Department of Pharmacology, College of Medicine, University of California, Irvine, California 92697-4625
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ABSTRACT |
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The cerebral vasculature is an important
target tissue for estrogen, as evidenced by significant effects of
estrogen on vascular reactivity and protein levels of endothelial
nitric oxide synthase and prostacyclin synthase. However, the presence,
localization, and regulation of estrogen receptors in the cerebral
vasculature have not been investigated. In this study, we identified
the presence of estrogen receptor- (ER-
) in female rat cerebral
blood vessels and localized this receptor to both smooth muscle and
endothelial cells by use of immunohistochemistry and confocal
microscopy. With immunoblot analysis, multiple forms of ER-
were
detected at 110, 93, 82, 50, and 45 kDa in addition to a relatively
weak band corresponding to the 66-kDa putative unmodified receptor. The
82-kDa band was identified as Ser118-phosphorylated ER-
,
whereas the 50-kDa band lacks the normal NH2 terminus,
suggestive of an ER-
splice variant. Lower molecular mass bands
persisted after in vivo inhibition of 26S proteasome activity with
lactacystin, whereas the 110- and 93-kDa bands increased. All forms of
ER-
in cerebral vessels were decreased after ovariectomy but
significantly increased after chronic estrogen exposure in vivo.
estrogen receptors; 26S proteasome; 17-estradiol; cerebral
circulation
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INTRODUCTION |
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A GROWING BODY OF
EVIDENCE suggests that estrogen may produce its protective
effects in the cerebral vasculature, in part by modulating the
synthesis and release of endothelial-derived vasodilators. Previous
work in our laboratory demonstrated that, in cerebral blood vessels,
chronic estrogen exposure leads to elevated protein levels of
endothelial nitric oxide synthase (eNOS), cyclooxygenase-1, and
prostacyclin synthase, enzymes that produce the vasodilators nitric
oxide (NO) and prostacyclin (26, 32). In contractile
studies that used rat and mouse cerebral arteries, effects of chronic
estrogen treatment depended entirely on an intact endothelium,
suggesting that estrogen modulates cerebrovascular reactivity through
an action on the endothelium, with no effect on smooth muscle
reactivity per se (9, 10). The effect of estrogen
treatment on levels of vasodilator-producing enzymes in cerebral
arteries is thought to be receptor mediated, because estrogen treatment
of estrogen receptor- (ER-
) knockout mice does not elevate these
proteins (11). In addition, preincubation of female rat
cerebral blood vessels with the ER antagonist ICI-182780 or partial
agonist tamoxifen completely prevents estrogen-dependent increases in
eNOS protein in vitro (27).
Actions of estrogen in target tissues are mediated by two defined
receptor subtypes, ER- and ER-
. These receptors and their respective signal transduction pathways and regulatory mechanisms are
therefore a subject of intense research. ER-
, first cloned in 1986, was believed to mediate all of the physiological effects of estrogen
until 1995, when ER-
was cloned from rat prostate (12, 13, 23,
31). Both subtypes of ER have been detected in vascular tissue
(41). Various estrogen target tissues have been probed for
the presence of ER-
by immunoblot analysis, and most tissues display
the putative 66-kDa receptor. However, additional bands of ER-
immunoreactivity have been detected at molecular masses ranging from 28 to 112 kDa (3, 4, 15, 25, 28, 36, 37). These bands,
distinct from 66 kDa, have been attributed to unique states or splice
variants of ER-
or to proteolysis products, either from proteases or
proteasomal degradation. However, the functional significance of these
multiple bands in any estrogen target tissue is not yet well understood.
The cerebral circulation is regulated by a variety of mechanisms,
including myogenic responses, pH and PCO2,
metabolic factors, and vascular innervation (6). It is
also clear that estrogen treatment has a profound effect on the
function of this vascular bed (34). However, little is
known concerning the presence, localization, and regulation of ERs in
the cerebral vasculature. Because there is evidence that ER- is
important for estrogen modulation of cerebral vascular reactivity, we
used confocal microscopy to characterize the cell-type localization of
ER-
. We also used immunoblot analysis to characterize ER-
proteins in cerebral vessels and assess possible regulation of these
receptors by estrogen treatment.
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METHODS |
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In vivo treatments.
