Estrogen Modulates Endothelial and Neuronal Nitric Oxide Synthase Expression via an Estrogen Receptor ?-Dependent Mechanism in Hypothalamic
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内分泌学杂志 2005年第7期
Center for Neuroscience and Department of Cell Biology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Canada T6G 2H7
Address all correspondence and requests for reprints to: Dr. Teresa L. Krukoff, Center for Neuroscience and Department of Cell Biology, 5-31 Medical Sciences Building, University of Alberta, Edmonton, Alberta, Canada T6G 2H7. E-mail: teresa.krukoff@ualberta.ca.
Abstract
Although it is evident that estrogen has important physiological effects in the brain, the signaling mechanisms mediating these effects remain unclear. We recently showed that estrogen mediates attenuated blood pressure responses to psychological stress in ovariectomized female rats through brain nitric oxide (NO). An area likely to mediate these effects is the hypothalamic paraventricular nucleus (PVN), because here NO exerts inhibitory effects on autonomic output to the periphery. Because little is known about how estrogen acts on the NO system in the PVN, our aim was to study the effects of estrogen on the NO system in the PVN of hypothalamic slices cultures. We show that 17?-estradiol (E2; 1 nM) increases endothelial NO synthase (eNOS) protein expression and decreases the numbers of neuronal NOS (nNOS)-positive neurons in the PVN after 8 and 24 h, respectively. Using the nonselective estrogen receptor (ER) antagonist, ICI 182,780 (10 nM), we determined that E2-induced changes in NOS expression in the PVN are ER dependent. Using the ER? agonist, genistein (0.1 μM), we determined that activation of ER? induces increased eNOS expression and a decreased number of nNOS-positive neurons. We used the selective ER agonist, propyl-pyrazole-triol (10 nM), and antagonist, methyl-piperidino-pyrazole (1 μM), to exclude the possibility that ER is involved in the E2-induced increase in eNOS and nNOS in the PVN. These results demonstrate that E2 induces changes in NOS expression in the PVN and that these effects are ER? dependent.
Introduction
HORMONE REPLACEMENT THERAPY (HRT) is widely used in women to relieve menopausal symptoms and until recently was believed to protect aging women from cardiovascular disease. However, the 2002 Women’s Health Initiative Study on HRT created a great deal of controversy when it was halted abruptly because of risks associated with HRT, including increased incidence of stroke (1, 2). These findings dramatically demonstrate that not only are the effects of HRT in women poorly understood, but estrogen’s effects on normal physiological processes are also still unclear.
We recently reported that ovariectomized, estrogen-treated (OVX-E) rats display attenuated blood pressure (BP) responses to psychological stress compared with OVX, vehicle-treated (OVX-V) rats (3). In agreement with our results, peri- and postmenopausal women receiving estrogen exhibit lower BP responses to mental stress compared with those not receiving estrogen (4, 5). A potential mediator of estrogen’s effect on cardiovascular responses to stress is nitric oxide (NO), a neurotransmitter involved in maintaining homeostasis and regulating autonomic activity (6, 7). Indeed, we found that estrogen acts through brain NO to attenuate BP responses to psychological stress (3).
Studies performed in both peripheral and brain tissues have demonstrated that estrogen influences the NO system. For example, estrogen increases NO production (8, 9), endothelial nitric oxide synthase (eNOS) expression (8, 10), and eNOS activity (8, 9) in cultured endothelial cells. Estrogen also increases eNOS protein expression in cerebral microvessels (11, 12, 13, 14). Finally, estrogen increases NO production (15, 16) and eNOS protein expression (17) in ex vivo preparations of the median eminence. The effects of estrogen on neuronal NOS (nNOS) in the brain appear to be regionally specific. Estrogen has been shown to increase the numbers of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase (nNOS) neurons (18, 19) and nNOS mRNA (20, 21) in select brain regions of OVX rats. Others report that estrogen does not change nNOS mRNA levels or numbers of nNOS neurons in other brain regions (20, 22). The specific mechanism(s) by which estrogen acts on NO in the brain remains to be elucidated.
Because the paraventricular nucleus (PVN) of the hypothalamus is an autonomic center that regulates neuroendocrine and autonomic functions (7), we hypothesize that estrogen acts on the NO system in the PVN to alter central autonomic activity. Our first aim was to investigate estrogen’s effects on NOS expression in the PVN. Because estrogen acts on two receptors, estrogen receptor- (ER) and ER?, and the PVN expresses only ER? (23, 24, 25, 26), our second aim was to investigate the role of ER? in estrogen’s effects on NOS expression in the PVN. Using hypothalamic slice cultures, we investigated the effects of 17?-estradiol (E2) on eNOS and nNOS expression in the PVN. We used the ER antagonist, ICI 182,780, to determine whether E2 affects NOS expression through an ER-dependent pathway. We also used the selective ER? agonist, genistein, the selective ER agonist, propyl-pyrazole-triol (PPT), and the selective ER antagonist, methyl-piperidino-pyrazole (MPP), to determine whether E2 acts through an ER?-dependent mechanism to affect NOS expression.
Materials and Methods
Hypothalamic slice culture
Hypothalamic slice cultures were prepared as described by others (27). Briefly, 5- to 7-d-old Sprague Dawley rat pups (Biological Sciences, University of Alberta, Edmonton, Canada) were quickly decapitated. Blocks of hypothalamic tissue were cut from the brains and sectioned at a thickness of 400 μm using a McIlwain tissue chopper (Mickle Laboratory Engineering, Gomshall, UK). Slices were immediately placed into Gey’s balanced salt solution (4 C) enriched with 5 mg/liter glucose. A total of five coronal slices from each brain were placed on a single Millicell-CM filter insert (Millipore Corp., Bedford, MA; pore size, 0.4 μm; diameter, 30 mm). Each filter insert was placed into a 35-mm petri dish containing 1.1 ml serum-containing medium (SCM); each dish, representing n = 1, was exposed to a single treatment as described below. Slice cultures were maintained in a humidified incubator at 35 C in 5% CO2 and 95% air.
Culture conditions
Hypothalamic slice cultures were maintained in SCM for 7 d. SCM was composed of 50% basal Eagle’s medium (Invitrogen Life Technologies, Inc., Carlsbad, CA), 25% heat-inactivated horse serum (Invitrogen Life Technologies, Inc.), 25% Hanks’ balanced salt solution (Invitrogen Life Technologies, Inc.), 1 mM L-glutamine (Invitrogen Life Technologies, Inc.), 5 mg/liter glucose, 25 μg/ml penicillin-streptomycin, 50 μg/ml neomycin (Invitrogen Life Technologies, Inc.), and 25 μg/ml ciliary neurotropic factor (Cedarlane Laboratories, Hornby, Canada). Culture medium was then changed to serum-free medium (SFM) on d 8. SFM was composed of 95% phenol red-free Neurobasal-A medium (Invitrogen Life Technologies, Inc.) supplemented with 2% B27, 1 mM sodium pyruvate, 2 mM Glutamax, 10 mM HEPES, 0.075% sodium bicarbonate (all from Invitrogen Life Technologies, Inc.), 5 mg/ml glucose, 25 μg/ml penicillin-streptomycin, and 50 μg/ml neomycin. All media were changed three times a week. Slice cultures were subjected to different experimental conditions on d 9.
NOS expression: immunohistochemistry and immunofluorescence
To determine which NOS isoforms are present in hypothalamic slice cultures, immunohistochemistry for eNOS, nNOS, and inducible NOS (iNOS) was performed after 8 d in culture. To verify that eNOS expression was localized in blood vessels, slice cultures were double-labeled with eNOS and RECA-1, a marker for rat endothelial cells (28). Because iNOS was not expected to be basally expressed in hypothalamic slice cultures, a positive control for iNOS expression was performed in which slice cultures were treated with lipopolysaccharide (LPS; 1 μg/ml) for 24 h.
