Estradiol increases urethral tone through the local inhibition of neuronal nitric oxide synthase expression.
ABSTRACT Estrogens are known to modulate lower urinary tract (LUT) trophicity and neuronal nitric oxide synthase (nNOS) expression in several organs. The aim of this study was to explore the effects of endogenous and supraestrus levels of 17beta-estradiol (E2) on LUT and urethral nNOS expression and function. LUT function and histology and urethral nNOS expression were studied in adult female mice subjected either to sham surgery, surgical castration, or castration plus chronic E2 supplementation (80 microg.kg(-1).day(-1), i.e., pregnancy level). The micturition pattern was profoundly altered by long-term supraestrus levels of E2 with decreased frequency paralleled by increased residual volumes higher than those of ovariectomized mice. Urethral resistance was increased twofold in E2-treated mice, with no structural changes in urethra, supporting a pure tonic mechanism. Acute nNOS inhibition by 7-nitroindazole decreased frequency and increased residual volumes in ovariectomized mice but had no additive effect on the micturition pattern of long-term supraestrus mice, showing that long-term supraestrus E2 levels and acute inhibition of nNOS activity had similar functional effects. Finally, E2 decreased urethral nNOS expression in ovariectomized mice. Long-term supraestrus levels of E2 increased urethral tone through inhibition of nNOS expression, whereas physiological levels of E2 had no effect.
- [show abstract] [hide abstract]
ABSTRACT: Selective estrogen receptor modulators (SERMs) include a relatively large number of compounds, each with different profiles of estrogenic/antiestrogenic actions on the genital tract. The aim of this review was to systematically evaluate all the available data from randomized, controlled studies on the effects of these compounds on pelvic organ prolapse and urinary incontinence. Literature searches were performed using three computerized databases to identify the result of all randomized, controlled trials performed with SERMs having any effects on pelvic floor as an outcome. A manual search was performed on all related articles. We have identified only one randomized, placebo-controlled trial specifically designed to assess the effect of raloxifene and tamoxifen on the urogenital tract. Most of the data on genitourinary effects of various compounds derive from either questionnaires or adverse events reported during phase III clinical trials. Both tamoxifen and raloxifene appear to increase the incidence of pelvic floor prolapse in one trial, although this was not apparent from the licensing studies data for either of the drugs. Raloxifene does not appear to increase the incidence of urinary incontinence. Levormeloxifene and idoxifene, on the contrary, were noted to increase uterine prolapse and incontinence during phase III trials that prematurely terminated. No data are available on the genitourinary effect of toremifene and on the newer SERMs currently undergoing phase III trials: basedoxifene, lasofoxifene, and arzoxifene. Contrary to their effects in bone, SERMs do not have a class-specific effect on the genitourinary tract. In fact, compounds that are more estrogenic on the uterus such as levormeloxifene and idoxifene also increase the risk of prolapse and incontinence. SERMs can adversely affect the pelvic floor and incontinence but data from urodynamic studies are not yet available. Data on prolapse are contradictory. Given the increased incidence of prolapse and incontinence observed in several licensing trials, more focused research on the effect of these molecules on pelvic floor function is needed.Climacteric 10/2005; 8(3):214-20. · 1.96 Impact Factor
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ABSTRACT: Estrogen is known to have important effects on both reproductive and non-reproductive tissues. Moreover, there is increasing interest in developing compounds that may have selective effects on bone versus reproductive tissues. Since mouse models are often used in these studies, we administrated increasing doses of estradiol (E2) (0 to 500 microg/kg/day) by slow release pellets to ovariectomized 6-month-old C57BL/6 mice and assessed skeletal and uterine responses following 2 months of treatment. The mice lost bone at multiple sites following ovariectomy (OVX); however, while the lowest E2 dose of 5 microg/kg/day completely prevented loss of cancellous bone (at the lumbar spine and tibial metaphysis), it had no stimulatory effects on the uterus. Higher doses of E2 resulted in further increases in bone mineral density, with eventual stimulation of the uterus at a dose of 40 microg/kg/day. By contrast, when 3-month-old C57BL/6 mice were administered the same doses of E2 and studied after 1 month, the 5 microg/kg/day dose resulted in uterine hypertropy, but was not able to prevent loss of cancellous bone. Thus these results (i) provide data on the dose-response for the effects of E2 on mouse bone and (ii) indicate that the relative effects of E2 on bone versus the uterus are highly dependent on the particular experimental conditions used. This issue needs to be considered in evaluating agents with potential 'selective' effects on bone versus reproductive tissues.European Journal of Endocrinology 11/2004; 151(4):503-10. · 3.14 Impact Factor
