Activation of estrogen receptor α enhances bradykinin signaling in peripheral sensory neurons of female rats

Abstract

Numerous studies have demonstrated that females have a higher risk of experiencing several pain disorders with either greater frequency or severity than males. Although the mechanisms that underlie this sex disparity remain unclear, several studies have shown an important role for sex steroids, such as estrogen, in the modulation of nociception. Receptors for estrogen are present in primary afferent neurons in the trigeminal and dorsal root ganglia, and brief exposure to estrogen increases responses to the inflammatory mediator bradykinin (BK). However, the mechanism for estrogen-mediated enhancement of BK signaling is not fully understood. The aim of the present study was to evaluate the relative contributions of estrogen receptor α (ERα), ERβ, and G protein-coupled estrogen receptor 1 (GPER) to the enhanced signaling of the inflammatory mediator BK by 17β-estradiol (17β-E2) in primary sensory neurons from female rats in culture (ex vivo) and in behavioral assays of nociception in vivo. The effects of 17β-E2 on BK responses were mimicked by ERα-selective agonists and blocked by ERα-selective antagonists and by small interfering RNA knockdown of ERα. The data indicate that ERα is required for 17β-E2-mediated enhancement of BK signaling in peripheral sensory neurons in female rats.

Fig. 1.

Local application of 17β-E₂ rapidly and dose-dependently enhances bradykinin-induced thermal allodynia in OVX rats. (A) OVX rats received intraplantar injections (50 µl) of 17β-E₂ (doses indicated) or vehicle 15 minutes before injection with a subthreshold dose of BK (1 µg). PWL in response to a radiant heat stimulus applied to the ventral surface of the hind paw was measured in duplicate at 5-minute intervals before (baseline) and following each injection. Data are expressed as the change (seconds) from individual preinjection baselines (10 ± 2 seconds) and represent the mean ± S.E.M. of four to six animals per group. **P < 0.01, ***P < 0.001 versus vehicle by two-way analysis of variance (ANOVA) with Bonferroni post-hoc analysis. (B) Dose-response curve for 17β-E₂ enhancement of BK-induced thermal allodynia. Data points are from (A) at the time of peak BK response (5 minutes post injection) as a function of 17β-E₂ dose. ED₅₀ = 8.85 ng. BL, baseline.

Fig. 2.

Local application of 17β-estradiol rapidly and dose-dependently enhances BK-stimulated PLC activity in cultures of peripheral sensory neurons from female rats. Peripheral sensory neuron cultures, prepared from OVX rats, were treated with vehicle [Veh; 0.1% dimethylsulfoxide (DMSO)] or 17β-E₂ (concentrations indicated) for 15 minutes (37°C) before addition of BK (1 nM) or vehicle (HBSS). Total IP accumulation after 25 minutes was measured as described under Materials and Methods. Data are expressed as the total counts (dpm) and represent the mean ± S.E.M. of three experiments.

Fig. 3.

BK-induced thermal allodynia is enhanced by the ERα-selective agonist PPT but not by the ERβ-selective agonist DPN or the GPER-selective agonist G-1, in OVX rats. Separate groups of OVX animals received intraplantar injections of PPT (2 or 20 ng), DPN (4 or 40 ng), G-1 (0.1 or 1 µg), or vehicle (Veh; 0.1% DMSO, 2% Tween-20) 15 minutes before injection with a subthreshold dose of BK (1 µg). PWL was measured in duplicate at 5-minute intervals before (baseline) and after each injection. Data are expressed as the change (seconds) from individual preinjection baseline (10 ± 2 seconds) and represent the mean ± S.E.M. of four to six animals per group. ***P < 0.001 versus Veh by two-way ANOVA with Bonferroni post-hoc analysis. BL, baseline.

Fig. 4.

Activation of ERα enhances BK-stimulated PLC activity in female peripheral sensory neuron cultures. Peripheral sensory neuron cultures from OVX rats were treated with vehicle (0.1% DMSO), 17β-E₂, PPT, DPN, or G-1 (concentrations indicated) for 15 minutes (37°C) before addition of BK (1 nM) and further incubation for 25 minutes (37°C). Total IP accumulation was determined as described under Materials and Methods. Data are expressed as the percentage of the BK response and represent the mean ± S.E.M. of six to eight experiments. Basal IP accumulation = 310 ± 19 dpm; BK-stimulated IP accumulation = 408 ± 25 dpm (31% over basal).