Hypothalamic slice cultures were fixed for 1 h in ice-cold 4% paraformaldehyde (PFA, in PBS) and then incubated for 48 h at 4 C in primary antibody (in 0.4% Triton/PBS). The following primary antibodies were used: rabbit anti-eNOS (SC-654; 1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), rabbit anti-nNOS (AB 5380; 1:8000; Chemicon International, Temecula, CA), and rabbit anti-iNOS (SA-200; 1:2500; BIOMOL, Plymouth Meeting, PA). Slice cultures were incubated in biotinylated goat antirabbit antibody (BA-1000; 1:300; Vector Laboratories, Inc., Burlingame, CA; in 0.4% Triton/PBS) for 1 h at room temperature. Slice cultures were then incubated in avidin-biotin-peroxidase complex solution (Vectastain Elite ABC kit, Vector Laboratories, Inc.) for 1 h at room temperature. The reaction product was visualized with a 5- to 10-min incubation in the chromagen, 3,3'diaminobenzidine tetrahydrochloride (Sigma-Aldrich Corp., St. Louis, MO; 5 mg/10 ml, in PBS).
Slice cultures labeled by immunofluorescence were incubated for 48 h at 4 C in the following primary antibodies: rabbit anti-eNOS (SC-654; 1:1000; Santa Cruz Biotechnology, Inc.) and mouse anti-RECA-1 (MCA970; 1:100; Serotec, Oxford, UK). Slice cultures were incubated in the following secondary antibodies: CY3-conjugated donkey antirabbit IgG and fluorescein isothiocyanate-conjugated donkey antimouse IgG (1:2000; Jackson ImmunoResearch Laboratories, West Grove, PA).
Individual slices were gently removed from filters with a fine-tipped paintbrush and mounted onto slides. Slides were then rinsed in water, allowed to dry thoroughly, and coverslipped using Cytoseal (Richard Allen Scientific, Kalamazoo, MI).
NOS quantification
NOS expression in the PVN was quantified from one hypothalamic slice per culture dish (which translates to one slice per animal). Photographs were acquired with Image-Pro Plus software (Media Cybernetics, Sliver Spring, MD) using a DC-330 camera (Dage-MTI, Michigan City, IN) mounted onto a Leica microscope (Leica Microsystems, Deerfield, IL).
Quantification of eNOS expression was performed using Scion Image software (National Institutes of Health, Bethesda, MD). Images were converted into gray scale and thresholded to remove background; the density of eNOS-immunoreactive (eNOS-IR) elements in the PVN was determined by counting the numbers of pixels above threshold in a defined area, as previously described by others (29). When the PVN was analyzed as a whole, eNOS expression was quantified within a standardized box that encompassed one side of the PVN, and values for both sides were summed to obtain a value for the entire PVN; when the PVN was divided into regions (see below), a separate standardized box was used for each region. nNOS-IR neurons were counted either within the entire PVN or within regions (see below).
The PVN of the hypothalamus is an autonomic center comprised of two functionally distinct regions: the parvocellular region, which regulates the autonomic and neuroendocrine systems (7, 30), and the magnocellular region, which regulates posterior pituitary function (30, 31). To determine whether E2 affects the NO system differentially within the parvocellular and/or magnocellular regions, NOS expression in these regions was analyzed separately in some experiments. The parvocellular and magnocellular regions were delineated based on anatomical location; eNOS expression and numbers of nNOS-IR neurons within each region of the PVN were interpreted as proportions of the total signal within the PVN. To determine whether E2 affects eNOS expression exclusively in the PVN within the slice, eNOS expression was quantified in an area adjacent to the PVN delineated by a 400-μm2 box placed 100 μm away from the ventrolateral edge of the parvocellular region of the PVN.
ER expression
To study the expression of ER and ER? in the PVN of hypothalamic slice cultures, in situ hybridization and immunohistochemistry for ER and ER?, respectively, were performed after 8 d in culture. In situ hybridization was performed as follows (32). Hypothalamic slice cultures were fixed for 1 h in ice-cold 4% PFA (in PBS), rinsed, mounted onto slides, and allowed to dry thoroughly. Slices were then immersed in ice-cold 4% PFA for 10 min, rinsed twice in PBS, incubated in proteinase K buffer for 8 min, rinsed in PBS, and treated with 4% PFA for 4 min. Slices were incubated in acetic anhydride (in Tris-EDTA) for 10 min, followed by 70% ethanol in sodium acetate, 80% ethanol in sodium acetate, 95% ethanol, and 100% ethanol, and allowed to air dry. Slices were hybridized overnight at 53 C in a humid chamber with a 35S-labeled antisense RNA probe for ER generated from a 365-bp cDNA fragment (provided by Dr. Martha Campbell-Thompson, University of Florida, Gainesville, FL) and transcribed with T7 polymerase from a plasmid linearized with BamHI. After hybridization, sections were rinsed twice in 2x standard saline citrate (SSC), followed by incubation in ribonuclease A in sodium-Tris-EDTA buffer at 37 C for 30 min, 1x SSC at 45 C for 45 min, and 0.1x SSC at 65 C for 50 min, and then air dried. Slices were exposed to x-ray film (Kodak Biomax MR film, Eastman Kodak Co., Rochester, NY) for 3 d and then dipped in NTB-2 Kodak photographic emulsion (diluted 1:1 with water). Autoradiograms were exposed for 14 d, and after processing, sections were stained with 0.5% cresyl violet (Sigma-Aldrich Corp.).
For ER? immunohistochemistry, hypothalamic slice cultures were fixed for 1 h in 3% acrolein (in 4% PFA), rinsed in PBS, incubated in 1% sodium borohydride for 20 min, rinsed in PBS, and incubated in 0.2% Triton X/PBS for 20 min, then in 0.1 M glycine for 30 min. Slices were then incubated in blocking solution (5% rabbit serum and 1% H2O2 in 0.2% Triton/PBS) for 30 min and in anti-ER? primary antibody (provided by Dr. J. A. Gustafsson, Huddinge, Sweden; 503 IgY, 1:1000 dilution in 0.2% Triton/PBS containing 5% rabbit serum) for 72 h at 4 C. Slice cultures were incubated in biotinylated rabbit antichicken antibody (1:200; Jackson ImmunoResearch Laboratories, Inc.) for 1 h at room temperature. Slice cultures were then incubated in avidin-biotin-peroxidase complex solution (Vectastain Elite ABC kit, Vector Laboratories, Inc.) for 1 h at room temperature. The reaction product was visualized with a 5- to 10-min incubation in the chromagen, 3,3'diaminobenzidine tetrahydrochloride (Sigma-Aldrich Corp.; 5 mg/10 ml, in PBS) containing 5 mg nickel ammonium sulfate. Individual slices were then mounted and coverslipped as described above.
Experimental design
Dose-response experiments were performed to observe the effects of E2 (Sigma-Aldrich Corp.), genistein (Sigma-Aldrich Corp.), and PPT (Tocris Cookson, Ellisville, MO) on eNOS and nNOS protein expression in the PVN. Hypothalamic slices were incubated in either E2 (0.1–100 nM; in SFM), genistein [1 nM to 1 μM; in SFM containing 0.1% dimethylsulfoxide (DMSO)], or PPT (1 nM to 1 μM; in SFM containing 0.1% DMSO) for 48 h; controls received vehicle (SFM or SFM containing 0.1% DMSO).
Based on dose-response data, slice cultures were incubated in 1 nM E2 or 0.1 μM genistein and were processed for eNOS and nNOS by immunohistochemistry at 0, 1, 8, 24, and 48 h. NOS expression was measured within the entire PVN at all time points, whereas NOS expression in the parvo- and magnocellular regions of the PVN was measured only at 24 h, because this time point was used in subsequent experiments. Because PPT did not affect NOS expression, time-course experiments using PPT were not performed.
To investigate whether E2 or genistein alters NOS expression through an ER-dependent or -independent mechanism, the nonselective ER antagonist, ICI 182,780 (Tocris Cookson), was used to block the effects of E2 or genistein on NOS expression. Slice cultures were incubated in ICI 182,780 (10 nM) or ICI 182,780 plus E2 (1 nM) for 24 h while other slice cultures were incubated in ICI 182,780 (1 nM to 1 μM) or ICI 182,780 plus genistein (0.1 μM) for 24 h.
To investigate the role of ER in the effects of E2 and genistein on eNOS expression in the PVN, the selective ER antagonist, MPP (Obiter Research, Urbana, IL), was used. Slice cultures were incubated with MPP (10 nM to 10 μM), MPP plus E2 (1 nM), or MPP plus genistein (0.1 μM) for 24 h. Because ER is present in blood vessels, but not in neurons, of the PVN (23, 24, 33), we chose to investigate the effect of MPP only on eNOS expression.
