| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
ARTICLES |
Institute of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy
Address all correspondence and requests for reprints to: Lina Puglisi, Institute of Pharmacological Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy. E-mail: Lina.Puglisi{at}unimi.it
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and
ERß are expressed in rat aorta SMC grown in culture; 2) low
concentrations of hormone modulate NOS II activity; 3) the specific
ER antagonist ICI182,780 completely blocks 17ß-estradiol effect.
On the other hand, progesterone is deprived of any effect on NOS II
content or activity, proving the specificity of 17ß-estradiol effect.
In addition, we show that 17ß-estradiol can counteract the increase
in NOS II activity following cytokine treatment. The observation could
indicate a novel mechanism for the protective effects exerted by these
hormones in cardiovascular diseases and atherosclerosis in particular. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
(ER) has been demonstrated both in
vascular smooth muscle and in endothelial cells (13, 14, 15). It is now
known that the two ERs so far described (named ER and ERß) (16)
are hormone-dependent transcription factors that modulate the
transcription of selected genes by binding to consensus regulatory
sequences, referred to as EREs (estrogen responsive elements) located
in the promoter of the target genes (17). Recent studies indicate that 17ß-estradiol up-regulates nitric oxide synthase (NOS) III messenger RNA (mRNA) in cultured endothelial cells (18, 19, 20), and this may represent a possible mechanism for the protective role of estrogens on the vessel wall. NO is the most potent endogenous vasodilator. In the vessel wall, its synthesis is regulated by two major types of NOS: a constitutive form (cNOS, NOS III), normally expressed in endothelial cells, and an inducible form (iNOS, NOS II), mostly expressed in smooth muscle cells after exposure to inflammatory stimuli (21, 22, 23). Excessive NOS II production induced by proinflammatory stimuli (cytokines and lipopolysaccharides) has been associated with atherosclerosis, whereas reduced NO synthesis due to impaired NOS III activity may be responsible for hypertension and vascular pathologies (21, 24).
The aim of the present study was to investigate the effects of 17ß-estradiol and progesterone on NOS II protein content and activity in primary cultures of smooth muscle cells from rat aorta. The results here presented demonstrate that 17ß-estradiol, but not progesterone, decreases NOS II protein content and activity, suggesting that estrogens may contribute to an anti-inflammatory action in these cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-estradiol and progesterone,
as well as the antiestrogen tamoxifen were obtained from Sigma Chemical Co. (Milano, Italy). The antiestrogen ICI 182,780 was
kindly provided by Zeneca Pharmaceuticals (Macclesfield,
UK). Antirabbit NOS II polyclonal antibody was purchased from
Transduction Laboratories, Inc. (Lexington, KY).
Oligodeoxynucleotides were synthesized by Pharmacia Biotech (Cambridge, UK).
Cell cultures
Smooth muscle cells (SMC) were obtained from the intimal-medial
layers of aorta of male Sprague Dawley rats (Charles River Laboratories, Inc., Calco, Italy) (200–250 g), according to
Ross (25). Cells were grown in monolayers in medium 199 (M199)
supplemented with 10% (vol/vol) FCS, 50 U/ml penicillin, 50 µg/ml
streptomycin, 2 mM glutamine, and 2.2 g/liter
NaHCO3 (referred to as "M199 + FCS") and were
incubated at 37 C in a humidified atmosphere with 5%
CO2. SMC were characterized for growth and morphology and
by immunostaining with monoclonal antibodies recognizing the actin
-isoform typically expressed by SMC (26). For the experiments here
described, cells grown at passages 2–6 were seeded into either 60-mm
Petri dishes (2 x 105 cells/dish) or 24-well plates
(5 x 104 cells/well). Cells were grown in M199 + FCS
for 48 h; then, the medium was replaced with phenol red-free M199
with 10% FCS (wM199 + FCS). At 16–24 h before the hormonal treatment,
cells were incubated with phenol red-free M199 without serum (wM199).
Pharmacological agents were added for 24 h, as specified in each
figure legend. Typically, the treatments were done with confluent
cells.