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ABSTRACT: Estrogen depletion markedly exacerbates hypertension in female congenic mRen2. Lewis rats, a model of tissue renin overexpression. Because estrogen influences nitric oxide synthase (NOS) and NO may exert differential effects on blood pressure, the present study investigated the functional expression of NOS isoforms in the kidney of ovariectomized (OVX) mRen2. Lewis rats. OVX-mRen2. Lewis exhibited an increase in systolic blood pressure (SBP) of 171 +/- 5 vs. 141 +/- 7 mmHg (P < 0.01) for intact littermates. Renal cortical mRNA and protein levels for endothelial NOS (eNOS) were reduced 50-60% (P < 0.05) and negatively correlated with blood pressure. In contrast, cortical neuronal NOS (nNOS) mRNA and protein levels increased 100 to 300% (P < 0.05). In the OVX kidney, nNOS immunostaining was more evident in the macula densa, cortical tubules, and the medullary collecting ducts compared with the intact group. To determine whether the increase in renal nNOS expression constitutes a compensatory response to the reduction in renal eNOS, we treated both intact and OVX mRen2. Lewis rats with the selective nNOS inhibitor L-VNIO from 11 to 15 wk of age. The nNOS inhibitor reduced blood pressure in the OVX group (185 +/- 3 vs. 151 +/- 8 mmHg, P < 0.05), but pressure was not altered in the intact group (146 +/- 4 vs. 151 +/- 4 mmHg). In summary, exacerbation of blood pressure in the OVX mRen2. Lewis rats was associated with the discoordinate regulation of renal NOS isoforms. Estrogen sensitivity in this congenic strain may involve the influence of NO through the regulation of both eNOS and nNOS.AJP Regulatory Integrative and Comparative Physiology 02/2007; 292(2):R819-26. · 3.28 Impact Factor
ESTRADIOL INCREASES URETHRAL TONE THROUGH THE LOCAL INHIBITION
OF NEURONAL NITRIC OXIDE SYNTHASE EXPRESSION
Xavier Gamé, Julien Allard *, Ghislaine Escourrou $, Pierre Gourdy*, Ivan Tack *, Pascal
Rischmann, Jean-François Arnal *, Bernard Malavaud
Service d’ Urologie, Transplantation Rénale et Andrologie, CHU Rangueil
* : INSERM U858 et Laboratoire de Physiologie, CHU Rangueil
$ : Laboratoire D’ Anatomie Pathologique, CHU Rangueil
Toulouse , France
Address for correspondence: Dr Xavier Gamé, Service d’ Urologie, Andrologie et
Transplantation Rénale, CHU Rangueil, TSA 50032, 31059 Toulouse, France
Phone: +335 61 323 229
Fax: + 335 61 323 230
Word count : 3636
Keywords: estrogen, neurourology, nitric oxide synthase, urethra
Page 1 of 26
Articles in PresS. Am J Physiol Regul Integr Comp Physiol (January 9, 2008). doi:10.1152/ajpregu.00467.2007
Copyright © 2008 by the American Physiological Society.
Objectives: Estrogens are known to modulate lower urinary tract (LUT) trophicity as well as
neuronal NO synthase (nNOS) expression in several organs. The aim of this study was to
explore the impact of endogenous and of supraestrus levels of estradiol (E2) on LUT and
urethral nNOS expression and function.
Material and methods: LUT function and histology and urethral nNOS expression were
studied in adult female mice subjected either to sham surgery, surgical castration or castration
plus chronic E2 supplementation (80 µg.kg-1.day-1, i.e. pregnancy level).
Results: The micturition pattern was profoundly altered by long-term supraestrus levels of E2
with decreased frequency paralleled by increased residual volumes higher as compared to
ovariectomized mice. Urethral resistance was increased two-fold in E2-treated mice, with no
structural changes in urethra supporting a pure tonic mechanism.
Acute nNOS inhibition by 7-nitroindazole decreased frequency and increased residual
volumes in ovariectomized mice but had no additive effect on the micturition pattern of long-
term supraestrus mice, showing that long-term supraestrus E2 levels and acute inhibition of
nNOS activity had similar functional effects. Finally, E2 decreased urethral nNOS expression
in ovariectomized mice.
Conclusion: Long-term supraestrus levels of E2 increased urethral tone through inhibition of
nNOS expression whereas physiological levels of E2 had no effect.
Page 2 of 26
Up to 50 % of women are reported to suffer from urinary incontinence or overactive bladder
(Norton and Brubaker 2006). Lower urinary tract function is intimately interrelated to
physiological estradiol variations as illustrated by the transitory increase in urethral pressure
at the peak of estradiol secretion at midcycle (Van Geelen et al. 1981), its gradual increase
during pregnancy (Iosif et al. 1980) or the prevalence of urinary bothers after menopause
(Cardozo et al. 1998).