Statistical analysis
All data are presented as the mean ± SEM (n = 6–14); each measurement was obtained from a different animal and from two or three independent experiments. Significant differences were determined using either one- or two-way ANOVA, followed by the post hoc Student-Newman-Keuls method or Tukey’s test, respectively. Differences were considered statistically significant at P < 0.05.
Results
eNOS and nNOS are expressed in the PVN of hypothalamic slices
eNOS protein expression was seen throughout the slices in blood vessel-like structures. These positively stained structures were verified to be blood vessels based on double-labeling for eNOS and RECA-1, a marker for endothelial cells (28) (Fig. 1, E–G). The PVN displayed a higher density of eNOS-IR vessels compared with surrounding areas (Fig. 1A). Most nNOS-IR neurons were localized in the PVN (Fig. 1B). Untreated hypothalamic slice cultures did not express iNOS (Fig. 1C). Because others have shown that LPS treatment induces iNOS expression in hippocampal slice cultures (34, 35), LPS treatment was used as a positive control. Hypothalamic slice cultures treated with LPS (1 μg/ml) for 24 h expressed iNOS within the PVN (Fig. 1D).
FIG. 1. Representative photographs of the PVN in control hypothalamic slice cultures (A–C and E–I) and in a culture treated with LPS (D). A, eNOS expression in blood vessel-like structures; B, nNOS-positive neurons. The inset in B shows a higher magnification of the outlined region. C, Lack of iNOS expression in control slices. D, iNOS expression in a LPS-treated slice (1 μM, 24 h). E, eNOS expression. F, Expression of RECA-1, a marker for endothelial cells. G, Merged image showing that blood vessels are double-labeled with eNOS and RECA-1. H, ER? expression (immunohistochemistry) in the PVN. I, Lack of ER mRNA (in situ hybridization) in the PVN. J, ER mRNA in the ventral medial hypothalamus (VMH). V, Third ventricle. Scale bars: A–D, 200 μm; inset in B, 100 μm; E–G, 50 μm; H–J, 100 μm.
ER? is expressed in the PVN
ER?-positive neurons were found in the PVN of hypothalamic slices (Fig. 1H). ER mRNA was not expressed in the PVN (Fig. 1I), but was found in the ventral medial hypothalamus (Fig. 1J).
E2 alters NOS expression
Slice cultures treated with E2 (0.1–10 nM) for 48 h displayed a dose-dependent increase in eNOS protein expression in the PVN, with a 36% increase when treated with 1 nM E2, compared with control cultures (Fig. 2, A and B, and Fig. 3A). E2 at 10 nM did not change eNOS expression compared with controls. All concentrations of E2 significantly decreased the numbers of nNOS-positive neurons in the PVN. E2 at 1 nM decreased the numbers of nNOS-IR neurons by 28% compared with control slice cultures (Fig. 2, D and E, and Fig. 3B).
FIG. 2. Representative photographs of eNOS (A–C) and nNOS (D–F) expression in the PVN of hypothalamic slice cultures treated with vehicle (A and D), 1 nM E2 (B and E), or 0.1 μM genistein (C and F) for 48 h. V, Third ventricle. Scale bar, 100 μm.
FIG. 3. Dose-response effects of E2 (A and B), genistein (C and D), and PPT (E and F) on eNOS (A, C, and E) and nNOS (B, D, and F) expression in the PVN of hypothalamic slice cultures. Cultures were treated with E2 (0.1–10 nM), genistein (0.01–10 μM), or PPT (1–1000 nM) for 48 h. Results are expressed as the mean ± SEM (n 6 for each group). *, P < 0.05; **, P < 0.001 (vs. control).
Based on dose-response data, 1 nM E2 was used in all subsequent experiments, because it was the lowest concentration that significantly altered eNOS and nNOS protein expression. Compared with time-matched controls, 1 nM E2 significantly increased eNOS expression in the PVN at 8, 24, and 48 h (31%, 34%, and 41%, respectively; Fig 4A). E2 decreased the numbers of nNOS-IR neurons in the PVN at 24 and 48 h (23% and 32%, respectively) compared with time-matched control cultures (Fig. 4C).
FIG. 4. Time-course effects of E2 (A and C) and genistein (B and D) on eNOS-IR (A and B) and numbers of nNOS-IR neurons (C and D). Cultures were treated with E2 (1 nM) or genistein (0.1 μM) for 0, 1, 8, 24, or 48 h. Results are expressed as the mean ± SEM (n 6 for each group). *, P < 0.05; **, P < 0.001 (vs. time-matched control).
Genistein alters NOS expression
Slice cultures treated with genistein (10 nM to 10 μM; 48 h) displayed a dose-dependent increase in eNOS expression, with an increase of 42% when treated with 0.1 μM genistein compared with control cultures (Fig. 2, A and C, and Fig. 3C). The numbers of nNOS-IR neurons were decreased by approximately 28% at all concentrations of genistein compared with controls (Fig. 2, D and F, and Fig. 3D).
Based on the dose-response data, 0.1 μM genistein was used in all subsequent experiments, because it was the lowest concentration that significantly altered eNOS and nNOS protein expression. Compared with time-matched controls, 0.1 μM genistein significantly increased eNOS expression at 8, 24, and 48 h (21%, 32%, and 31%, respectively; Fig. 4B). Genistein decreased the numbers of nNOS-IR neurons in the PVN at 8, 24, and 48 h (27%, 28%, and 22%, respectively) compared with time-matched controls (Fig. 4D).
E2 and genistein alter NOS expression equally in the parvo- and magnocellular regions of the PVN
E2 and genistein altered eNOS and nNOS expression in the parvo- and magnocellular regions of the PVN. However, E2 and genistein treatments did not change eNOS or nNOS expression in the parvo- and magnocellular regions as a proportion of total expression in the entire PVN (Table 1). In all subsequent experiments, therefore, the PVN was analyzed as a single entity.
TABLE 1. Effects of E2 (1 nM) and genistein (0.1 μM) treatment (24 h) on NOS expression in the parvocellular and magnocellular regions of the PVN as a proportion of NOS expression in the entire PVN
E2 and genistein alter eNOS expression exclusively in the PVN of slice cultures
Compared with their vehicle-treated controls, neither E2 (1 nM) nor genistein (0.1 μM) treatment for 24 h affected eNOS expression in an area adjacent to the PVN: E2, 654 ± 20 pixels vs. 665 ± 14 (controls); genistein, 662 ± 28 pixels vs. 676 ± 22 (controls).
PPT does not alter NOS expression
Compared with controls, slice cultures treated with PPT (1 nM to 1 μM) for 48 h did not display any change in either eNOS (Fig. 3E) or nNOS (Fig. 3F) expression in the PVN.
Effects of E2 and genistein on NOS expression are ER dependent
To determine whether the changes in eNOS and nNOS expression in the PVN stimulated by E2 or genistein are mediated by an ER-dependent mechanism, E2 (1 nM) or genistein (0.1 μM) was applied to slice cultures for 24 h in the presence or absence of the nonselective ER antagonist, ICI 182,780. ICI 182,780 (10 nM) blocked the E2-induced effects on eNOS expression and on numbers of nNOS-IR neurons in the PVN (Fig. 5, A and C). All concentrations of ICI 182,780 (1 nM to 1 μM) blocked the genistein-induced effects on eNOS and nNOS expression in the PVN (Fig. 5, B and D, illustrates the results for ICI 182,780 at 10 nM).
FIG. 5. Effects of ICI 182,780 on E2-induced (A and C) and genistein-induced (B and D) changes in eNOS (A and B) and nNOS (C and D) expression in the PVN of hypothalamic slice cultures. Cultures were treated with E2 (1 nM) or genistein (0.1 μM) for 24 h in the presence or absence of ICI 182,780 (10 nM). Results are expressed as the mean ± SEM (n 8 for each group). *, P < 0.05.
Effects of E2 and genistein on eNOS expression are not ER dependent
To determine whether the increases in eNOS expression in the PVN stimulated by E2 and genistein are mediated by an ER-dependent mechanism, E2 or genistein was applied to cultures for 24 h in the presence or absence of the selective ER antagonist, MPP (100 nM to 10 μM). None of the concentrations of MPP used altered the effect of E2 or genistein on eNOS expression (Fig. 6 shows the results for MPP at 1 μM).