RT-PCR
Total cell RNA was isolated from rat uterus, used as a positive
control for ER mRNA expression, and VSMC cells with the
Bio/RNA-X Cell kit (Bio/Gene, Kimbolton Cambridge, UK), using 1 ml of
RNA-X reagent for 1 g of tissue or for 10 x 106
cells. 1 µg of total RNA was denatured at 68 C with 10 pmol
oligo-d(T)12–18 (Perkin Elmer, Milan, Italy)
and reverse-transcribed using MuMLV reverse transcriptase (HT Biotechnology Ltd., Cambridge, UK) as previously described (27).
One-tenth of the complementary DNA (cDNA) reaction was amplified
containing 2.5 U of DynaZyme-DNA polymerase (Finenzyme OY, Espo,
Finland), the buffer provided by Finenzyme, 0.2 mM dNTP and
100 pmol PCR primers in 100 µl final volume. Amplification of ER-
mRNA was obtained as previously described (28). Briefly, the region of
254 bp, from nucleotide (nt) 530 to nt 784, was amplified using as
primers ER-1a (5'-AGCGTGTCGCCGAGTCCG-3') and ER-1ß
(5'-AGCACAGTAGCGAGTCTC-3'). After denaturing at 94 C for 5 min, PCR
amplification was performed for 30 cycles (94 C for 15 sec, 48 C for 20
sec, and 72 C for 30 sec) followed by a final extension step (72 C for
3 min). The duration and temperature of the PCR cycles were
experimentally optimized to fall into the exponential phase of the
amplification (28). At the 30th cycle, the reaction was
terminated by addition of 50 µl of DNA dye (50% glycerol and 0.25%
wt/vol xylene cyanol). Fifteen microliters of the incubation mixture
were then loaded onto a 3% agarose gel, together with the appropriate
molecular weight markers. The bands corresponding to the amplified DNA
were then visualized by ethidium bromide staining.
For amplification of the rat ER-ß, a region of 262 bp, from nt 39 to
nt 301, was amplified by primers ERß-1
(5'-TTCCCGGCAGCACCAGTAACC-3') and ERß-1ß
(5'-TCCCTCTTTGCGTTTGGACTA-3'). After denaturing at 94 C for 5 min, PCR
amplification was performed for 40 cycles (94 C for 1 min, 50 C for 1
min, and 72 C for 1 min). A subsequent Southern blot and hybridization
with a rat ER-ß probe was done to prove the specificity of the
signal. No amplification product was detected in samples in which
either the RT or the cDNA were omitted.
Southern blot
Probe preparation. Plasmid pCMV5-ratER-ß (a gift from
Jan-Ake Gustaffson) was used as template for the preparation of
nonradioactively labeled DNA probe by means of PCR; probe corresponded
to nt 39 to nt 301 of the rat ER-ß. PCR reaction mix contained 15 ng
plasmid DNA, 0.8 mM ERß-1 and ERß-1ß primers, 0.35
mM Dig-11 dUTP (Boehringer Mannheim, Mannheim,
Germany), 0.65 mM dTTP and 1 mM of each dATP,
dCTP dGTP, and 2 U Taq DNA polymerase (Perkin Elmer) in 20 µl final volume. PCR reaction profile was as
follows: 95 C for 5 min, then 30 cycles at 92 C for 1 min, 50 C for 1
min and 72 C for 1 min. The 262-bp amplification product was purified
on a 1% agarose gel in Tris-acetate EDTA.