In woman, estrogen therapy has been shown to prevent post-menopausal cystitis (Cardozo et
al. 2001), urinary atrophy and overactive bladder (Cardozo et al. 1998). Moreover, clinical
data suggest that estrogens partly improve lower urinary tract functioning by improving local
trophicity (Hextall and Cardozo 2001). However, regarding urinary incontinence, estrogens
either failed to show any impact on stress urinary incontinence (Robinson and Cardozo 2003)
or actually increased in a large multicentric study the incidence and severity of all types of
urinary incontinence (Hendrix et al. 2005).
The intricate relationship between urine storage and micturition involves a reciprocal balance
in the muscle tone of bladder and urethra, which are under spinal and supraspinal controls.
Autonomic regulation of the lower urinary tract physiology is driven by all three components
of the autonomic nervous system. Nitric Oxide (NO), the key neurotransmitter of the
nonadrenergic, noncholinergic nerves of the peripheral nervous system, is produced by the
neuronal isoform of NO synthase (nNOS). In the lower urinary tract, nNOS is mainly
expressed in nerves located in the muscular and inner lamina propria layers of the urethral
wall and only sparsely present in the detrusor muscle (Burnett et al. 1997; Ho et al. 1999). In
line with the ubiquitous relaxing effect of NO (Moncada et al. 1991), variations in local NO
production are suspected to play a physiologic role in urethral sphincter relaxation during
micturition (Mamas et al. 2003).
Page 3 of 26
Because, estrogens are known to modulate the expression of nNOS in several target organs,
such as the hypothalamus (Warembourg et al. 1999) and genital tract (Traish et al. 2003), we
explored the impact of supraestrus levels of estradiol on LUT function and morphology and
urethral nNOS expression, as well as the consequences of acute inhibition of nNOS activity
by 7-nitro indazole.
Page 4 of 26
Material and Methods
Female C57/Bl6 mice (Charles Rivers, Les Oncins, France) were housed in stainless steel
cages in a temperature-controlled facility on a 12-hour light-dark cycle and fed normal
laboratory mouse chow diet. All experiments were conducted in conformity with the guiding
principles in the care and use of laboratory animals published by the US National Institutes of
Health (NIH Publication n°8523, revised 1985).
For all surgical procedures, mice were anesthetized by intraperitoneal injection of a mixture
of 150 mg.kg-1 ketamine (PanPharma, Luitré-Fougères, France) and xylazine (Bayer,
Leverkusen, Germany). Body temperature was maintained at 37°C by means of a rectal probe
connected to a homeothermic blanket.
In mice, 17βestradiol (E2) levels vary three-fold with the estrus cycle within a range of 0.1
(proestrus) to 0.3 nM (estrus) with intermediate values (0.2 nM) observed in non-cycling
animals (Stelck et al. 2005). After ovariectomy, attempts to restore with exogen depots the
physiology of endogenous estradiol have shown some limitations. For instance, Modder
reported that physiological E2 serum levels were reached with 5 and 10 µg.kg-1.d-1 pellets,
but that higher dosages 20-40 µg.kg-1.d-1 and supra-physiological serum levels were needed
to restore the weight of such a select target organ as the uterus (Modder et al. 2004). In
laboratory animals, we therefore opted for classical sham operated animals as physiological
controls to study the influence of estradiol on the lower urinary tract.
Mice were sham-operated or ovariectomized at 4 weeks of age. Two weeks later, the
ovariectomized animals were subcutaneously implanted with either a placebo or a E2-60 day
Page 5 of 26
time release pellets (0.1 mg 17ß-estradiol releasing 80 µg.kg-1.d-1, Innovative Research of
America, Sarasota, FL). This dose was previously reported to induce plasma levels of 0.3 nM
(80 pg/ml), a concentration encountered during pregnancy and about 10 times higher than that
found during the estrus cycle (Elhage et al. 1997). Six weeks later, the mice received either an
intraperitoneal injection of 7- nitro-indazole (7-NI, ACROS Organics, NJ), a potent selective
competitive inhibitor of nNOS without significant effect on eNOS in mice (Moore et al.
1993), sonicated in peanut oil at a dose of 50 mg/kg or the vehicle alone.
Micturition behaviour was then recorded in the animals (6 per group), and two days later leak
point pressures were measured. A separate set of animals (6 per group) was followed after
hormonal manipulation (ovariectomy, sham surgery, ovariectomy plus E2 pellets every 2
months hereater reported as E2-treated mice) until 4 months of age when they were
euthanized for histology and Western blot analysis. In all conditions, the hormonal status was
verified by the combination of uterus weights and serum estradiol levels.
In all animals, serum samples were taken prior to sacrifice at 12 weeks (micturition behavior)
or 16 weeks (histology) of age. Estradiol was first extracted under diethyl ether. Plasma 17β-
estradiol was measured using a commercially available double-antibody radioimmunoassay
immulite-kit (Coat-A-Count Estradiol-6, Diagnostic Products Corporation, Los Angeles, CA,
USA). The inter-assay coefficient of variation (CV) and intra-assay CV for this kit are
reported to be 4.1-15.3% and 3.5-7.6%, respectively. Assay sensitivity was 7.4 pg.mL-1 and
the cross-reactivity with other estrogenic compounds negligible.