FIG. 6. Effects of MPP on E2-induced (A) and genistein-induced (B) changes in eNOS-IR in hypothalamic slice cultures. Cultures were treated with either E2 (1 nM) or genistein (0.1 μM) for 24 h in the presence or absence of MPP (1 μM). Results are expressed as the mean ± SEM (n 7 for each group). *, P < 0.05.
Discussion
In the present study we investigated the effects of E2 on the NO system in hypothalamic slice cultures. We show that E2 increases eNOS expression and decreases the numbers of nNOS-positive neurons in the PVN. Using a selective ER? agonist, a selective ER agonist, and a selective ER antagonist, we show that E2-induced changes in NOS expression in the PVN of hypothalamic slice cultures are ER? dependent.
Little is known about how estrogen affects the NO system in autonomic centers of the brain and, in particular, the PVN. In vivo studies are often difficult to interpret due to physiological influences from other brain regions. For this reason, we used hypothalamic slice cultures to investigate estrogen’s effect on the NO system, because this model eliminates influences from other brain regions and maintains the cellular morphology and organization of the hypothalamus (36, 37). Although the hypothalamic tissues used in these cultures were obtained from postnatal rats, a comprehensive study that investigated the expression of ERs in postnatal rat brain (postnatal d 3, 7, and 14) revealed that, with the exception of the arcuate nucleus and the supramammillary complex, postnatal and adult hypothalamic tissues exhibit the same expression patterns of ERs (38). Furthermore, we show that ER? (but not ER) is expressed in the PVN from hypothalamic slice cultures, as is the case in adult hypothalamus in vivo. Finally, we also show that, like the hypothalamus in vivo, hypothalamic slice cultures express basal levels of eNOS and nNOS, but not iNOS. Therefore, based on these data, we conclude that hypothalamic slice cultures constitute an appropriate model for investigating the effects of E2 on the NO system of the PVN.
We demonstrate that E2 stimulates increased eNOS expression in the PVN of hypothalamic slices in a time-dependent manner. These results agree with other studies that have shown that estrogen stimulates eNOS expression and activity in cultured endothelial cells of the aorta and umbilical cord (8, 9, 10, 39). Furthermore, estrogen treatment in OVX rats has been shown to stimulate eNOS protein expression in cerebral microvessel fractions and isolated pial arterioles (11, 12, 13, 14) and in ex vivo preparations of the median eminence (17). Our data indicate that the E2-stimulated increases in eNOS expression are, in fact, due to increases in the density of eNOS-IR blood vessels, suggesting that E2 promotes angiogenesis in the PVN. Estrogen treatment has also been shown to stimulate angiogenesis in OVX mice, as demonstrated by increased vascularization of a subdural gel plug (40). Interestingly, we show that within our hypothalamic slice cultures, E2 appears to stimulate eNOS expression exclusively within the PVN. This finding emphasizes the importance of this nucleus as a target for estrogen’s effects on eNOS expression in the hypothalamus. Finally, we found that the effect of E2 on eNOS expression is concentration dependent, because 10 nM E2 did not alter eNOS expression. The reasons for this result are not known, but it is possible that 10 nM E2 treatment has adverse effects on the hypothalamic tissue, because it has been shown that E2 at this concentration induces oxidative DNA damage in a variety of cells and tissues, including MCF-7 breast cancer cells, kidney, and liver tissue (41, 42).
We show that E2 decreases the numbers of nNOS-positive neurons in the PVN in vitro. In vivo studies have shown that estrogen increases levels of nNOS mRNA in the ventral medial nucleus (20) and the hippocampus (21), but reports on estrogen’s effect on nNOS expression in the PVN are conflicting. Some groups report no changes in nNOS mRNA levels (20) or numbers of NADPH-diaphorase neurons (20, 22) in the PVN of OVX-E rats, whereas another group reported that OVX decreases the numbers of NADPH-diaphorase neurons in the PVN and that E2 treatment reverses this effect (43). Finally, estrogen decreases levels of nNOS protein expression in nerves and ganglia of the vagina and clitoris of rabbits (44). The significance of these divergent findings is unclear at this time, but they may be due to the fact that different parameters were measured in each of the studies.
Multiple pathways of estrogen signaling have been described; estrogen mediates genomic or nongenomic effects through receptor-dependent and -independent pathways (39, 45, 46, 47, 48). ER-dependent genomic mechanisms involve binding of an estrogen-ER complex to the estrogen response element of the target gene promoter to regulate nuclear transcription (39, 45), whereas ER-dependent, nongenomic mechanisms involve membrane ERs and rapid activation of signaling cascades, such as mitogen-activated protein and protein kinase C pathways (39, 45, 49, 50, 51). ER-independent mechanisms appear to include nonclassical membrane receptors that act on intracellular signaling pathways and are not blocked by the nonselective ER antagonist, ICI 182,780 (50). Our data show that E2 stimulates increased eNOS expression after 8 h, suggesting that E2 acts through a slow, genomic pathway to alter transcription of the eNOS gene. Indeed, the eNOS promoter contains an estrogen response element (52). Our data also show that E2-induced changes in NOS expression are ER dependent, because ICI 182,780, used at concentrations (1 nM to 1 μM) shown by others to effectively block ERs (13, 53), inhibits the effects of E2 on eNOS and nNOS. Our findings are consistent with other studies that show that estrogen increases eNOS expression by a genomic ER-dependent mechanism in isolated cerebral vessels from adult rats (13). We and others have reported nongenomic or membrane ER-dependent up-regulation of eNOS activity and/or NO production by estrogen in cultured uterine endothelial cells (54), neuroblastoma cells (53), and pedal ganglia of molluscs (55).
Due to homologous regions in the genes for ER and ER? (25, 26), few selective ligands for each receptor exist, and to date, there are no selective ER? antagonists commercially available. Therefore, to study activation of ER?, we used genistein, because it confers estrogenic actions at concentrations between 1 nM and 1 μM (31, 56), is 20 times more selective for ER? than ER (31, 56, 57, 58), and does not activate ER-dependent gene expression in the hypothalamus in vivo (58). We did not use higher concentrations of genistein (upper micromolar concentration range) because they inhibit protein tyrosine kinases (59, 60). Our data show that genistein increases eNOS expression in the PVN and decreases the numbers of nNOS-positive neurons in the PVN at all concentrations used. In agreement with our results, E2 increased eNOS promoter activity in cardiac myocytes, and this effect was blocked with a selective ER? antagonist developed by this group (61). These results support our hypothesis that ER? mediates the E2-induced changes in NOS expression in the PVN.
Although neurons of the PVN express ER? and no ER (23, 24, 25), cerebral blood vessels express both ER? and ER in a ratio of 3:1 (33). Therefore, to investigate the possibility that activation of ER alters NOS expression, we used the highly selective ER agonist, PPT (62, 63). Our data show that activation of ER has no effect on either eNOS or nNOS expression in the PVN. We also used the selective ER antagonist, MPP (62, 64), to exclude the possibility that ER contributes to the E2-induced increase in eNOS expression. Our data show that ER is not involved in the E2- or genistein-induced increases in eNOS expression in hypothalamic slice cultures. Interestingly, OVX-E ER-knockout mice apparently do not display altered levels of eNOS expression in cerebral microvessels compared with OVX-V ER-knockout mice (65). However, this study did not account for the finding that ER-knockout mice expressed higher levels of eNOS compared with wild-type mice. Furthermore, developmental differences between ER-knockout mice and wild-type mice may also contribute to the differences between the results of this study and ours.
In conclusion, we show that E2 stimulates increases in eNOS protein expression in the PVN and decreases the numbers of nNOS-positive neurons in the PVN of hypothalamic slice cultures. Furthermore, our time-course data suggest that E2 alters NOS expression via a slow, genomic effect. Finally, our morphological data in combination with our use of a variety of selective ER agonists and antagonists provide strong evidence that ER? is responsible for E2’s effect on NOS expression in the PVN. We previously showed that estrogen treatment attenuates blood pressure responses to psychological stress in OVX rats through brain NO produced by eNOS and/or nNOS (3). Therefore, our current results raise the intriguing possibility that NO produced by eNOS in blood vessels of the PVN via an ER?-dependent mechanism acts on neighboring neurons to influence autonomic pathways originating from the PVN.
