Estrogen receptor-beta agonist diarylpropionitrile: biological activities of R- and S-enantiomers on behavior and hormonal response to stress

Abstract

Estrogens have been shown to have positive and negative effects on anxiety and depressive-like behaviors, perhaps explained by the existence of two distinct estrogen receptor (ER) systems, ERalpha and ERbeta. The ERbeta agonist, diarylpropionitrile (DPN) has been shown to have anxiolytic properties in rats. DPN exists as a racemic mixture of two enantiomers, R-DPN and S-DPN. In this study, we compared R-DPN and S-DPN for their in vitro binding affinity, ability to activate transcription in vitro at an estrogen response element, and in vivo endocrine and behavioral responses. In vitro binding studies using recombinant rat ERbeta revealed that S-DPN has a severalfold greater relative binding affinity for ERbeta than does R-DPN. Furthermore, cotransfection of N-38 immortalized hypothalamic cells with an estrogen response element-luc reporter and ERbeta revealed that S-DPN is a potent activator of transcription in vitro, whereas R-DPN is not. Subsequently, we examined anxiety-like behaviors using the open-field test and elevated plus maze or depressive-like behaviors, using the forced swim test. Ovariectomized young adult female Sprague Dawley rats treated with racemic DPN, S-DPN, and the ERbeta agonist, WAY-200070, showed significantly decreased anxiety-like behaviors in both the open-field and elevated plus maze and significantly less depressive-like behaviors in the forced swim test compared with vehicle-, R-DPN-, or propylpyrazoletriol (ERalpha agonist)-treated animals. In concordance with the relative binding affinity and transcriptional potency, these results demonstrate that the S-enantiomer is the biologically active form of DPN. These studies also indicate that estrogen's positive effects on mood, including its anxiolytic and antidepressive actions, are due to its actions at ERbeta.

Figure 1

Separation of DPN enantiomers by reverse-phase chiral HPLC. A, Chromatogram of S-DPN. B, Chromatogram of R-DPN.

Figure 2

Effects of ER ligands on ERE-luc promoter activity mediated by ERβ (A and C) or ERα (B). N-38 cells were cotransfected with an ERE-tk-luc reporter construct and an expression vector containing ERα or ERβ. After transfection, cells were treated with vehicle (0.001% EtOH), estradiol, DPN, S-DPN, or R-DPN for 16 h. Data are represented as percent change in relative light units (RLU) from vehicle-treated empty vector controls (set at 100%). a, Significant differences (P < 0.05) from vehicle-treated controls; b, significant differences (P < 0.05) within treatment groups. Solid horizontal lines indicate constitutive activity of receptor in absence of ligand. A, Effect of 1, 10, and 100 nm concentrations of ligands on ERE-luc promoter activity in cells cotransfected with ERβ. B, Effect of 1, 10, and 100 nm concentrations of ligands on ERE-luc promoter activity in cells cotransfected with ERα. C, Effect of ligands (1 nm) on ERE-luc promoter activity in cells cotransfected with ERβ in the presence or absence of the ERβ antagonist PHTPP (10 μm).

Figure 3

Effects of ER ligands on anxiety-related behavior in the OF (A) and EPM (B), and learned helplessness in the FST (C). Behaviors measured include time spent in the middle squares, novel item interactions, and rearing in the OF; open arm entries, time spent in open arm, and rearing in the EPM; head dips, time spent immobile, and time spent struggling in the FST. Data are represented as mean ± sem (n = 9 animals per treatment group). *, Significant difference (P < 0.05) compared with vehicle treatment.

Figure 4

Effects of ER ligands on plasma CORT after the FST. Plasma samples were obtained 20 min after the start of the swim test. Data are represented as mean plasma CORT ± sem (n = 6 animals per treatment group). *, Significant difference (P < 0.05) compared with vehicle treatment.

Figure 5

Effects of ERβ ligands on PVN c-fos mRNA levels after the FST as measured by ISH. A, Relative levels of c-fos mRNA expression. Data are represented as mean arbitrary density units (ADU) ± sem (n = 6 animals per treatment group). *, Significant difference (P < 0.05) compared with vehicle treatment. B–E, Representative film autoradiograms for vehicle (B), S-DPN (C), R-DPN (D), and WAY-200070 (E)-treated animals.