Electrophoresis, blotting, and hybridization. Ten microliters of PCR mix were loaded on 2% agarose gel in Tris-borate EDTA and subjected to electrophoresis performed at 100 V. The gel was denatured at room temperature for 30 min in 0.5 M NaOH and 1 M NaCl and neutralized in 0.5 M Tris, pH 7.7, 1.5 M NaCl, and 1 mM EDTA for 15 min at room temperature. DNA was transferred onto nylon membrane (Hybond-N, Amersham Pharmacia Biotech, Milan, Italy) by capillarity blotting o/n in 20 x SSC (3 M NaCl, 0.3 M Na3 citrate·2H2O) and then fixed to the membrane by UV irradiation and baking at 80 C for 48 h. After two subsequent prehybridizations of 1 h at 65 C in "pre 1" solution (250 mM Na-posphate buffer pH 7.2, 7% SDS, 1% BSA and 1 mM EDTA) and "pre2" solution (5 x SSC, 50% formamide, 0.2% SDS, 1% Sarcosyl and Blocking reagent (Boehringer Mannheim) dissolved in 0.01 M Maleic acid, 0.015 M NaCl), heat-denatured Dig-11dUTP-labeled probe was annealed at 42 C o/n and washed at room temperature twice in 5 x SSC for 30 min, once in 1 x SSC containing 0.1% SDS for 30 min and finally, twice in 0.1 x SSC with 0.1% SDS for 15 min. The subsequent enhanced chemiluminescent reaction was performed as specified by the manufacturers (Boehringer Mannheim).
Western blot
Confluent cells were incubated for 24 h either with a
cytokine cocktail [interleukin-1ß (IL-1ß) 10 ng/ml,
interferon 
(INF-) 10 ng/ml, tumor necrosis factor
(TNF)- 25 ng/ml and lipopolysaccharides (LPS) 10 µg/ml] as
positive control and/or with steroid and nonsteroid ligands
(17ß-estradiol, progesterone, 17-estradiol, tamoxifen, and ICI
182,780) as described in the figure legends. The incubation was ended
by addition of sample buffer (0.125 M Tris-HCl pH 6.8, 40
mM EDTA, 4.6% SDS, 20% glycerol). Protein concentration
was measured according to Lowry’s method with BSA as a standard (29)
and twenty-eight-microgram proteins were separated on
SDS-denaturating PAGE using discontinuous gradient gels (10–5%) (30).
Proteins were electrophoretically transferred to nitrocellulose
membranes. Immunoblotting was performed with the polyclonal antirabbit
NOS II antibody (1:1000 dilution in BSA 5%, Transduction Laboratories, Inc.) and with the secondary peroxidase conjugated
goat antirabbit antibody (1:2000 dilution in BSA 5%, Bio-Rad Laboratories, Inc.), which was then revealed by enhanced
chemiluminescence (ECL, Amersham). Antibody specificity
was assayed by testing extracts from cytokine-stimulated rat peritoneal
macrophages. To compare the NOS II content in the experimental samples,
scanning densitometry was performed; care was taken to ensure that the
OD of all the bands considered were in the range of linearity
previously assessed.
Nitrite assay
Confluent cells were stimulated for 24 h with the cytokines
cocktail in the presence or absence of either 17ß-estradiol or
progesterone at different concentrations. Nitrite production was
determined in the cell culture supernatants by the Griess’ reaction
(31). Briefly, 200 µl of the supernatant were placed in each well of
a 96-well plate. Subsequently, 20 µl of 6.5 M
hydrochloric acid and 20 µl of 37.5 mM sulfanilic acid
were added. After incubation for 10 min at room temperature, 20 µl of
12.5 mM N(1-napthyl)ethylendiamine was added.
Thirty minutes later, the OD was read at 550 nm and compared with a
standard curve which showed a linearity range between 0.045 and 2.0
µmol/mg protein. Protein content was determined on cell extracts by
the Bradford’s method (32) and used to normalize the nitrite
values.
Statistical analysis
For NOS II protein accumulation, values are expressed as
densitometric units. Data are obtained from three different
experiments, and each value represents the mean ± SD.
In experiments in which NOS II activity was measured, statistical
analysis was performed with one-way ANOVA, followed by Fisher’s test.