Acknowledgments
We thank Dr. Robert Campenot for graciously providing some of the facilities required for these experiments.
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Address all correspondence and requests for reprints to: Dr. Teresa L. Krukoff, Center for Neuroscience and Department of Cell Biology, 5-31 Medical Sciences Building, University of Alberta, Edmonton, Alberta, Canada T6G 2H7. E-mail: teresa.krukoff@ualberta.ca.
Abstract
Although it is evident that estrogen has important physiological effects in the brain, the signaling mechanisms mediating these effects remain unclear. We recently showed that estrogen mediates attenuated blood pressure responses to psychological stress in ovariectomized female rats through brain nitric oxide (NO). An area likely to mediate these effects is the hypothalamic paraventricular nucleus (PVN), because here NO exerts inhibitory effects on autonomic output to the periphery. Because little is known about how estrogen acts on the NO system in the PVN, our aim was to study the effects of estrogen on the NO system in the PVN of hypothalamic slices cultures. We show that 17?-estradiol (E2; 1 nM) increases endothelial NO synthase (eNOS) protein expression and decreases the numbers of neuronal NOS (nNOS)-positive neurons in the PVN after 8 and 24 h, respectively. Using the nonselective estrogen receptor (ER) antagonist, ICI 182,780 (10 nM), we determined that E2-induced changes in NOS expression in the PVN are ER dependent. Using the ER? agonist, genistein (0.1 μM), we determined that activation of ER? induces increased eNOS expression and a decreased number of nNOS-positive neurons. We used the selective ER agonist, propyl-pyrazole-triol (10 nM), and antagonist, methyl-piperidino-pyrazole (1 μM), to exclude the possibility that ER is involved in the E2-induced increase in eNOS and nNOS in the PVN. These results demonstrate that E2 induces changes in NOS expression in the PVN and that these effects are ER? dependent.
Introduction
HORMONE REPLACEMENT THERAPY (HRT) is widely used in women to relieve menopausal symptoms and until recently was believed to protect aging women from cardiovascular disease. However, the 2002 Women’s Health Initiative Study on HRT created a great deal of controversy when it was halted abruptly because of risks associated with HRT, including increased incidence of stroke (1, 2). These findings dramatically demonstrate that not only are the effects of HRT in women poorly understood, but estrogen’s effects on normal physiological processes are also still unclear.
We recently reported that ovariectomized, estrogen-treated (OVX-E) rats display attenuated blood pressure (BP) responses to psychological stress compared with OVX, vehicle-treated (OVX-V) rats (3). In agreement with our results, peri- and postmenopausal women receiving estrogen exhibit lower BP responses to mental stress compared with those not receiving estrogen (4, 5). A potential mediator of estrogen’s effect on cardiovascular responses to stress is nitric oxide (NO), a neurotransmitter involved in maintaining homeostasis and regulating autonomic activity (6, 7). Indeed, we found that estrogen acts through brain NO to attenuate BP responses to psychological stress (3).
Studies performed in both peripheral and brain tissues have demonstrated that estrogen influences the NO system. For example, estrogen increases NO production (8, 9), endothelial nitric oxide synthase (eNOS) expression (8, 10), and eNOS activity (8, 9) in cultured endothelial cells. Estrogen also increases eNOS protein expression in cerebral microvessels (11, 12, 13, 14). Finally, estrogen increases NO production (15, 16) and eNOS protein expression (17) in ex vivo preparations of the median eminence. The effects of estrogen on neuronal NOS (nNOS) in the brain appear to be regionally specific. Estrogen has been shown to increase the numbers of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase (nNOS) neurons (18, 19) and nNOS mRNA (20, 21) in select brain regions of OVX rats. Others report that estrogen does not change nNOS mRNA levels or numbers of nNOS neurons in other brain regions (20, 22). The specific mechanism(s) by which estrogen acts on NO in the brain remains to be elucidated.
Because the paraventricular nucleus (PVN) of the hypothalamus is an autonomic center that regulates neuroendocrine and autonomic functions (7), we hypothesize that estrogen acts on the NO system in the PVN to alter central autonomic activity. Our first aim was to investigate estrogen’s effects on NOS expression in the PVN. Because estrogen acts on two receptors, estrogen receptor- (ER) and ER?, and the PVN expresses only ER? (23, 24, 25, 26), our second aim was to investigate the role of ER? in estrogen’s effects on NOS expression in the PVN. Using hypothalamic slice cultures, we investigated the effects of 17?-estradiol (E2) on eNOS and nNOS expression in the PVN. We used the ER antagonist, ICI 182,780, to determine whether E2 affects NOS expression through an ER-dependent pathway. We also used the selective ER? agonist, genistein, the selective ER agonist, propyl-pyrazole-triol (PPT), and the selective ER antagonist, methyl-piperidino-pyrazole (MPP), to determine whether E2 acts through an ER?-dependent mechanism to affect NOS expression.
Materials and Methods
Hypothalamic slice culture
Hypothalamic slice cultures were prepared as described by others (27). Briefly, 5- to 7-d-old Sprague Dawley rat pups (Biological Sciences, University of Alberta, Edmonton, Canada) were quickly decapitated. Blocks of hypothalamic tissue were cut from the brains and sectioned at a thickness of 400 μm using a McIlwain tissue chopper (Mickle Laboratory Engineering, Gomshall, UK). Slices were immediately placed into Gey’s balanced salt solution (4 C) enriched with 5 mg/liter glucose. A total of five coronal slices from each brain were placed on a single Millicell-CM filter insert (Millipore Corp., Bedford, MA; pore size, 0.4 μm; diameter, 30 mm). Each filter insert was placed into a 35-mm petri dish containing 1.1 ml serum-containing medium (SCM); each dish, representing n = 1, was exposed to a single treatment as described below. Slice cultures were maintained in a humidified incubator at 35 C in 5% CO2 and 95% air.
Culture conditions
Hypothalamic slice cultures were maintained in SCM for 7 d. SCM was composed of 50% basal Eagle’s medium (Invitrogen Life Technologies, Inc., Carlsbad, CA), 25% heat-inactivated horse serum (Invitrogen Life Technologies, Inc.), 25% Hanks’ balanced salt solution (Invitrogen Life Technologies, Inc.), 1 mM L-glutamine (Invitrogen Life Technologies, Inc.), 5 mg/liter glucose, 25 μg/ml penicillin-streptomycin, 50 μg/ml neomycin (Invitrogen Life Technologies, Inc.), and 25 μg/ml ciliary neurotropic factor (Cedarlane Laboratories, Hornby, Canada). Culture medium was then changed to serum-free medium (SFM) on d 8. SFM was composed of 95% phenol red-free Neurobasal-A medium (Invitrogen Life Technologies, Inc.) supplemented with 2% B27, 1 mM sodium pyruvate, 2 mM Glutamax, 10 mM HEPES, 0.075% sodium bicarbonate (all from Invitrogen Life Technologies, Inc.), 5 mg/ml glucose, 25 μg/ml penicillin-streptomycin, and 50 μg/ml neomycin. All media were changed three times a week. Slice cultures were subjected to different experimental conditions on d 9.
NOS expression: immunohistochemistry and immunofluorescence
To determine which NOS isoforms are present in hypothalamic slice cultures, immunohistochemistry for eNOS, nNOS, and inducible NOS (iNOS) was performed after 8 d in culture. To verify that eNOS expression was localized in blood vessels, slice cultures were double-labeled with eNOS and RECA-1, a marker for rat endothelial cells (28). Because iNOS was not expected to be basally expressed in hypothalamic slice cultures, a positive control for iNOS expression was performed in which slice cultures were treated with lipopolysaccharide (LPS; 1 μg/ml) for 24 h.
Hypothalamic slice cultures were fixed for 1 h in ice-cold 4% paraformaldehyde (PFA, in PBS) and then incubated for 48 h at 4 C in primary antibody (in 0.4% Triton/PBS). The following primary antibodies were used: rabbit anti-eNOS (SC-654; 1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), rabbit anti-nNOS (AB 5380; 1:8000; Chemicon International, Temecula, CA), and rabbit anti-iNOS (SA-200; 1:2500; BIOMOL, Plymouth Meeting, PA). Slice cultures were incubated in biotinylated goat antirabbit antibody (BA-1000; 1:300; Vector Laboratories, Inc., Burlingame, CA; in 0.4% Triton/PBS) for 1 h at room temperature. Slice cultures were then incubated in avidin-biotin-peroxidase complex solution (Vectastain Elite ABC kit, Vector Laboratories, Inc.) for 1 h at room temperature. The reaction product was visualized with a 5- to 10-min incubation in the chromagen, 3,3'diaminobenzidine tetrahydrochloride (Sigma-Aldrich Corp., St. Louis, MO; 5 mg/10 ml, in PBS).