All animal procedures were approved by the Institutional Animal Care
and Use Committee. Fischer 344 female rats (3 mo old, Harlan
Sprague Dawley) were unoperated (INT.F), ovariectomized (OVX), or
ovariectomized and treated with 17-estradiol (OE), as described
previously (32). Rats were anesthetized by intraperitoneal injection of 46 mg/kg ketamine and 4.6 mg/kg xylazine for all surgical
procedures. OE animals received a subcutaneous silicone implant (placed
dorsally at the neck) filled with 17
-estradiol at the time of
ovariectomy. Implants were left in place for 4 wk; animals were then
anesthetized by CO2 and killed by decapitation. Brains and
ovaries were isolated and used immediately or frozen at
80°C. We
have previously demonstrated by this procedure that serum estrogen
levels in OE animals are within the physiological range
(32). Body weights were 184 ± 1 g for OVX and
164 ± 1 g for OE. Uterine weights were 33 ± 2 mg for
OVX and 127 ± 4 mg for OE (P
0.05). In some
cases, OVX and OE rats received a single intraperitoneal injection of 1 mg/kg lactacystin (Affinity Bioreagents) in 0.9% saline vehicle
(17). Animals were euthanized at 2, 6, or 12 h after
injection, as indicated, and cerebral vessels were isolated as
described in the following section. Cerebrovascular proteasome activity
was measured using a kit from BIOMOL.
Cerebral vessel isolation. Pial arteries were dissected from the surface of the brain. Alternatively, blood vessels were isolated from whole brain as described previously (26, 32). Briefly, four brains from each treatment group were pooled, gently homogenized in a Dounce tissue grinder in ice-cold phosphate-buffered saline (PBS), and then centrifuged at 720 g for 5 min at 4°C. The supernatant was discarded, and the pellet was resuspended and washed in cold PBS twice, followed by 720 g centrifugation for 5 min at 4°C. The resuspended pellet was then layered over 16% dextran (35-45 kDa, Sigma) followed by centrifugation at 4,500 g for 20 min at 4°C to pellet the blood vessels. The supernatant was discarded, and the parenchymal tissue layer was resuspended in PBS and layered again over dextran, followed by centrifugation at 4,500 g for 20 min at 4°C. The pellets were combined in cold PBS, and blood vessels were harvested and washed over a 50-µm nylon mesh. This preparation contains both pial and intraparenchymal vessels that, examined with light microscopy, are a mixture of arteries, arterioles, capillaries, veins, and venules.
Confocal microscopy.
Cerebral blood vessels were dissected from the surface of the brain,
cut into small segments, fixed in 3% formaldehyde for 30 min, and
permeabilized using Triton X-100 (0.1%) for 5 min. Vessels were then
incubated for 30 min in 1% bovine serum albumin (BSA)-PBS and then
incubated overnight at 4°C in primary antibody at 1:50 dilution
[rabbit anti-ER- H-184 (Santa Cruz) and mouse anti-eNOS-610296
(Transduction Laboratories)]. Vessels were then washed for 30 min in
PBS (with change in buffer every 10 min) and incubated overnight at
4°C with the secondary antibodies at 10 µg/ml (goat anti-rabbit
Oregon green 488 and goat anti-mouse Texas red, Molecular Probes),
followed by a final wash for 30 min (buffer change every 10 min) in
PBS. Vessels were then laid on slides and covered with mounting medium
(VectaShield, Vector Laboratories), and coverslips were applied. Images
were obtained using a Bio-Rad model 1024 laser scanning confocal
microscope equipped with standard and UV lasers.
Immunoblot analysis.
Blood vessels were glass homogenized at 4°C in lysis buffer (50 mM
-glycerophosphate, 100 µM NaVO3, 2 mM
MgCl2, 1 mM EGTA, 0.5% Triton X-100, 1 mM
DL-dithiothreitol, 20 µM pepstatin, 20 µM leupeptin,
0.1 U/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride) and
incubated on ice for 20 min. Samples were then centrifuged for 10 min
at 4,500 g at 4°C, and the supernatant was collected. Protein concentrations were determined using the bicinchoninic acid
protein assay kit (Pierce, Rockford, IL). Lysates were used immediately
or stored at
80°C. In all immunoblot experiments, equal amounts of
protein [50 µg in 1× SDS sample buffer (Invitrogen), boiled for 4 min] were loaded in each lane of an 8% Tris-glycine gel (Novex) and
separated by SDS-PAGE. Positive controls consisted of recombinant human
ER-
(Panvera) and rat ovary lysate. Molecular masses of
immunoreactive bands were determined by loading biotinylated molecular
mass standards (Bio-Rad). After electrophoresis, protein was
transferred to nitrocellulose membranes (Amersham) and subsequently incubated at 4°C overnight in blocking buffer (0.01 M PBS, 0.1% Tween 20, and 6.5% nonfat dry milk). Membranes were then incubated with primary antibodies (HC-20, H-184, p-Ser118, Santa
Cruz; PA1-308, Affinity BioReagents) at dilutions from 1:500 to
1:200 for 3 h at room temperature, followed by 5× 5-min washes in
0.1% Tween 20-PBS (T-PBS). Blots were then incubated in secondary
antibody [goat-anti-rabbit IgG-horseradish peroxidase (HRP) for H-184,
PA1-308, and HC-20; donkey-anti-goat IgG-HRP for
p-Ser118, Santa Cruz] at 1:10,000 dilution for 1 h at
room temperature. Biotinylated standards were incubated in 1:7,500
streptavidin-HRP (Sigma). Both segments of the membrane were then
washed 5× 5 min in T-PBS. Membranes were then incubated with enhanced
chemiluminescence reagent (Amersham) for 1 min and exposed to Hyperfilm
(Amersham) for 15 s to 10 min, depending on signal strength.