Each value represents the mean ± SEM of three
different experiments (run in triplicate).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, lane 1). However, these cells express
a high concentration of NOS II protein in response to inflammatory
stimuli, such as 24 h treatment with the cytokine cocktail (10
ng/ml interleukin-1ß, 10 ng/ml interferon , 25 ng/ml tumor
necrosis factor , and 10 µg/ml lipopolysaccharides),
which leads to an increase in NOS immunoreactivity of about 25–70 fold
with respect to controls. Interestingly, when SMC were grown in the
absence of phenol red and serum (wM199), NOS II content was
significantly increased (of about 50%) with respect to
cytokine-stimulated cells (Fig. 1B, compare lanes 2 and 3). To verify
whether this increase was due to the well described estrogen-like
activity of phenol red, we tested the effect of estrogen addition. Fig. 1B shows that the presence of 17ß-estradiol at concentration as low
as 0.01 nM is sufficient to reduce the NOS II protein
content to the levels measured in complete medium (M199 + FCS). The
effect is maintained at 1 nM 17ß-estradiol.
|
Analysis of estrogen receptor mRNA content in SMC
To prove that the SMC grown in vitro were expressing
the estrogen receptor isoforms and ß, total RNA from cells at the
4th and 9th passage of growth was
retrotranscribed and amplified by PCR with specific primer sets. Fig. 2A shows that cells at the
4th passage express ER mRNA (lane 2), while, at the
conditions used in this assay, no amplification product was observed
from cells grown at the 9th passage (lane 3). In another
series of experiments, we could demonstrate that in SMC grown in
vitro the expression of ER decreases progressively with time in
culture and, by the 8th passage, it falls below the
detection limits of our assay. As a consequence, in our experiments we
used only cells between the 2nd and 6th passage
in culture (data not shown).
|
shows the
autoradiography of RT-PCR and Southern blot for ER-ß; a specific
amplification signal of the expected size of 262 bp is detected in SMC
cDNA (lane 4), whereas a control sample, in which the reverse
transcriptase was omitted from the cDNA reaction, did not show any
amplification product, suggesting that no DNA contamination was present
in any step of the experimental procedure (lane 5).
NOS II content in SMC is controlled by the state of activation of
ER
To verify whether activation of the ER was mediating the
hormone-dependent decrease in NOS II content previously described, we
investigated the effect of two antagonists of ER, ICI 182,780
and tamoxifen, which block the estrogen-induced transcriptional
activity of the receptor. Figure 3 shows
that in cells grown in medium without estrogen (phenol red-free medium,
wM199) NOS II protein immunoreactivity is relatively high, and
treatment with 0.01 nM 17ß-estradiol reduces its levels
of about 90%. A dose of 10 nM tamoxifen was able to
block 17ß-estradiol effect, leading NOS II protein content to a level
very close to that of cells grown in wM199 alone. Interestingly, in the
presence of ICI 182,780 the levels of NOS II protein were even higher
than in wM199 alone. This observation is not reconcilable with a
blockade of the effect of estrogen-like compounds, which might still be
present in wM199, because in all of our experiments we failed to see
any effect of ICI 182,780 or tamoxifen alone (data not shown). The
double band recognized by NOS II antibody, in samples where NOS is
present at high concentrations, could result from the recognition of
heterogeneous NOS subunits, as reported by other authors (33).
|
).
|
, when nitrites were measured in
unstimulated cells, the treatment with estradiol did not have any
significant consequence; we believe that this is principally due to the
lack of sensitivity of the methodology used. In fact, we did not detect
any difference in NO production even in cells grown in M199 + FCS or in
wM199, whereas the NOS enzyme levels were significantly different (see
results shown in Fig. 1, lanes 1 and 3). Estradiol, however, at
concentrations as low as 0.01 nM, significantly diminished
the nitrites in the medium of cytokine-stimulated cells. This
observation clearly suggests that estradiol significantly reduces the
extent of cytokine-induced production of NO. Consistent with our
previously data, progesterone was capable of decreasing the nitrite
content in the medium only at pharmacological concentrations.
|
shows that estradiol at nanomolar
concentrations significantly reduced the cytokine-induction of NOS II
protein, thus confirming the results obtained with the nitrite
assay.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and ER-ß are
both expressed in these cells indicate that the effect here reported
might be due to the activation of either receptors. Previous studies
proved that estradiol increases NOS III mRNA and activity in vascular
endothelial cells (19, 20) and in a variety of other tissues (34). In
the present study, for the first time in a well defined cell system, we
provide evidence of an effect of estradiol on NOS II. These results are
in agreement with in vivo studies by Kauser et
al. (35). On the other hand, ex vivo studies by Binko
(36) and Kauser (37), have shown contrasting results in NOS II protein
following estradiol treatment of endothelium-denuded aortic rings. The
apparent discrepancies might be related to the different protocols of
NOS II stimulation and hormone concentration used. However, it is worth
to underline that in the above studies the hormonal effect on NOS II
protein levels were evaluated in the presence of endothelial cells.