Slice cultures labeled by immunofluorescence were incubated for 48 h at 4 C in the following primary antibodies: rabbit anti-eNOS (SC-654; 1:1000; Santa Cruz Biotechnology, Inc.) and mouse anti-RECA-1 (MCA970; 1:100; Serotec, Oxford, UK). Slice cultures were incubated in the following secondary antibodies: CY3-conjugated donkey antirabbit IgG and fluorescein isothiocyanate-conjugated donkey antimouse IgG (1:2000; Jackson ImmunoResearch Laboratories, West Grove, PA).
Individual slices were gently removed from filters with a fine-tipped paintbrush and mounted onto slides. Slides were then rinsed in water, allowed to dry thoroughly, and coverslipped using Cytoseal (Richard Allen Scientific, Kalamazoo, MI).
NOS quantification
NOS expression in the PVN was quantified from one hypothalamic slice per culture dish (which translates to one slice per animal). Photographs were acquired with Image-Pro Plus software (Media Cybernetics, Sliver Spring, MD) using a DC-330 camera (Dage-MTI, Michigan City, IN) mounted onto a Leica microscope (Leica Microsystems, Deerfield, IL).
Quantification of eNOS expression was performed using Scion Image software (National Institutes of Health, Bethesda, MD). Images were converted into gray scale and thresholded to remove background; the density of eNOS-immunoreactive (eNOS-IR) elements in the PVN was determined by counting the numbers of pixels above threshold in a defined area, as previously described by others (29). When the PVN was analyzed as a whole, eNOS expression was quantified within a standardized box that encompassed one side of the PVN, and values for both sides were summed to obtain a value for the entire PVN; when the PVN was divided into regions (see below), a separate standardized box was used for each region. nNOS-IR neurons were counted either within the entire PVN or within regions (see below).
The PVN of the hypothalamus is an autonomic center comprised of two functionally distinct regions: the parvocellular region, which regulates the autonomic and neuroendocrine systems (7, 30), and the magnocellular region, which regulates posterior pituitary function (30, 31). To determine whether E2 affects the NO system differentially within the parvocellular and/or magnocellular regions, NOS expression in these regions was analyzed separately in some experiments. The parvocellular and magnocellular regions were delineated based on anatomical location; eNOS expression and numbers of nNOS-IR neurons within each region of the PVN were interpreted as proportions of the total signal within the PVN. To determine whether E2 affects eNOS expression exclusively in the PVN within the slice, eNOS expression was quantified in an area adjacent to the PVN delineated by a 400-μm2 box placed 100 μm away from the ventrolateral edge of the parvocellular region of the PVN.
ER expression
To study the expression of ER and ER? in the PVN of hypothalamic slice cultures, in situ hybridization and immunohistochemistry for ER and ER?, respectively, were performed after 8 d in culture. In situ hybridization was performed as follows (32). Hypothalamic slice cultures were fixed for 1 h in ice-cold 4% PFA (in PBS), rinsed, mounted onto slides, and allowed to dry thoroughly. Slices were then immersed in ice-cold 4% PFA for 10 min, rinsed twice in PBS, incubated in proteinase K buffer for 8 min, rinsed in PBS, and treated with 4% PFA for 4 min. Slices were incubated in acetic anhydride (in Tris-EDTA) for 10 min, followed by 70% ethanol in sodium acetate, 80% ethanol in sodium acetate, 95% ethanol, and 100% ethanol, and allowed to air dry. Slices were hybridized overnight at 53 C in a humid chamber with a 35S-labeled antisense RNA probe for ER generated from a 365-bp cDNA fragment (provided by Dr. Martha Campbell-Thompson, University of Florida, Gainesville, FL) and transcribed with T7 polymerase from a plasmid linearized with BamHI. After hybridization, sections were rinsed twice in 2x standard saline citrate (SSC), followed by incubation in ribonuclease A in sodium-Tris-EDTA buffer at 37 C for 30 min, 1x SSC at 45 C for 45 min, and 0.1x SSC at 65 C for 50 min, and then air dried. Slices were exposed to x-ray film (Kodak Biomax MR film, Eastman Kodak Co., Rochester, NY) for 3 d and then dipped in NTB-2 Kodak photographic emulsion (diluted 1:1 with water). Autoradiograms were exposed for 14 d, and after processing, sections were stained with 0.5% cresyl violet (Sigma-Aldrich Corp.).
For ER? immunohistochemistry, hypothalamic slice cultures were fixed for 1 h in 3% acrolein (in 4% PFA), rinsed in PBS, incubated in 1% sodium borohydride for 20 min, rinsed in PBS, and incubated in 0.2% Triton X/PBS for 20 min, then in 0.1 M glycine for 30 min. Slices were then incubated in blocking solution (5% rabbit serum and 1% H2O2 in 0.2% Triton/PBS) for 30 min and in anti-ER? primary antibody (provided by Dr. J. A. Gustafsson, Huddinge, Sweden; 503 IgY, 1:1000 dilution in 0.2% Triton/PBS containing 5% rabbit serum) for 72 h at 4 C. Slice cultures were incubated in biotinylated rabbit antichicken antibody (1:200; Jackson ImmunoResearch Laboratories, Inc.) for 1 h at room temperature. Slice cultures were then incubated in avidin-biotin-peroxidase complex solution (Vectastain Elite ABC kit, Vector Laboratories, Inc.) for 1 h at room temperature. The reaction product was visualized with a 5- to 10-min incubation in the chromagen, 3,3'diaminobenzidine tetrahydrochloride (Sigma-Aldrich Corp.; 5 mg/10 ml, in PBS) containing 5 mg nickel ammonium sulfate. Individual slices were then mounted and coverslipped as described above.
Experimental design
Dose-response experiments were performed to observe the effects of E2 (Sigma-Aldrich Corp.), genistein (Sigma-Aldrich Corp.), and PPT (Tocris Cookson, Ellisville, MO) on eNOS and nNOS protein expression in the PVN. Hypothalamic slices were incubated in either E2 (0.1–100 nM; in SFM), genistein [1 nM to 1 μM; in SFM containing 0.1% dimethylsulfoxide (DMSO)], or PPT (1 nM to 1 μM; in SFM containing 0.1% DMSO) for 48 h; controls received vehicle (SFM or SFM containing 0.1% DMSO).
Based on dose-response data, slice cultures were incubated in 1 nM E2 or 0.1 μM genistein and were processed for eNOS and nNOS by immunohistochemistry at 0, 1, 8, 24, and 48 h. NOS expression was measured within the entire PVN at all time points, whereas NOS expression in the parvo- and magnocellular regions of the PVN was measured only at 24 h, because this time point was used in subsequent experiments. Because PPT did not affect NOS expression, time-course experiments using PPT were not performed.
To investigate whether E2 or genistein alters NOS expression through an ER-dependent or -independent mechanism, the nonselective ER antagonist, ICI 182,780 (Tocris Cookson), was used to block the effects of E2 or genistein on NOS expression. Slice cultures were incubated in ICI 182,780 (10 nM) or ICI 182,780 plus E2 (1 nM) for 24 h while other slice cultures were incubated in ICI 182,780 (1 nM to 1 μM) or ICI 182,780 plus genistein (0.1 μM) for 24 h.
To investigate the role of ER in the effects of E2 and genistein on eNOS expression in the PVN, the selective ER antagonist, MPP (Obiter Research, Urbana, IL), was used. Slice cultures were incubated with MPP (10 nM to 10 μM), MPP plus E2 (1 nM), or MPP plus genistein (0.1 μM) for 24 h. Because ER is present in blood vessels, but not in neurons, of the PVN (23, 24, 33), we chose to investigate the effect of MPP only on eNOS expression.