Software for electrophoresis analysis, UN-SCAN-IT (Silk Scientific),
was used for densitometric analysis of immunoreactive bands.
Statistics.
Values for body and uterine weights are means ± SE. Statistical
differences in immunoblot band densities were determined by one-way
ANOVA followed by Dunnett's Multiple Comparison Test (GraphPad Prism
2.0 software) and displayed as a fold difference relative to OVX or
vehicle. In all cases, statistical significance was set at
P 0.05.
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RESULTS |
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Localization of ER- in cerebral blood vessels.
To observe the presence and localization of ER-
in cerebral vessels,
a rabbit polyclonal antibody generated against the first 185 amino
acids of the NH2 terminus of ER-
(H-184) was used to probe small segments of pial vessels isolated from female rat brain.
The nuclear stain 4',6-diamidino-2-phenylindole dihydrochloride was
used to identify smooth muscle cell nuclei (concentric to the vessel
circumference) or endothelial cell nuclei (parallel to direction of
blood flow) (2). With use of an anti-rabbit secondary
antibody conjugated to Oregon green 488 and an anti-mouse secondary
antibody conjugated to Texas red, laser scanning confocal microscopy
revealed intense immunoreactivity for ER-
(green) in smooth muscle
cells (Fig. 1B). No reactivity
for eNOS (red), a marker for endothelial cells, was detected in smooth
muscle (Fig. 1, A and C). When the vessels were
visualized at the level of the endothelium near the internal elastic
lamina, strong immunoreactivity for eNOS (red) was detected (Fig.
1D). Strong ER-
immunofluorescence (green) was detected
in the same focal plane (Fig. 1E). Figure 1F
shows the merged image of Fig. 1, D and E,
revealing strong colocalization of ER-
with eNOS in the endothelial
layer (orange).
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Western blot analysis with various antibodies to ER-.
Two different cerebral vessel preparations were compared by using
immunoblot analysis for ER-
with both COOH-terminal (HC-20) and
NH2-terminal (H-184) antibodies. Pial vessels from freshly isolated brains were quickly dissected and immediately homogenized at
4°C in lysis buffer and compared with lysate from vessels isolated from whole brain with the density centrifugation procedure. When run in
adjacent lanes for comparison, no differences in the location or number
of ER-
immunoreactive bands were observed between surface vessels
isolated by rapid dissection and whole brain vessels isolated by
density centrifugation with either antibody (Fig. 2, COOH-terminal antibody shown). With either vascular
preparation, little to no immunoreactivity was observed at 66 kDa,
which corresponds to the unmodified ER-
. Detection of
immunoreactivity at 66 kDa was found to require loading
50 µg of
total protein, an antibody dilution of 1:100-1:200, and lengthy
film exposure; therefore, this band was not consistently seen. However,
strong immunoreactivity was consistently observed in both preparations
at ~110, 93, 82, and 50 kDa under normal immunoblot conditions.
Protease inhibitors were present in the lysis buffer in both cases, and
immediate boiling/freezing of protein lysates in SDS sample buffer did
not alter the immunoreactive bands detected. Multiple freeze-thaw cycles of recombinant human (r) ER-
also did not show any change in
bands detected (data not shown). Furthermore, as an additional control,
rER-
was added to cerebrovascular protein lysate and run under
SDS-PAGE conditions adjacent to an equal amount of rER-
alone and to
protein lysate without rER-
. Addition of rER-
produced a strong
band at 66 kDa, consistent with the rER-
control, but other bands in
the lysate were unchanged (data not shown). Therefore, we concluded
that the major band detected at ~50 kDa was not due to cleavage of
the native receptor by proteases or to freeze-thawing of protein
lysates.
