upregulation of SERCA2b, via activation of the classic ERα and ERβ pathway.

Keywords: Ca2+-ATPase pump; artery; endothelium; oestrogen; protein kinase G; sarcoplasmic reticulum.

Fig. 1

(a) The effect of estrogen on the protein expression of SERCA2b. The data represent the ratio of the band intensity for the EtOH and estrogen (E2) groups compared to the untreated group for each Western blot. The insert shows a representative raw Western blot for SERCA2b and the loading control, β-actin. * indicates P < 0.05, n = 6. (b) SERCA2b expression in the presence of estrogen and the estrogen receptor α/β antagonist, ICI 182,780. The data represent the ratio of the band intensity for the experimental groups compared to the EtOH group for each Western blot. * indicates P < 0.05, n = 4. In both (a) and (b) the endothelium was intact.

Fig. 2

The effect of estrogen on protein kinase A (PKA) expression in arteries with an intact endothelium. The data represent the ratio of the band intensity for the EtOH and estrogen (E2) groups compared to the untreated group for each Western blot. The insert shows a representative raw Western blot for PKA and the loading control, β-actin. * indicates P < 0.05, n = 6.

Fig. 3

Protein kinase G (PKG) expression in the presence of physiological concentrations of estrogen. The data represent the ratio of the band intensity for the EtOH and estrogen (E2) groups compared to the untreated group for each Western blot. The insert shows a representative raw Western blot for PKG and the loading control, β-actin. The data are from endothelial intact arteries. * indicates P < 0.05, n = 5.

Fig. 4

(a) Inhibition of protein kinase G (PKG) attenuates the estrogen (E2)-induced increase in SERCA2b. The data represent the ratio of the band intensity for the E2 and E2 + KT5823 groups. (b) The protein kinase A inhibitor, KT5720, did not significantly inhibit the E2-mediated increase in SERCA2b expression. The data represent the ratio of the band intensity for the E2 and E2 + KT5720 groups. For both (a) and (b) there was no difference (P > 0.05) between the vehicle solvents (EtOH and DMSO) for E2 and the kinase inhibitors. Both sets of data are from endothelial intact arteries. * indicates P < 0.05, n = 6.

Fig. 5

Scanning electron micrograph image of the luminal surface of a coronary artery. (A) The arterial endothelium was left intact and several red blood cells are visible. (B) The endothelium was rubbed off using a wooden toothpick and the elongated smooth muscle cells are clearly present.

Fig. 6

The effect of the endothelium on SERCA2b and protein kinase G (PKG) expression in the presence of estrogen (E2). The data for SERCA2b (a) and PKG (b) represent the ratio of the band intensity for E2 compared to EtOH in the presence and absence of the endothelium. * indicates P < 0.05, n=7 (a) and n=6 (b).