Nontranscriptional activation of PI3K/Akt signaling mediates hypotensive effect following activation of estrogen receptor β in the rostral ventrolateral medulla of rats

Abstract

Background: Estrogen acts on the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons are located, to elicit vasodepressor effects via an estrogen receptor (ER)β-dependent mechanism. We investigated in the present study nontranscriptional mechanism on cardiovascular effects following activation of ERβ in the RVLM, and delineated the involvement of phosphatidylinositol 3-kinase (PI3K)/serine/threonine kinase (Akt) signaling pathway in the effects.

Methods: In male Sprague-Dawley rats maintained under propofol anesthesia, changes in arterial pressure, heart rate and sympathetic neurogenic vasomotor tone were examined after microinjection bilaterally into RVLM of 17β-estradiol (E2β) or a selective ERα or ERβ agonist. Involvement of ER subtypes and PI3K/Akt signaling pathway in the induced cardiovascular effects were studied using pharmacological tools of antagonists or inhibitors, gene manipulation with antisense oligonucleotide (ASON) or adenovirus-mediated gene transfection.

Results: Similar to E2β (1 pmol), microinjection of ERβ agonist, diarylpropionitrile (DPN, 1, 2 or 5 pmol), into bilateral RVLM evoked dose-dependent hypotension and reduction in sympathetic neurogenic vasomotor tone. These vasodepressive effects of DPN (2 pmol) were inhibited by ERβ antagonist, R,R-tetrahydrochrysene (50 pmol), ASON against ERβ mRNA (250 pmol), PI3K inhibitor LY294002 (5 pmol), or Akt inhibitor (250 pmol), but not by ERα inhibitor, methyl-piperidino-pyrazole (1 nmol), or transcription inhibitor, actinomycin D (5 or 10 nmol). Gene transfer by microinjection into bilateral RVLM of adenovirus encoding phosphatase and tensin homologues deleted on chromosome 10 (5 × 10(8) pfu) reversed the vasodepressive effects of DPN.

Conclusions: Our results indicate that vasodepressive effects following activation of ERβ in RVLM are mediated by nongenomic activation of PI3K/Akt signaling pathway. This study provides new insight in the intracellular signaling cascades involved in central vasodepressive functions of estrogen.

Figure 1

Cardiovascular effects following microinjection bilaterally into the RVLM of E2β, ERα or ERβ agonist. Time-course of the changes in mean systemic arterial pressure (MSAP), heart rate (HR) and total power density of vasomotor components (0–0.8 Hz) of systemic arterial pressure (SAP) spectrum in anaesthetized rats that received microinjection bilaterally into the rostral ventrolateral medulla (RVLM, at time 0) of artificial cerebrospinal fluid (aCSF), 17β-estradiol (E2β, 1 pmol), diarylpropionitrile (DPN, 1, 2 or 5 pmol) or 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1 H-pyrazole (PPT, 5 pmol). Values are presented as the mean ± SEM; n = 6-8 animals per experimental group. ^*P < 0.05 versus aCSF group in the post hoc Scheffé multiple-range test.

Figure 2

Effects of ER antagonist on the E2β agonist-induced vasodepressive responses. Time-course of the changes in MSAP and total power density of vasomotor components (0–0.8 Hz) of SAP spectrum in anaesthetized rats that received microinjection bilaterally into the RVLM (at time 0) of aCSF, or DPN (2 pmol) given together with ICI 182780 (ICI, 0.25 or 0.5 pmol), R,R-THC (50 pmol), MPP (1 nmol) or 5 % DMSO. Values are presented as the mean ± SEM; n = 6-8 animals per experimental group. ^*P < 0.05 versus aCSF group and ^#P < 0.05 versus DPN + DMSO group in the post hoc Scheffé multiple-range test.