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Inhibition of the 26S proteasome.
Several in vitro studies have shown that ER- is ubiquitinated and
subsequently degraded by the 26S proteasome (1, 15, 21,
30). To validate this degradation pathway for cerebral vessels
and also to test the hypothesis that the strongly immunoreactive band
at 50 kDa resulted from proteasomal degradation, intact female rats
received a single intraperitoneal injection of the 26S proteasome inhibitor lactacystin (1 mg/kg) and were euthanized 2, 6, or 12 h
later. To verify that in vivo lactacystin treatment inhibited cerebrovascular proteasome activity, an assay for proteasome activity was performed. Cerebrovascular proteasome activity was inhibited by
94% 2 h after intraperitoneal injection of lactacystin
[vehicle = 21,524 ± 212 arbitrary fluorescence units (AFU);
2-h treated = 1,197 ± 39 AFU; n = 3]. As
shown in Fig. 5, immunoreactivity of both
the 93- and 110-kDa bands significantly increased after lactacystin
treatment. This effect was seen within 2 h of lactacystin injection and was maintained, with minimal further increase, at 6 and
12 h. The 110-kDa band displayed a robust sixfold increase, whereas an approximately twofold increase was seen in the
immunoreactive band at 93 kDa. Interestingly, the density of the 82-kDa
band showed only a minor increase, which was not significantly
different from vehicle-treated levels. No increase in immunoreactivity
could be detected for the putative receptor at 66 kDa; any band at this molecular mass was below the limit of detection in all blots from lactacystin-treated vessels. However, a very faint band was detected at
66 kDa in vessels from the vehicle-treated animals. In contrast, the
density of the 50-kDa band was not reduced but was maintained at 6 and
12 h after proteasomal inhibition, indicating that it is not a
degradation product of the 66-kDa ER-
.
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Effect of 17-estradiol exposure on ER-
.
As shown in Fig. 6, lysates from animals
chronically exposed to estrogen (OE and intact females) contained
significantly greater amounts of ER-
immunoreactive protein compared
with OVX. As shown in Fig. 3, A and B, this
difference was seen using antibodies against either the COOH or
NH2 termini in all immunoreactive bands and was not limited
to an effect of estrogen on the putative receptor at 66 kDa. The two
identified bands, 66 and 82 kDa, were quantified. Figure 6A
shows that, in cerebral vessels from both intact females and OE
animals, there is an ~1.5-fold greater level of protein at 66 kDa, as
detected with the NH2-terminal antibody, compared with
vessels from OVX animals. When the antibody specific for the
Ser118-phosphorylated state of ER-
was used, there also
was an ~1.5-fold greater level of immunoreactivity in vessel protein
from animals exposed to estrogen compared with OVX (Fig.
6B).
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DISCUSSION |
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In this study, we have shown that ER- is present in cerebral
blood vessels and is localized to both smooth muscle and the endothelium. Surprisingly, very little unmodified, 66-kDa receptor could be detected in female rat cerebral vessels by use of immunoblot analysis. However, major immunoreactive bands for ER-
were detected at ~45, 50, 82, 93, and 110 kDa. Immunoreactivity at 82 kDa
corresponded to the Ser118-phosphorylated state of ER-
.
The ~50-kDa band is not a degradation product but does lack the
normal ER-
NH2 terminus, suggestive of an ER-
splice
variant (35). Furthermore, we have shown that estrogen
exposure elevates protein levels of all ER-
immunoreactive bands. In
addition, we demonstrate the importance of the 26S proteasome in
mediating degradation of ER-
in cerebral vessels.
The presence of ER- in smooth muscle and endothelial cells of
cerebral vessels was expected, as estrogen has been shown to have
direct effects on the vasculature (27). These effects
include increased protein levels of the enzymes eNOS and prostacyclin synthase (26, 32), which produce the vasodilatory
molecules NO and prostacyclin, respectively. Although not demonstrated
in the cerebral vasculature, estrogen is also known to act on vascular smooth muscle cells to inhibit proliferation (29). The
aforementioned effects of estrogen on vascular tissue are likely the
result of classic nuclear hormone receptor-induced transcriptional
activation of genes, often referred to as "genomic" effects.