Because it is well known that these latter cells are sensitive to
estrogen action, their initial presence in the preparation could
influence the SMC response.
Unlike the other forms of NOS so far described (NOS I and
NOS III) (23), NOS II is considered as the high throughput path
for NO production, which is reserved for mechanisms involving host
defense, like infection and inflammation. The production of NOS II by
SMC may play a role in the formation of the atherosclerotic plaque
dependent on sustained inflammatory reactions occurring within
the vessel wall. The estrogen-mediated prevention of cytokine-induced
production of NO and NOS II by SMC shown in Figs. 5 and 6 suggests the
potential for an antiinflammatory role played by this hormone, which
might be of great interest also with regard to a possible protective
role exerted by estrogen in other pathologies, such as multiple
sclerosis (38) and Alzheimer’s disease (39, 40, 41), in which immune
responses play a relevant role. Interestingly, recent studies carried
out in macrophages, and our unpublished observations in microglial
cells, demonstrated that progesterone (42) and estradiol (43) decrease
NO production following cytokine stimulation.
The question as to how estrogen negatively regulates NOS II is still
open. It is known that NOS II can be transcriptionally regulated, at
least in rodent cells (23). Studies with IL-1ß in rat vascular smooth
muscle cells (44) and with IL-1ß or cAMP-elevating agents (45) in rat
glomerular cells prove that NOS II transcriptional control is not
restricted to a single cell type or regulator. The complexity of NOS II
promoter suggests that a substantial number of factors may participate
in its regulation. The fact that we did not identify any consensus
binding site for the ER does not eliminate the possibility of a direct
interaction between ER and NOS II promoter. By interacting with other
factors, such as AP-1 or NF-kB components, estrogen could directly
influence NOS II mRNA synthesis (46). It has been shown that
NF-
B-mediated induction of the IL-6 promoter, which does not contain
any ERE consensus sequence, can be blocked by estrogen through the
interaction of the ER with members of the NF-B family of
transcription factors (47, 48, 49).
On the other hand, our study does not exclude the possibility of a posttranscriptional control of NOS II mRNA or protein; several reports proved that NOS II can be regulated by mRNA destabilization or by posttranslational mechanisms also in rat SMC (50).
In conclusion, our study might suggest that estradiol exerts an antiinflammatory activity in rat SMC through a decreased NO production. This effect is not observed with progesterone. Thus, the negative regulation of NOS II enzyme may represent another mechanism associated with the beneficial effects of estrogen in cardiovascular disorders.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received August 17, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B/Rel in induction of nitric
oxide synthase. J Biol Chem 269:4705–4708B. J Biol Chem 271:6039–6044This article has been cited by other articles:
 |
|
 |
|
  A. Cignarella, C. Bolego, V. Pelosi, C. Meda, A. Krust, C. Pinna, R. M. Gaion, E. Vegeto, and A. Maggi Distinct Roles of Estrogen Receptor-{alpha} and {beta} in the Modulation of Vascular Inducible Nitric-Oxide Synthase in Diabetes J. Pharmacol. Exp. Ther., January 1, 2009; 328(1): 174 - 182. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W.-H. Kan, J.-T. Hsu, Z.-F. Ba, M. G. Schwacha, J. Chen, M. A. Choudhry, K. I. Bland, and I. H. Chaudry p38 MAPK-dependent eNOS upregulation is critical for 17{beta}-estradiol-mediated cardioprotection following trauma-hemorrhage Am J Physiol Heart Circ Physiol, June 1, 2008; 294(6): H2627 - H2636. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Ohtani, A. Garcia, A. B. Rogers, Z. Ge, N. S. Taylor, S. Xu, K. Watanabe, R. P. Marini, M. T. Whary, T. C. Wang, et al. Protective role of 17 -estradiol against the development of Helicobacter pylori-induced gastric cancer in INS-GAS mice Carcinogenesis, December 1, 2007; 28(12): 2597 - 2604. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Tenenbaum, A. N. Azab, and J. Kaplanski Effects of estrogen against LPS-induced inflammation and toxicity in primary rat glial and neuronal cultures Innate Immunity, June 1, 2007; 13(3): 158 - 166. [Abstract] [PDF]
