The selective beta1-adrenoceptor antagonist nebivolol is a potential oestrogen receptor agonist with neuroprotective abilities

Abstract

Background and purpose: Nebivolol, a selective beta(1)-adrenoceptor antagonist mediating rapid vasodilating effects, is used clinically to treat hypertension. Recently, it was reported that nebivolol also acts as an oestrogen receptor (ER) agonist. To investigate the neuroprotective potential of oestrogens, we assessed the oestrogenic effects of nebivolol in several in vitro neuronal models.

Experimental approach: Human neuroepithelioma SK-N-MC cells stably transfected with human ER alpha and beta, and mouse N2A neuroblastoma cells expressing human APP695(SWE)[N2Aswe, stably transfected with the Swedish mutation form of the Alzheimer-associated amyloid precursor protein (APPswe, K670M/N671L)] were incubated with different concentrations of nebivolol and 17beta-oestradiol (E2) for 24-48 h. ER activation was detected in a specific reporter assay, and ER-dependent gene expression was measured by quantitative real-time PCR (qRT PCR). Furthermore, cell survival rates were determined, and oxidative stress was induced by hydrogen peroxide and paraquat. Amyloid beta protein precursor (APP) processing was investigated, and the cleavage fragments sAPPalpha and Abeta were quantified via alpha-, beta- and gamma-secretase activity assays. Alterations of secretase expression levels were determined by qRT PCR.

Key results: Nebivolol induces oestrogen-dependent gene transcription, and protects neuronal cells against oxidative stress even at low and physiological concentrations (10(-8) M). Moreover, nebivolol modulates processing of APP in mouse neuronal N2Aswe cells by increasing alpha-secretase activity, ultimately leading to enhanced release of soluble non-amyloidogenic sAPPalpha.

Conclusions and implications: We showed that nebivolol acts as ER agonist in neuronal cell lines, and suggest oestrogen-like neuroprotective effects mediated by nebivolol.

Figure 1

ER binding competition for nebivolol in N2A cells. In this assay, 2 × 10⁻⁹ M [³H]-E2 was competing for binding to the ER with increasing concentrations of nebivolol from 1 × 10⁻⁶ M to 2 × 10⁻² M. A 100-fold molar excess of unlabelled E2 was used to determine the specific displaceable binding. Specific binding was calculated based on the percentage of remaining isotopic activity. Results are expressed as the mean of ±SEM of three independent measurements. The nebivolol concentration at 50% inhibition of [³H]-E2 binding (IC₅₀) = 0.8 × 10⁻⁵ M.

Figure 2

ER reporter assay with N2A and N2Aswe (A) and ER stable transfected SK-N-MC cells (B). Activation of the ER response element was plotted as fold induction of luciferase activity normalized to untreated cells (n.t.). Treatment: n.t., untreated; ICI, ICI 182780 at final concentration (f.c) of 10⁻⁶ M; E-7, E2 f.c. 10⁻⁷ M; E-8, oestrogen f.c. 10⁻⁸ M, N-6, nebivolol f.c. 10⁻⁶ M. The results shown represent more than three independent studies. Significance is defined as follows: *P < 0.05; ***P < 0.001 compared with untreated cells (anova).

Figure 3

Nebivolol and E2 increased the survival of N2Aswe cells after H₂O₂ or PQ-induced oxidative stress. Cells were pretreated with oestrogen (f.c. 10⁻⁸ M), different nebivolol concentrations as indicated, ICI 182780 (ICI, f.c. 10⁻⁶ M), l-NMMA (f.c. 10⁻⁶ M) and oestrogen and nebivolol together with ICI 182780 and l-NMMA, followed by exposure to H₂O₂ (A) and PQ (0–1000 µM) (B). Data show mean cell survival from a representative experiment (n = 9). *P < 0.05, compared with untreated cells (n.t) (anova).

Figure 4

Relative mRNA expression ratios of E2 and nebivolol-regulated genes in N2Aswe and SK-N-MC ERα and ERβ over-expressing cells. Cells were treated with E2 (f.c. 10⁻⁸ M), nebivolol (10⁻⁶ M and 10⁻⁸M) and oestrogen and nebivolol (10⁻⁸M) together with ICI 182780 for 18 h. Genes were considered differentially expressed when the expression levels between untreated and treated cells (expressed as a ratio) were more than 1.5-fold difference (dotted lines) and a P value <0.05. Fold change of gene expression levels was based on the ΔΔCt method with normalization of the raw data to housekeeping genes by using the relative expression software tool (REST-MCS V2.0).

Figure 5

Modulation of the generation of sAPPα and Aβswe induced by the enhanced α-secretase activity observed after nebivolol and E2 treatment. (A) Quantification of sAPPα (elisa). (B) Quantification of Aβ swe (elisa). (C) α-Secretase activity assay; activity was plotted as fold induction normalized to untreated cells (n.t.). Treatments: n.t., untreated; ICI, ICI 182780 at final concentration (f.c) of 10⁻⁶ M; E-7, oestrogen f.c. 10⁻⁷ M; E-8, oestrogen f.c. 10⁻⁸ M, N-6, nebivolol f.c. 10⁻⁶ M, N-7, nebivolol f.c. 10⁻⁷ M, N-8, nebivolol f.c. 10⁻⁸ M. The histograms represent results from more than three independent studies. **P < 0.005, compared with untreated cells of the same kind (anova).