However, there is a growing body of evidence suggesting that estrogen
may also exert "nongenomic" effects on vascular tissue, defined as
effects that occur too rapidly to be explained by the genomic mechanism (7, 14, 22, 37-39). These nongenomic effects have
been seen primarily in endothelial cells in culture and include rapid
production and release of NO and activation of the mitogen-activated
protein (MAP) kinase pathway. It is interesting to note that, in all
cases cited, the ER antagonist ICI-182780 and partial agonist tamoxifen blocked these rapid effects, further validating that the effects of
estrogen on vascular tissue are mediated by one of the two previously
characterized estrogen receptors,
or
, or a receptor(s) highly
homologous to ER-
and -
.
Multiple bands of ER- immunoreactivity.
The predicted molecular mass of the cloned unmodified ER-
is 66 kDa
(31). Very little unmodified ER-
was detected in
cerebral blood vessels. Instead, multiple bands of both higher (82, 93, and 110 kDa) and lower (45 and 50 kDa) molecular masses were observed in pial and whole brain vessel preparations. Use of multiple antibodies and specific blocking peptides further indicate that the multiple immunoreactive bands were due to specific ER-
antibody interactions. Unfortunately, many published blots of ER-
do not show the entire molecular mass range; therefore, it is impossible to know whether these
authors have observed, or ignored, some of the bands we have reported.
However, several studies in estrogen target tissues, such as mouse
uterus, rabbit uterine microsomes, breast tumor cells, and mouse
cerebral cortex, have reported ER-
immunoreactive bands at a wide
range of molecular masses, including 45-50, 66, 92-94, and
110-112 kDa (3, 15, 25, 28, 36). Many of these bands
appear at molecular masses identical to those we found in cerebral
blood vessels. Therefore, additional experiments were performed to
attempt to characterize some of the ER-
immunoreactive bands found
in cerebral vessels.
Serine118-phosphorylated ER-.
One band consistently detected by immunoblot analysis in cerebral
vessels runs at an apparent molecular mass of 82 kDa. Our data indicate
that this band corresponds to the Ser118-phosphorylated
state of ER-
. The electrophorectic shift of
Ser118-phosphorylated ER-
appears greater in our blots
than that described previously (18). A possible
explanation is that ER-
may be polyubiquitinated after
phosphorylation, a mechanism that has been described for progesterone
receptors (24). Although increased phosphorylation of
ER-
in response to estradiol is well documented (31),
phosphorylation of Ser118 and subsequent enhancement of
transcription by the activiated AF-1 region were originally reported as
being estrogen independent (31). The literature currently
reflects that both estrogen and activators of the MAP kinase pathway
can lead to phosphorylation of ER-
at Ser118
(31). The mechanism by which estrogen increases
Ser118-phosphorylated ER-
in cerebral vessels is
unknown. However, one possible explanation is that the increase seen in
total ER-
protein levels in the presence of estrogen simply
increases the amount of receptor available for phosphorylation and is
not necessarily due to induction of phosphorylation by estrogen of the
MAP kinase pathway. A similar conclusion has been drawn with respect to
phosphorylation of the androgen receptor (20).
Immunoreactivity at ~50 kDa.
One of the most prominent ER- immunoreactive bands in cerebral
arteries appeared at ~50 kDa. Our positive control, rat ovary lysate,
showed all of the same ER-
immunoreactive bands as in cerebral
vessels, including a dense band at 50 kDa. Other workers, using a
COOH-terminal antibody for ER-
and human umbilical vein endothelial
cells, also showed strong immunoreactivity at 45 kDa and weak
reactivity at 66 kDa, a ratio similar to that seen in our study
(37). In many studies, ER-
immunoreactive bands lower than 66 kDa have been suggested to result from proteolysis. In our
study, we took care to prevent proteolysis by routine use of protease
inhibitors, and we also demonstrated that our method of tissue
isolation did not contribute to the appearance of additional ER-
immunoreactive bands. Our original hypothesis to explain this ~50-kDa
band was that unmodified (66-kDa) ER-
was being cleaved in the
NH2-terminal region by the proteasome, removing the first
~140-170 amino acids. Our data indicate, however, that neither
the strong immunoreactive band at ~50 kDa nor the ~45 kDa band is a
product of proteasomal degradation, as they remained unchanged after
12 h of in vivo proteasome inhibition. Additionally, no
concomitant increase in the 66-kDa band was detected at any measured
time point after lactacystin exposure.
Proteasomal degradation.
Our findings suggest that there is a steady-state degradation of ER-
in cerebral vessels. These results indicate that half-lives of some
forms of ER-
in cerebral vessels may be on the order of ~2-6
h, as maximal or near-maximal increases in both 110- and 93-kDa ER-
immunoreactive bands were seen after 2 or 6 h of proteasome inhibition with lactacystin. However, longer time points may be required to see increases in both the 82- and 50-kDa bands, because in
our study protein levels of these two bands from lactacystin-treated vessels were not significantly different from levels in vehicle-treated controls.