Fig. 5.

17β-Estradiol, PPT, and 17β-E₂–BSA enhancement of BK-induced thermal allodynia is blocked by the ER antagonist ICI 182780. Separate groups of OVX animals received intraplantar injections of the nonselective ER antagonist ICI 182780 (ICI; 1 µg) or vehicle (Veh; 0.1% EtOH) 15 minutes prior to injection with 17β-E₂ (100 ng), PPT (2 ng), or 17β-E₂–BSA (30 µg). Fifteen minutes later, animals received intraplantar injections of BK (1 µg). PWL was measured in duplicate at 5-minute intervals before (baseline) and after each injection. Data are expressed as the change from individual preinjection baselines and represent the mean ± S.E.M. of four to six animals per group. ***P < 0.001 respective Veh versus ICI by two-way ANOVA with Bonferroni post-hoc analysis. BL, baseline.

Fig. 6.

17β-E₂ and 17β-E₂–BSA-mediated enhancement of BK-induced thermal allodynia is blocked by the ERα antagonist MPP, but not by antagonists of ERβ, cyclofenil, or GPER, G-15. Separate groups of OVX animals received intraplantar injections of MPP (1 µg), cyclofenil (1 ng), G-15 (1 µg), or vehicle (Veh; 0.1% EtOH) 15 minutes prior to injection with 17β-E₂ (100 ng) or 17β-E₂–BSA (30 µg). Fifteen minutes later, animals were injected with BK (1 µg). PWL was measured in duplicate at 5-minute intervals before (baseline) and after each injection. Data are expressed as the change (seconds) from individual preinjection baselines and represent the mean ± S.E.M. of four to six animals per group. ***P < 0.001 versus all other groups by two-way ANOVA with Bonferroni post-hoc analysis. BL, baseline.

Fig. 7.

17β-E₂–mediated enhancement of BK-stimulated PLC signaling is blocked by the nonselective antagonist ICI 182780 and by the ERα-selective antagonist MPP but not by the ERβ- or the GPER-selective antagonists cyclofenil and G-15, respectively. Cultures of peripheral sensory neurons from OVX rats were treated with vehicle (Veh; 0.1% DMSO) or antagonist [ICI 182780 (ICI), 30 nM; MPP, 300 nM; cyclofenil (CYC), 10 nM; G-15, 2 µM] 15 minutes before treatment with vehicle (0.1% DMSO) or 17β-E₂ (50 nM). Fifteen minutes after 17β-E₂ addition, cells were treated with BK (1 nM) for 25 minutes, and total IP accumulation was determined as described under Materials and Methods. Data are expressed as a percentage of BK stimulation and represent the mean ± S.E.M. of five to six experiments. *P < 0.05 by one-way ANOVA with Bonferroni post-hoc analysis. Basal dpm 470 ± 23.1, BK dpm 578 ± 34.4 (23% stimulation over basal).

Fig. 8.

ERα is required for 17β-E₂–mediated enhancement of BK-stimulated PLC activity in female peripheral sensory neuron cultures. Peripheral sensory neuron cultures from OVX rats were incubated with 50 nM siRNA or siGLO control for 48 hours prior to experiments as described under Materials and Methods. Total mRNA was isolated, amplified by reverse-transcription polymerase chain reaction, and separated by agarose gel electrophoresis as described under Materials and Methods. (A) Representative agarose gel bands acquired using primers specific to ERα (top row), ERβ (middle row), or β-actin (bottom row) following treatment with siGLO (left two columns), ERα siRNA (middle two columns), or ERβ siRNA (right two columns). (B) Quantification of band densities normalized to β-actin. Data represent the mean ± S.E.M. of three experiments. **P < 0.01, ***P < 0.001 versus vehicle (Veh) by one-way ANOVA with Bonferroni. (C) Following siRNA treatment, peripheral sensory neuron cultures were treated with vehicle (0.1% DMSO) or 17β-E₂ (50 nM) for 15 minutes (37°C) before addition of BK (1 nM) and further incubation for 25 minutes. Total IP accumulation was determined as described under Materials and Methods. Data are expressed as a percentage of BK stimulation and represent the mean ± S.E.M. of eight experiments. ***P < 0.001 by one-way ANOVA with Bonferroni post-hoc analysis.