Estrogen receptor-β in the paraventricular nucleus and rostroventrolateral medulla plays an essential protective role in aldosterone/salt-induced hypertension in female rats

Abstract

The identification of the specific estrogen receptor (ER) subtypes that are involved in estrogen protection from hypertension and their specific locations in the central nervous system is critical to our understanding and design of effective estrogen replacement therapies in women. Using selective ER agonists and recombinant adeno-associated virus (AAV) carrying small interference (si) RNA to silence either ERα (AAV-siRNA-ERα) or ERβ (AAV-siRNA-ERβ), the present study investigated regional specificity of different ER subtypes in the protective actions of estrogen in aldosterone (Aldo)-induced hypertension. Intracerebroventricular infusions of either diarylpropionitrile, a selective ERβ agonist, or propyl-pyrazole-triol, a selective ERα agonist, attenuated Aldo/NaCl-induced hypertension in ovariectomized rats. In contrast, intracerebroventricular injections of siRNA-ERα or siRNA-ERβ augmented Aldo-induced hypertension in intact females. Site-specific paraventricular nucleus (PVN) or rostroventrolateral medulla (RVLM) injections of siRNA-ERβ augmented Aldo-induced hypertension. However, rats with PVN or RVLM injections of siRNA-ERα did not significantly increase blood pressure induced by Aldo. Real-time polymerase chain reaction analyses of the PVN and RVLM of siRNA-injected rat confirmed a marked reduction in the expression of ERα and ERβ. In cultured PVN neurons, silencing either ERα or ERβ by culturing PVN neurons with siRNA-ERα or siRNA-ERβ enhanced Aldo-induced reactive oxygen species production. Ganglionic blockade after Aldo infusion showed an increase in sympathetic activity in ERβ knockdown rats. These results indicate that both PVN and RVLM ERβ, but not ERα in these nuclei, contribute to the protective effects of estrogen against Aldo-induced hypertension. The brain regions responsible for the protective effects of estrogen interaction with ERα in Aldo-induced hypertension still need to be determined.

Keywords: aldosterone; blood pressure; central nervous system; estrogen receptors.

Figure 1

Icv infusion of PPT or DPN for 21 days attenuated Aldo/NaCl-induced hypertension in ovariectomized (OVX) rats. A. Daily mean arterial pressures (MAP) before and during systemic infusion of Aldo in icv vehicle, PPT or DPN treated OVX rats. B. Average changes in MAP across days induced by Aldo infusion in all groups. (* p<0.05 vs. baseline; # p<0.05 vs. icv vehicle plus Aldo infusion)

Figure 2

Both icv injections of siRNA-ERα and siRNA-ERβ augmented Aldo/NaCl-induced hypertension as comparison with siRNA-scamble (SCM) injected intact female rats. A. Daily mean arterial pressures (MAP) before and during systemic infusion of Aldo in icv siRNA-SCM, siRNA-ERα or siRNA-ERβ treated intact females. B. Average changes in MAP across days induced by Aldo infusion in all groups. (* p<0.05 vs. baseline; # p<0.05 vs. icv siRNA-SCM plus Aldo infusion)

Figure 3

Bilateral PVN microinjections of siRNA-ERβ, but not siRNA-ERα, augmented Aldo-induced hypertension in intact female rats. A. Daily mean arterial pressures (MAP) before and during systemic infusion of Aldo in intact females with PVN microinjection of siRNA-SCM, siRNA-ERα or siRNA-ERβ. B. Average changes in MAP across days induced by Aldo infusion in all groups. (* p<0.05 vs. baseline; # p<0.05 vs. PVN siRNA-SCM plus Aldo infusion)

Figure 4

Bilateral RVLM microinjection of siRNA-ERβ, but not siRNA-ERα, augmented Aldo-induced hypertension in intact female rats. A. Daily mean arterial pressures (MAP) before and during systemic infusion of Aldo in intact females with RVLM microinjection of siRNA-SCM, siRNA-ERα or siRNA-ERβ. B. Average changes in MAP across days induced by Aldo infusion in all groups. (* p<0.05 vs. baseline; # p<0.05 vs. RVLM siRNA-SCM or siRNA-ERα plus Aldo infusion)

Figure 5

Mean arterial pressure (MAP) in response to ganglionic blockade with hexamethonium before beginning aldosterone (Aldo) infusion and 1% NaCl access and on day 21 of Aldo infusion in all groups. (* P<0.05 vs. control; † p<0.05 vs. icv PPT or DPN treated OVX females; # p<0.05 vs. intact females with siRNA-SCM or siRNA-ERα injections)

Figure 6

mRNA expressions of ERα or ERβ in the PVN or RVLM were significantly knocked down in intact females with siRNA-ERα or siRNA-ERβ microinjections as comparison with those with siRNA-SCM injections. A. viral delivery of the transgene for green fluorescent protein (GFP) results in robust transgene expression localized to the PVN (a) or RVLM (c). Panel b and d are higher magnification of a portion of Panel a and c, respectively. GFP expression could not be detected in the SFO (e) and AP (f) in the same animal indicating that the injected siRNA did not leak into the ventricle. B and C. RT-PCR analysis of ERα or ERβ mRNA expressions in the PVN or RVLM from intact females receiving PVN or RVLM microinjections of siRNA-SCM, siRNA-ERα or siRNA-ERβ. (* p<0.05 vs. intact females with siRNA-SCM injections)

Figure 6

mRNA expressions of ERα or ERβ in the PVN or RVLM were significantly knocked down in intact females with siRNA-ERα or siRNA-ERβ microinjections as comparison with those with siRNA-SCM injections. A. viral delivery of the transgene for green fluorescent protein (GFP) results in robust transgene expression localized to the PVN (a) or RVLM (c). Panel b and d are higher magnification of a portion of Panel a and c, respectively. GFP expression could not be detected in the SFO (e) and AP (f) in the same animal indicating that the injected siRNA did not leak into the ventricle. B and C. RT-PCR analysis of ERα or ERβ mRNA expressions in the PVN or RVLM from intact females receiving PVN or RVLM microinjections of siRNA-SCM, siRNA-ERα or siRNA-ERβ. (* p<0.05 vs. intact females with siRNA-SCM injections)

Figure 7

Knock-down ERα or ERβ enhanced Aldo-induced ROS production in neurons cultured from the PVN. A. Representative confocal images of DHE-loaded neurons cultured from the PVN showing the effects of Aldo on ROS production in neurons treated with siRNASCM, siRNA-ERα or siRNA-ERβ. Green in the cytoplasm represents GFP, a marker for virus transfection. Red in the cytoplasm represents DHE, a marker for ROS production. B. Summary of standardized emission intensity of DHE fluorescence in PVN neurons with overnight treatment of vehicle or Aldo. Neurons were treated with Aldo 24 hours after treatment with siRNA-SCM, siRNA-ERα or siRNA-ERβ. The change in ROS production was standardized by neurons treated with siRNA-SCM and vehicle. (* p<0.05 vs. neurons with siRNA-SCM plus vehicle; # p <0.05 vs. neurons with siRNA-SCM plus Aldo)