Effect of estrogen on ER-.
A surprising finding in this study is that chronic estrogen exposure
increased ER-
protein levels. All forms of ER-
were decreased by
ovariectomy and increased by estrogen treatment. Although
desensitization and downregulation are common mechanisms involving many
ligand-activated receptors, regulation of nuclear hormone receptors is
not so well understood. For example, androgen receptor (AR) half-life
is stabilized over sixfold in the presence of ligand in AR-transfected
COS cells (20). In female mice, ovariectomy reduced ER-
levels in cortex (3). However, both progesterone receptors
and ER-
have been reported to show a decreased half-life or
downregulation in the presence of ligand (1, 21, 24).
Interestingly, a recent study suggested that, in the absence of
estrogen, ER-
is highly ubiquitinated, offering another explanation for the differences seen in ER-
levels in OVX vs. OE animals in our
study (40). Analysis of Fig. 7 might suggest that estrogen has no effect on levels of ER-
in the absence of proteasomal degradation. However, this experiment was designed to determine only
maximal effects of lactacystin treatment; therefore, further experiments are needed to verify this point. In fact, if estrogen binding stabilizes ER-
, protecting it from degradation, this could
account for the increased ER-
immunoreactivity observed in vessels
from estrogen-treated animals. Most work of this nature has been done
in cell culture, where gene regulation and signal transduction may not
reflect in vivo conditions. Our findings are important, because they
reflect in vivo effects of chronic estrogen exposure on the regulation
and state of ER-
in cerebral blood vessels.
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ACKNOWLEDGEMENTS |
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We thank Jonnie Stevens for technical assistance with surgical procedures and Anabel Martinez and Amin Boroujerdi for help with cerebral vessel isolation.
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FOOTNOTES |
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This work was supported in part by National Heart, Lung, and Blood Institute Grant RO1-HL-50775 and by a grant from the American Heart Association.
Address for reprint requests and other correspondence: D. N. Krause, Dept. of Pharmacology, College of Medicine, Code 4625, Univ. of California, Irvine, CA 92697-4625 (E-mail: dnkrause{at}uci.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
September 11, 2002;10.1152/ajpendo.00165.2002
Received 18 April 2002; accepted in final form 4 September 2002.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alarid, ET,
Bakopoulos N,
and
Solodin N.
Proteasome-mediated proteolysis of estrogen receptor: a novel component in autologous down-regulation.
Mol Endocrinol
13:
1522-1534,
1999
2.
Arribas, SM,
Vila E,
and
McGrath JC.
Impairment of vasodilator function in basilar arteries from aged rats.
Stroke
28:
1812-1820,
1997
3.
Asaithambi, A,
Mukherjee S,
and
Thakur MK.
Expression of 112-kDa estrogen receptor in mouse brain cortex and its autoregulation with age.
Biochem Biophys Res Commun
231:
683-685,
1997[ISI][Medline].
4.
Barraille, P,
Chinestra P,
Bayard F,
and
Faye JC.
Alternative initiation of translation accounts for a 67/45 kDa dimorphism of the human estrogen receptor ER.
Biochem Biophys Res Commun
257:
84-89,
1999[ISI][Medline].
5.
Drummond, AE,
Baillie AJ,
and
Findlay JK.
Ovarian estrogen receptor alpha and beta mRNA expression: impact of development and estrogen.
Mol Cell Endocrinol
149:
153-161,
1999[ISI][Medline].
6.
Edvinsson, L,
and
Krause DN.
Cerebral Blood Flow and Metabolism. Philadelphia, PA: Lippincott, Williams and Wilkins, 2002.
7.
Figtree, GA,
Webb CM,
and
Collins P.
Tamoxifen acutely relaxes coronary arteries by an endothelium-, nitric oxide-, and estrogen receptor-dependent mechanism.
J Pharmacol Exp Ther
295:
519-523,
2000
8.
Flouriot, G,
Brand H,
Denger S,
Metivier R,
Kos M,
Reid G,
Sonntag-Buck V,
and
Gannon F.
Identification of a new isoform of the human estrogen receptor-alpha that is encoded by distinct transcripts and that is able to repress hER- activation function 1.
EMBO J
19:
4688-4700,
2000
9.
Geary, GG,
Krause DN,
and
Duckles SP.