Figure 3

Effects of ERα or ERβ ASON on the ERβ agonist-induced vasodepressive responses. Time-course of the changes in MSAP and total power density of vasomotor components (0–0.8 Hz) of SAP spectrum in anaesthetized rats that received microinjection bilaterally into the RVLM (at time 0) of aCSF, or to rats pretreated with ERα ASON (250 pmol), ERα SCR (250 pmol), ERβ ASON (250 pmol), ERβ SCR (250 pmol) or aCSF, administered into the bilateral RVLM 24 hours before DPN (2 pmol) microinjection. Values are presented as the mean ± SEM; n = 6-7 animals per experimental group. ^*P < 0.05 versus aCSF group and ^#P < 0.05 versus aCSF + DPN group in the post hoc Scheffé multiple-range test.

Figure 4

Effects of RNA synthesis inhibitor on the ERβ agonist-induced vasodepressive responses. Time-course of the changes in MSAP and total power density of vasomotor components (0–0.8 Hz) of SAP spectrum in anaesthetized rats that received microinjection bilaterally into the RVLM (at time 0) of aCSF, or to rats pretreated with actinomycin D (AMD, 5 or 10 nmol) or 5 % DMSO, administered into the bilateral RVLM 1 hour before DPN (2 pmol) microinjection. Values are presented as the mean ± SEM; n = 7-8 animals per experimental group. ^*P < 0.05 versus aCSF group in the post hoc Scheffé multiple-range test.

Figure 5

Effects of PI3K inhibitor, Akt inhibitor or AdPTEN on the ERβ agonist-induced vasodepressive responses. Time-course of the changes in MSAP and total power density of vasomotor components (0–0.8 Hz) of SAP spectrum in anaesthetized rats that received microinjection bilaterally into the RVLM (at time 0) of aCSF, or DPN (2 pmol) given together with LY294002 (LY, 5 pmol), Akt inhibitor (Akt I, 250 pmol) or 5 % DMSO (A), or with additional treatment with AdPTEN (5 × 10⁸ pfu) or AdGFP (5 × 10⁸ pfu), administered into the bilateral RVLM 7 days before DPN (2 pmol) microinjection (B). (C) Representative Western blots (insets) or densitometric analysis of the amount of PTEN protein relative to basal PTEN protein levels (control), detected from the RVLM 3, 7 or 14 days after animals received microinjection bilaterally into the RVLM of AdPTEN (5 × 10⁸ pfu) or AdGFP (5 × 10⁸ pfu). (D) Representative photomicrographs showing the distribution of PTEN-immunoreactivity (arrows) in RVLM on 7 days after animals received bilateral microinjection into the RVLM of aCSF (control), AdPTEN (5 × 10⁸ pfu) or AdGFP (5 × 10⁸ pfu). Calibration bar: 100 μm in low magnification and 50 μm in high magnification of inset. Values are presented as the mean ± SEM; n = 6-7 animals per experimental group. ^*P < 0.05 versus aCSF or control group and ^#P < 0.05 versus DPN + DMSO or AdGFP + DPN group in the post hoc Scheffé multiple-range test. NA, nucleus ambiguus; RVLM, rostral ventrolateral medulla.

Figure 6

Microinjection sites of E2β and DPN in the RVLM. Diagrammatic representations of two rostral-caudal levels of the RVLM (dotted line areas) with reference to the lambda illustrating the location of sites where bilateral microinjection of E2β (○, 1 pmol) and DPN (●, 2 pmol) elicited significant inhibitory or minimal effects (■, non-RVLM microinjection sites) on the MSAP and power density of vasomotor components of SAP spectrum. Numbers on right side indicate distance from the lambda. For the purpose of clarity, approximately 10 % of the total microinjection sites are included and are presented on both side of the diagram. ION, inferior olivary nucleus; NA, nucleus ambiguous; NTS, nucleus tractus solitarii; RVLM, rostral ventrolateral medulla; V, nucleus of the spinal trigeminal nerve; py, pyramidal tract.