Statistical analysis
All data are presented as the mean ± SEM (n = 6–14); each measurement was obtained from a different animal and from two or three independent experiments. Significant differences were determined using either one- or two-way ANOVA, followed by the post hoc Student-Newman-Keuls method or Tukey’s test, respectively. Differences were considered statistically significant at P < 0.05.
Results
eNOS and nNOS are expressed in the PVN of hypothalamic slices
eNOS protein expression was seen throughout the slices in blood vessel-like structures. These positively stained structures were verified to be blood vessels based on double-labeling for eNOS and RECA-1, a marker for endothelial cells (28) (Fig. 1, E–G). The PVN displayed a higher density of eNOS-IR vessels compared with surrounding areas (Fig. 1A). Most nNOS-IR neurons were localized in the PVN (Fig. 1B). Untreated hypothalamic slice cultures did not express iNOS (Fig. 1C). Because others have shown that LPS treatment induces iNOS expression in hippocampal slice cultures (34, 35), LPS treatment was used as a positive control. Hypothalamic slice cultures treated with LPS (1 μg/ml) for 24 h expressed iNOS within the PVN (Fig. 1D).
FIG. 1. Representative photographs of the PVN in control hypothalamic slice cultures (A–C and E–I) and in a culture treated with LPS (D). A, eNOS expression in blood vessel-like structures; B, nNOS-positive neurons. The inset in B shows a higher magnification of the outlined region. C, Lack of iNOS expression in control slices. D, iNOS expression in a LPS-treated slice (1 μM, 24 h). E, eNOS expression. F, Expression of RECA-1, a marker for endothelial cells. G, Merged image showing that blood vessels are double-labeled with eNOS and RECA-1. H, ER? expression (immunohistochemistry) in the PVN. I, Lack of ER mRNA (in situ hybridization) in the PVN. J, ER mRNA in the ventral medial hypothalamus (VMH). V, Third ventricle. Scale bars: A–D, 200 μm; inset in B, 100 μm; E–G, 50 μm; H–J, 100 μm.
ER? is expressed in the PVN
ER?-positive neurons were found in the PVN of hypothalamic slices (Fig. 1H). ER mRNA was not expressed in the PVN (Fig. 1I), but was found in the ventral medial hypothalamus (Fig. 1J).
E2 alters NOS expression
Slice cultures treated with E2 (0.1–10 nM) for 48 h displayed a dose-dependent increase in eNOS protein expression in the PVN, with a 36% increase when treated with 1 nM E2, compared with control cultures (Fig. 2, A and B, and Fig. 3A). E2 at 10 nM did not change eNOS expression compared with controls. All concentrations of E2 significantly decreased the numbers of nNOS-positive neurons in the PVN. E2 at 1 nM decreased the numbers of nNOS-IR neurons by 28% compared with control slice cultures (Fig. 2, D and E, and Fig. 3B).
FIG. 2. Representative photographs of eNOS (A–C) and nNOS (D–F) expression in the PVN of hypothalamic slice cultures treated with vehicle (A and D), 1 nM E2 (B and E), or 0.1 μM genistein (C and F) for 48 h. V, Third ventricle. Scale bar, 100 μm.
FIG. 3. Dose-response effects of E2 (A and B), genistein (C and D), and PPT (E and F) on eNOS (A, C, and E) and nNOS (B, D, and F) expression in the PVN of hypothalamic slice cultures. Cultures were treated with E2 (0.1–10 nM), genistein (0.01–10 μM), or PPT (1–1000 nM) for 48 h. Results are expressed as the mean ± SEM (n 6 for each group). *, P < 0.05; **, P < 0.001 (vs. control).
Based on dose-response data, 1 nM E2 was used in all subsequent experiments, because it was the lowest concentration that significantly altered eNOS and nNOS protein expression. Compared with time-matched controls, 1 nM E2 significantly increased eNOS expression in the PVN at 8, 24, and 48 h (31%, 34%, and 41%, respectively; Fig 4A). E2 decreased the numbers of nNOS-IR neurons in the PVN at 24 and 48 h (23% and 32%, respectively) compared with time-matched control cultures (Fig. 4C).
FIG. 4. Time-course effects of E2 (A and C) and genistein (B and D) on eNOS-IR (A and B) and numbers of nNOS-IR neurons (C and D). Cultures were treated with E2 (1 nM) or genistein (0.1 μM) for 0, 1, 8, 24, or 48 h. Results are expressed as the mean ± SEM (n 6 for each group). *, P < 0.05; **, P < 0.001 (vs. time-matched control).
Genistein alters NOS expression
Slice cultures treated with genistein (10 nM to 10 μM; 48 h) displayed a dose-dependent increase in eNOS expression, with an increase of 42% when treated with 0.1 μM genistein compared with control cultures (Fig. 2, A and C, and Fig. 3C). The numbers of nNOS-IR neurons were decreased by approximately 28% at all concentrations of genistein compared with controls (Fig. 2, D and F, and Fig. 3D).
Based on the dose-response data, 0.1 μM genistein was used in all subsequent experiments, because it was the lowest concentration that significantly altered eNOS and nNOS protein expression. Compared with time-matched controls, 0.1 μM genistein significantly increased eNOS expression at 8, 24, and 48 h (21%, 32%, and 31%, respectively; Fig. 4B). Genistein decreased the numbers of nNOS-IR neurons in the PVN at 8, 24, and 48 h (27%, 28%, and 22%, respectively) compared with time-matched controls (Fig. 4D).
E2 and genistein alter NOS expression equally in the parvo- and magnocellular regions of the PVN
E2 and genistein altered eNOS and nNOS expression in the parvo- and magnocellular regions of the PVN. However, E2 and genistein treatments did not change eNOS or nNOS expression in the parvo- and magnocellular regions as a proportion of total expression in the entire PVN (Table 1). In all subsequent experiments, therefore, the PVN was analyzed as a single entity.
TABLE 1. Effects of E2 (1 nM) and genistein (0.1 μM) treatment (24 h) on NOS expression in the parvocellular and magnocellular regions of the PVN as a proportion of NOS expression in the entire PVN
E2 and genistein alter eNOS expression exclusively in the PVN of slice cultures
Compared with their vehicle-treated controls, neither E2 (1 nM) nor genistein (0.1 μM) treatment for 24 h affected eNOS expression in an area adjacent to the PVN: E2, 654 ± 20 pixels vs. 665 ± 14 (controls); genistein, 662 ± 28 pixels vs. 676 ± 22 (controls).
PPT does not alter NOS expression
Compared with controls, slice cultures treated with PPT (1 nM to 1 μM) for 48 h did not display any change in either eNOS (Fig. 3E) or nNOS (Fig. 3F) expression in the PVN.
Effects of E2 and genistein on NOS expression are ER dependent
To determine whether the changes in eNOS and nNOS expression in the PVN stimulated by E2 or genistein are mediated by an ER-dependent mechanism, E2 (1 nM) or genistein (0.1 μM) was applied to slice cultures for 24 h in the presence or absence of the nonselective ER antagonist, ICI 182,780. ICI 182,780 (10 nM) blocked the E2-induced effects on eNOS expression and on numbers of nNOS-IR neurons in the PVN (Fig. 5, A and C). All concentrations of ICI 182,780 (1 nM to 1 μM) blocked the genistein-induced effects on eNOS and nNOS expression in the PVN (Fig. 5, B and D, illustrates the results for ICI 182,780 at 10 nM).
FIG. 5. Effects of ICI 182,780 on E2-induced (A and C) and genistein-induced (B and D) changes in eNOS (A and B) and nNOS (C and D) expression in the PVN of hypothalamic slice cultures. Cultures were treated with E2 (1 nM) or genistein (0.1 μM) for 24 h in the presence or absence of ICI 182,780 (10 nM). Results are expressed as the mean ± SEM (n 8 for each group). *, P < 0.05.
Effects of E2 and genistein on eNOS expression are not ER dependent
To determine whether the increases in eNOS expression in the PVN stimulated by E2 and genistein are mediated by an ER-dependent mechanism, E2 or genistein was applied to cultures for 24 h in the presence or absence of the selective ER antagonist, MPP (100 nM to 10 μM). None of the concentrations of MPP used altered the effect of E2 or genistein on eNOS expression (Fig. 6 shows the results for MPP at 1 μM).