Estrogen reduces myogenic tone through a nitric oxide-dependent mechanism in rat cerebral arteries.
Am J Physiol Heart Circ Physiol
275:
H292-H300,
1998
10.
Geary, GG,
Krause DN,
and
Duckles SP.
Estrogen reduces mouse cerebral artery tone through endothelial NOS- and cyclooxygenase-dependent mechanisms.
Am J Physiol Heart Circ Physiol
279:
H511-H519,
2000
11.
Geary, GG,
McNeill AM,
Ospina JA,
Krause DN,
and
Duckles SP.
Selected contribution: cerebrovascular NOS and cyclooxygenase are unaffected by estrogen in mice lacking estrogen receptor-alpha.
J Appl Physiol
91:
2391-2399,
2001
12.
Green, S,
Walter P,
Kumar V,
Krust A,
Bornert JM,
Argos P,
and
Chambon P.
Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A.
Nature
320:
134-139,
1986[ISI][Medline].
13.
Greene, GL,
Gilna P,
Waterfield M,
Baker A,
Hort Y,
and
Shine J.
Sequence and espression of human estrogen receptor complementary DNA.
Science
231:
1150-1154,
1986[ISI][Medline].
14.
Haynes, MP,
Sinha D,
Russell KS,
Collinge M,
Fulton D,
Morales-Ruiz M,
Sessa WC,
and
Bender JR.
Membrane estrogen receptor engagement activates endothelial nitric oxide synthase via the PI3-kinase-Akt pathway in human endothelial cells.
Circ Res
87:
677-682,
2000
15.
Horigome, T,
Ogata F,
Golding TS,
and
Korach KS.
Estradiol-stimulated proteolytic cleavage of the estrogen receptor in mouse uterus.
Endocrinology
123:
2540-2548,
1988[Abstract].
16.
Iafrati, MD,
Karas RH,
Aronovitz M,
Kim S,
Sullivan TR, Jr,
Lubahn DB,
O'Donnell TF, Jr,
Korach KS,
and
Mendelsohn ME.
Estrogen inhibits the vascular injury response in estrogen receptor alpha-deficient mice.
Nat Med
3:
545-548,
1997[ISI][Medline].
17.
Itoh, M,
Takaoka M,
Shibata A,
Ohkita M,
and
Matsamura Y.
Preventive effect of lactacystin, a selective proteasome inhibitor, on ischemic acute renal failure in rats.
J Pharmacol Exp Ther
298:
501-507,
2001
18.
Joel, PB,
Traish AM,
and
Lannigan DA.
Estradiol-induced phosphorylation of serine 118 in the estrogen receptor is independent of p42/p44 mitogen-activated protein kinase.
J Biol Chem
273:
13317-13323,
1998
19.
Karas, RH,
Hodgin JB,
Kwoun M,
Krege JH,
Aronovitz M,
Mackey W,
Gustafsson JA,
Korach KS,
Smithies O,
and
Mendelsohn ME.
Estrogen inhibits the vascular injury response in estrogen receptor -deficient female mice.
Proc Natl Acad Sci USA
96:
15133-15136,
1999
20.
Kemppainen, JA,
Lane MV,
Sar M,
and
Wilson EM.
Androgen receptor phosphorylation, turnover, nuclear transport, and transcriptional activation.
J Biol Chem
267:
968-974,
1992
21.
Khissiin, AE,
and
Leclerq G.
Implication of proteasome in estrogen receptor degradation.
FEBS Lett
448:
160-166,
1999[ISI][Medline].
22.
Kim, HP,
Lee JY,
Jeong JK,
Bae SW,
Lee HK,
and
Jo I.
Nongenomic stimulation of nitric oxide release by estrogen is mediated by estrogen receptor localized in caveolae.
Biochem Biophys Res Commun
263:
257-262,
1999[ISI][Medline].
23.
Kuiper, GG,
Enmark E,
Pelto-Huikko M,
Nilsson S,
and
Gustafsson JA.
Cloning of a novel receptor expressed in rat prostate and ovary.
Proc Natl Acad Sci USA
93:
5925-5930,
1996
24.
Lange, CA,
Shen T,
and
Horwitz KB.
Phosphorylation of human progesterone receptors at serine-294 by mitogen-activated protein kinase signals their degradation by the 26S proteasome.
Proc Natl Acad Sci USA
97:
1032-1037,
2000
25.
Maaroufi, Y,
Lacroix M,
Lespagnard L,
Journe F,
Larsimont D,
and
Leclercq G.
Estrogen receptor of primary breast cancers: evidence for intracellular proteolysis.