|
|
|
|
|
|
C. Pinna, C. Bolego, P. Sanvito, V. Pelosi, R. Baetta, A. Corsini, R. M. Gaion, and A. Cignarella Raloxifene Elicits Combined Rapid Vasorelaxation and Long-Term Anti-Inflammatory Actions in Rat Aorta J. Pharmacol. Exp. Ther., December 1, 2006; 319(3): 1444 - 1451. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Bolego, E. Vegeto, C. Pinna, A. Maggi, and A. Cignarella Selective Agonists of Estrogen Receptor Isoforms: New Perspectives for Cardiovascular Disease Arterioscler Thromb Vasc Biol, October 1, 2006; 26(10): 2192 - 2199. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Noguchi, T. Sato, M. Hirata, T. Hara, K. Ohama, and A. Ito Identification and Characterization of Extracellular Matrix Metalloproteinase Inducer in Human Endometrium during the Menstrual Cycle in Vivo and in Vitro J. Clin. Endocrinol. Metab., December 1, 2003; 88(12): 6063 - 6072. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Maggi, A. Cignarella, A. Brusadelli, C. Bolego, C. Pinna, and L. Puglisi Diabetes Undermines Estrogen Control of Inducible Nitric Oxide Synthase Function in Rat Aortic Smooth Muscle Cells Through Overexpression of Estrogen Receptor-{beta} Circulation, July 15, 2003; 108(2): 211 - 217. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Cuzzocrea, E. Mazzon, L. Dugo, T. Genovese, R. Di Paola, Z. Ruggeri, E. Vegeto, A. P. Caputi, F. A. J. Van de Loo, D. Puzzolo, et al. Inducible Nitric Oxide Synthase Mediates Bone Loss in Ovariectomized Mice Endocrinology, March 1, 2003; 144(3): 1098 - 1107. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Mershon, R. S. Baker, and K. E. Clark Estrogen increases iNOS expression in the ovine coronary artery Am J Physiol Heart Circ Physiol, September 1, 2002; 283(3): H1169 - H1180. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Pfeilschifter, R. Koditz, M. Pfohl, and H. Schatz Changes in Proinflammatory Cytokine Activity after Menopause Endocr. Rev., February 1, 2002; 23(1): 90 - 119. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Tolbert, J. A. Thompson, P. Bouchard, and S. Oparil Estrogen-Induced Vasoprotection Is Independent of Inducible Nitric Oxide Synthase Expression: Evidence From the Mouse Carotid Artery Ligation Model Circulation, November 27, 2001; 104(22): 2740 - 2745. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. K. Dubey and E. K. Jackson Estrogen-induced cardiorenal protection: potential cellular, biochemical, and molecular mechanisms Am J Physiol Renal Physiol, March 1, 2001; 280(3): F365 - F388. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. T. Littleton-Kearney, D. M. Agnew, R. J. Traystman, and P. D. Hurn Effects of estrogen on cerebral blood flow and pial microvasculature in rabbits Am J Physiol Heart Circ Physiol, September 1, 2000; 279(3): H1208 - H1214. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Al-Hijji, B. Larsson, and S. Batra Nitric Oxide Synthase in the Rabbit Uterus and Vagina: Hormonal Regulation and Functional Significance Biol Reprod, May 1, 2000; 62(5): 1387 - 1392. [Abstract] [Full Text]
|
|
|
|
 |
|
 S. Zervou, L.D. Klentzeris, and R.W. Old Nitric oxide synthase expression and steroid regulation in the uterus of women with menorrhagia Mol. Hum. Reprod., November 1, 1999; 5(11): 1048 - 1054. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