FIG. 6. Effects of MPP on E2-induced (A) and genistein-induced (B) changes in eNOS-IR in hypothalamic slice cultures. Cultures were treated with either E2 (1 nM) or genistein (0.1 μM) for 24 h in the presence or absence of MPP (1 μM). Results are expressed as the mean ± SEM (n 7 for each group). *, P < 0.05.
Discussion
In the present study we investigated the effects of E2 on the NO system in hypothalamic slice cultures. We show that E2 increases eNOS expression and decreases the numbers of nNOS-positive neurons in the PVN. Using a selective ER? agonist, a selective ER agonist, and a selective ER antagonist, we show that E2-induced changes in NOS expression in the PVN of hypothalamic slice cultures are ER? dependent.
Little is known about how estrogen affects the NO system in autonomic centers of the brain and, in particular, the PVN. In vivo studies are often difficult to interpret due to physiological influences from other brain regions. For this reason, we used hypothalamic slice cultures to investigate estrogen’s effect on the NO system, because this model eliminates influences from other brain regions and maintains the cellular morphology and organization of the hypothalamus (36, 37). Although the hypothalamic tissues used in these cultures were obtained from postnatal rats, a comprehensive study that investigated the expression of ERs in postnatal rat brain (postnatal d 3, 7, and 14) revealed that, with the exception of the arcuate nucleus and the supramammillary complex, postnatal and adult hypothalamic tissues exhibit the same expression patterns of ERs (38). Furthermore, we show that ER? (but not ER) is expressed in the PVN from hypothalamic slice cultures, as is the case in adult hypothalamus in vivo. Finally, we also show that, like the hypothalamus in vivo, hypothalamic slice cultures express basal levels of eNOS and nNOS, but not iNOS. Therefore, based on these data, we conclude that hypothalamic slice cultures constitute an appropriate model for investigating the effects of E2 on the NO system of the PVN.
We demonstrate that E2 stimulates increased eNOS expression in the PVN of hypothalamic slices in a time-dependent manner. These results agree with other studies that have shown that estrogen stimulates eNOS expression and activity in cultured endothelial cells of the aorta and umbilical cord (8, 9, 10, 39). Furthermore, estrogen treatment in OVX rats has been shown to stimulate eNOS protein expression in cerebral microvessel fractions and isolated pial arterioles (11, 12, 13, 14) and in ex vivo preparations of the median eminence (17). Our data indicate that the E2-stimulated increases in eNOS expression are, in fact, due to increases in the density of eNOS-IR blood vessels, suggesting that E2 promotes angiogenesis in the PVN. Estrogen treatment has also been shown to stimulate angiogenesis in OVX mice, as demonstrated by increased vascularization of a subdural gel plug (40). Interestingly, we show that within our hypothalamic slice cultures, E2 appears to stimulate eNOS expression exclusively within the PVN. This finding emphasizes the importance of this nucleus as a target for estrogen’s effects on eNOS expression in the hypothalamus. Finally, we found that the effect of E2 on eNOS expression is concentration dependent, because 10 nM E2 did not alter eNOS expression. The reasons for this result are not known, but it is possible that 10 nM E2 treatment has adverse effects on the hypothalamic tissue, because it has been shown that E2 at this concentration induces oxidative DNA damage in a variety of cells and tissues, including MCF-7 breast cancer cells, kidney, and liver tissue (41, 42).
We show that E2 decreases the numbers of nNOS-positive neurons in the PVN in vitro. In vivo studies have shown that estrogen increases levels of nNOS mRNA in the ventral medial nucleus (20) and the hippocampus (21), but reports on estrogen’s effect on nNOS expression in the PVN are conflicting. Some groups report no changes in nNOS mRNA levels (20) or numbers of NADPH-diaphorase neurons (20, 22) in the PVN of OVX-E rats, whereas another group reported that OVX decreases the numbers of NADPH-diaphorase neurons in the PVN and that E2 treatment reverses this effect (43). Finally, estrogen decreases levels of nNOS protein expression in nerves and ganglia of the vagina and clitoris of rabbits (44). The significance of these divergent findings is unclear at this time, but they may be due to the fact that different parameters were measured in each of the studies.
Multiple pathways of estrogen signaling have been described; estrogen mediates genomic or nongenomic effects through receptor-dependent and -independent pathways (39, 45, 46, 47, 48). ER-dependent genomic mechanisms involve binding of an estrogen-ER complex to the estrogen response element of the target gene promoter to regulate nuclear transcription (39, 45), whereas ER-dependent, nongenomic mechanisms involve membrane ERs and rapid activation of signaling cascades, such as mitogen-activated protein and protein kinase C pathways (39, 45, 49, 50, 51). ER-independent mechanisms appear to include nonclassical membrane receptors that act on intracellular signaling pathways and are not blocked by the nonselective ER antagonist, ICI 182,780 (50). Our data show that E2 stimulates increased eNOS expression after 8 h, suggesting that E2 acts through a slow, genomic pathway to alter transcription of the eNOS gene. Indeed, the eNOS promoter contains an estrogen response element (52). Our data also show that E2-induced changes in NOS expression are ER dependent, because ICI 182,780, used at concentrations (1 nM to 1 μM) shown by others to effectively block ERs (13, 53), inhibits the effects of E2 on eNOS and nNOS. Our findings are consistent with other studies that show that estrogen increases eNOS expression by a genomic ER-dependent mechanism in isolated cerebral vessels from adult rats (13). We and others have reported nongenomic or membrane ER-dependent up-regulation of eNOS activity and/or NO production by estrogen in cultured uterine endothelial cells (54), neuroblastoma cells (53), and pedal ganglia of molluscs (55).
Due to homologous regions in the genes for ER and ER? (25, 26), few selective ligands for each receptor exist, and to date, there are no selective ER? antagonists commercially available. Therefore, to study activation of ER?, we used genistein, because it confers estrogenic actions at concentrations between 1 nM and 1 μM (31, 56), is 20 times more selective for ER? than ER (31, 56, 57, 58), and does not activate ER-dependent gene expression in the hypothalamus in vivo (58). We did not use higher concentrations of genistein (upper micromolar concentration range) because they inhibit protein tyrosine kinases (59, 60). Our data show that genistein increases eNOS expression in the PVN and decreases the numbers of nNOS-positive neurons in the PVN at all concentrations used. In agreement with our results, E2 increased eNOS promoter activity in cardiac myocytes, and this effect was blocked with a selective ER? antagonist developed by this group (61). These results support our hypothesis that ER? mediates the E2-induced changes in NOS expression in the PVN.
Although neurons of the PVN express ER? and no ER (23, 24, 25), cerebral blood vessels express both ER? and ER in a ratio of 3:1 (33). Therefore, to investigate the possibility that activation of ER alters NOS expression, we used the highly selective ER agonist, PPT (62, 63). Our data show that activation of ER has no effect on either eNOS or nNOS expression in the PVN. We also used the selective ER antagonist, MPP (62, 64), to exclude the possibility that ER contributes to the E2-induced increase in eNOS expression. Our data show that ER is not involved in the E2- or genistein-induced increases in eNOS expression in hypothalamic slice cultures. Interestingly, OVX-E ER-knockout mice apparently do not display altered levels of eNOS expression in cerebral microvessels compared with OVX-V ER-knockout mice (65). However, this study did not account for the finding that ER-knockout mice expressed higher levels of eNOS compared with wild-type mice. Furthermore, developmental differences between ER-knockout mice and wild-type mice may also contribute to the differences between the results of this study and ours.
In conclusion, we show that E2 stimulates increases in eNOS protein expression in the PVN and decreases the numbers of nNOS-positive neurons in the PVN of hypothalamic slice cultures. Furthermore, our time-course data suggest that E2 alters NOS expression via a slow, genomic effect. Finally, our morphological data in combination with our use of a variety of selective ER agonists and antagonists provide strong evidence that ER? is responsible for E2’s effect on NOS expression in the PVN. We previously showed that estrogen treatment attenuates blood pressure responses to psychological stress in OVX rats through brain NO produced by eNOS and/or nNOS (3). Therefore, our current results raise the intriguing possibility that NO produced by eNOS in blood vessels of the PVN via an ER?-dependent mechanism acts on neighboring neurons to influence autonomic pathways originating from the PVN.
Acknowledgments
We thank Dr. Robert Campenot for graciously providing some of the facilities required for these experiments.
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