Breast Cancer Res
2:
444-454,
2000[ISI][Medline].
26.
McNeill, AM,
Kim N,
Duckles SP,
and
Krause DN.
Chronic estrogen treatment increases levels of endothelial nitric oxide synthase protein in rat cerebral microvessels.
Stroke
30:
2186-2190,
1999
27.
McNeill, AM,
Zhang C,
Stanczyk FZ,
Duckles SP,
and
Krause DN.
Estrogen increases endothelial nitric oxide synthase via estrogen receptors in rat cerebral blood vessels: effect preserved after concurrent treatment with medroxyprogesterone acetate or progesterone.
Stroke
33:
1685-1691,
2002
28.
Monje, P,
and
Boland R.
Characterization of membrane estrogen binding proteins from rabbit uterus.
Mol Cell Endocrinol
147:
75-84,
1999[ISI][Medline].
29.
Morey, AK,
Pedram A,
Razandi M,
Prins BA,
Hu RM,
Biesiada E,
and
Levin ER.
Estrogen and progesterone inhibit vascular smooth muscle proliferation.
Endocrinology
138:
3330-3339,
1997
30.
Nawaz, Z,
Lonard DM,
Dennis AP,
Smith CL,
and
O'Malley BW.
Proteasome-dependent degradation of the human estrogen receptor.
Proc Natl Acad Sci USA
96:
1858-1862,
1999
31.
Nilsson, S,
Makela S,
Treuter E,
Tujague M,
Thomsen J,
Andersson G,
Enmark E,
Pettersson K,
Warner M,
and
Gustafsson JA.
Mechanisms of estrogen action.
Physiol Rev
81:
1535-1565,
2001
32.
Ospina, JA,
Krause DN,
and
Duckles SP.
17-Estradiol increases rat cerebrovascular prostacyclin synthesis by elevating cyclooxygenase-1 and prostacyclin synthase.
Stroke
33:
600-605,
2002
33.
Pasqualini, C,
Guivarc'h D,
Barnier JV,
Guibert B,
Vincent JD,
and
Vernier P.
Differential subcellular distribution and transcriptional activity of E3,
E4,
E3-4 isoforms of the rat estrogen receptor-
.
Mol Endocrinol
15:
894-908,
2001
34.
Pelligrino, DA,
and
Galea E.
Estrogen and cerebrovascular physiology and pathophysiology.
Jpn J Pharmacol
86:
137-158,
2001[ISI][Medline].
35.
Pendaries, C,
Darblade B,
Krust A,
Chambon P,
Korach KS,
Bayard F,
and
Arnal JF.
The AF-1 activation-function of ER may be dispensable to mediate the effect of estradiol on endothelial NO production in mice.
Proc Natl Acad Sci USA
99:
2205-2210,
2002
36.
Powell, CE,
Soto AM,
and
Sonnenschein C.
Identification and characterization of membrane estrogen receptor from MCF-7 estrogen-target cells.
J Ster Biochem Mol Biol
77:
97-108,
2001[ISI][Medline].
37.
Russell, KS,
Haynes MP,
Sinha D,
Clerisme E,
and
Bender JR.
Human vascular endothelial cells contain membrane binding sites for estradiol, which mediate rapid intracellular signaling.
Proc Natl Acad Sci USA
97:
5930-5935,
2000
38.
Russell, KS,
Haynes MP,
Caulin-Glaser T,
Rosneck J,
Sessa WC,
and
Bender JR.
Estrogen stimulates heat shock protein 90 binding to endothelial nitric oxide synthase in human vascular endothelial cells.
J Biol Chem
275:
5026-5030,
2000
39.
Stefano, GB,
Prevot V,
Beauvillain JC,
Cadet P,
Fimiani C,
Welters I,
Fricchione GL,
Breton C,
Lassalle P,
Salzet M,
and
Bilfinger TV.
Cell-surface estrogen receptors mediate calcium-dependent nitric oxide release in human endothelia.
Circulation
101:
1594-1597,
1999
40.
Wijayaratne, AL,
and
McDonnell DP.
The human estrogen receptor- is a ubiquitinated protein whose stability is affected differentially by agonists, antagonists, and selective estrogen receptor modulators.
J Biol Chem
276:
35684-35692,
2001
41.
Zhang, L,
Fishman MC,
and
Huang PL.
Estrogen mediates the protective effects of pregnancy and chorionic gonadotropin in a mouse model of vascular injury.
Arterioscler Thromb Vasc Biol
19:
2059-2065,
1999