17 Beta-estradiol regulates the expression of endothelin receptor type B in the heart

Abstract

(1) Little is known about the interaction of 17beta-estradiol (E2) and the vasoactive endothelin system in the heart. Endothelin signaling is activated in a failing heart and may contribute to myocardial dysfunction and remodeling. Therefore, we investigated the regulation of proteins of the endothelin system (ppET-1, ECE and ETA-R and ETB-R) in the hearts of female spontaneously hypertensive rats (SHR) with respect to E2. (2) Relative expression levels of the respective cardiac mRNA obtained from sham-operated, ovariectomized and ovariectomized E2-substituted SHR were quantified by real-time PCR. Ovariectomy led to a significant upregulation of the ETB-R mRNA (2.6+/-0.8-fold) in the left ventricular myocardium, which was not attendant with an alteration of ETA-R, ECE and ppET-1 mRNA expression. (3) An upregulation of the relative expression level of ETB-R protein due to ovariectomy was also demonstrated by radioligand binding assay. (4) Upregulation of both ETB-R mRNA and ETB-R protein expression was completely inhibited by E2 replacement. (5) To confirm these results in in vitro experiments, we quantified the mRNA of ET-R subtypes from isolated cardiomyocytes in the presence and absence of E2 (10-8 m, 24 h). Our data showed a markedly downregulated level of ETB-R mRNA in cardiomyocytes stimulated with E2. ETB-R downregulation was not attendant with the alteration of ETA-R, ECE and ppET-1 mRNA expression. (6) Taken together, these data demonstrate that estrogen regulates the expression of ETB-R in rat ventricular myocardium in vivo and in vitro. These observations may help to understand gender-based differences found in cardiovascular disease.

Figure 1

Relative expression of ET_A-R, ET_B-R, ppET-1 and ECE in the left ventricular tissue obtained from the three groups (S=sham, Ov=ovariectomized, E2=E2 substituted-ovariectomized). Expression levels for each target gene have been normalized to the mean expression of sham-operated rats taken as a value of 1. Expression level of ET_B-R mRNA was significantly upregulated in estrogen-deficient rats, whereas there was no significant difference between the three groups concerning expression levels of ET_A-R, ECE and ppET-1 (^*P<0.005 vs S and vs E2, n=10 for each treatment group).

Figure 2

Representative competition curves of binding of ¹²⁵J-ET-1 to membrane preparations from the left ventricular tissue of SHR (here E2-substituted-ovariectomized SHR). Membranes were preincubated with the ET_A-R antagonist BQ123 (10⁻⁷ M, dotted line) or vehicle (control) at 21°C for 30 min. ¹²⁵J-ET-1 was competitively displaced by unlabeled ET-1. Each point represents the mean±s.e.m. from three experiments performed in triplicate.

Figure 3

Density of ET_B-R in the left ventricle of SHR. B_max values derived from computer-assisted nonlinear regression of specific bound vs free data in the presence of ET_A-R antagonist BQ123. Each column bar represents mean±s.e.m. of 10 animals per group (S=Sham, Ov=Ovariectomized, E2=E2 substituted-ovariectomized, ^*P<0.001).

Figure 4

Relative expression of ET_A-R, ET_B-R, ppET-1 and ECE in neonatal cardiomyocytes in response to E2. Cells were stimulated for 24 h with E2 (10⁻⁸ M) or vehicle alone (C=control). PCR results were normalized for each target gene to control cells in such a manner that the mean expression of control equals a value of 1. The expression level of ET_B-R mRNA was significantly downregulated due to E2 treatment, whereas there was no significant difference concerning expression levels of ET_A-R, ECE and ppET-1. Results are mean±s.d. from three experiments (n=3, ^*P<0.005 vs control).