Page 6 of 26
Micturition behavior was assessed as described by Burnett (Burnett et al. 1997). Briefly,
animals were housed individually in hanging stainless cages for 14 hours prior to the
experiment and provided with food and water ad libitum. Preweighed absorbent cage paper
was placed underneath each cage and weighed at 5-min intervals for 8 hours.
Urine output was calculated as the sum of the volume urinated and the residual volume per 8
Leak point pressure study
The bladder dome was exposed under operative microscopy through a lower midline
abdominal incision. A 22-gauge angiocatheter was inserted and fixed to the bladder wall with
cyanoacrylate glue. The bladder was then distended with room temperature saline at a filling
rate of 20 µl.min-1. The intravesical pressures were recorded with a TA400 pressure transducer
(Gould Electronics, Ballainvilliers, France) and the leak point pressure was defined as the
pressure recorded when the first drop was observed at the meatus.
Histological analysis and histomorphometry
Mice were euthanized at 16 weeks of age for histology analysis. The animals were put under
general anesthesia as previously described and injected intraperitoneally with 12.5 mg.kg-1
atropine sulfate (Laboratoires Renaudin, Itxassou, France) to prevent premortem bladder
contractions. The bladder was catheterized, emptied and distended to 50 µl of volume as
described for the leak point pressure study. Animals were euthanized by intraperitoneal
injection of 250 mg.kg-1 lidocaine (Astra-Zeneca, Rueil-Malmaison, France).
The upper and lower urinary tract was removed en-bloc, and the kidneys, bladder and uterus
were weighed separately. Bladder dome, mid-ureter and mid-urethra specimens were fixed in
Dubosc-Brazil-Bouin mixture and routinely processed for Masson’ s trichromic stain.
Page 7 of 26
Digital microscopic pictures of 4 µm thick slides (ureter x 400, bladder x 160, urethra x100)
were analyzed with the freeware NIH Image° software (http:/rsb.info.nih.gov/nih-image). For
the ureter and urethra specimens, the areas of the epithelial layer, submucosa and muscularis
layer were measured, as well as the overall area of the section. For the bladder specimens, the
mean thickness of the muscularis layer of a bladder section was assessed as the ratio of the
muscularis surface to the urothelium segment length.
Protein extraction and Western blot
Urethral specimens were removed en-bloc, rinsed with ice-cold saline buffer, frozen by
immersion in liquid nitrogen and stored at -80°C until used for the experiment (within 3
The frozen tissues were thawed and then homogenised in approximately 10 vol. buffer
containing 10 % glycerol, 20 mM Tris, 140 mM NaCl, 10 mM Na pyrophosphate, 10 mM
fluoride, 2 mM Na orthovanadate, 3 mM EDTA, 10 µg/ml trypsin inhibitor, 10 µg/ml
leupeptin and 2 µg/ml aprotinin with a Polytron homogenizer for four periods of 1 min. with
intermittent cooling pauses of 4 min. The homogenate was centrifuged at 40.000 g for 45
minutes and the pellet was discarded. The supernatant was saved.
Aliquots of 60 µl of supernatant were diluted 1:2 in electrophoresis sample buffer (1.8 M
Tris-HCL, pH 6.8, 10% glycerol, 10% SDS, 0.01% bromophenol blue and 5% β-
mercaptoethanol) to yield 60 µg of protein per lane. The samples were reduced by boiling for
5 min. Mouse cerebellum cortex was used as a positive control. Sodium dodecyl sulphate-
polyacryl-amide gel electrophoresis (SDS-PAGE) was carried out on a 10% polyacrylamide
gel at 125 V for 30 min.
After electrophoretic transfer to a PVDF membrane at 10– 15 V for 30 min, the blots were
blocked overnight with dry milk diluted in TBS-Tween at 4°C. The blots were then incubated
with primary polyclonal antibodies (nNOS N31030, Transduction Laboratories, Lexington,
Page 8 of 26
KY) diluted in TBS-Tween dry milk (nNOS 1:1000) for 1 h at room temperature. They were
then rinsed in TBS-Tween for 1-15 min followed by 3 - 5 min and incubated with secondary
anti-rabbit IgG-HRP (SC2030, Santacruz Biotechnology, CA) diluted in TBS-Tween (1:10
000) for 1 h at room temperature. They were then rinsed as above and subjected to enhanced
chemiluminescence (ECL) system. Autoradiographic film was applied to the blot until
satisfactory exposure was obtained. After scanning, films were analyzed by NIH Image°
software to quantitate nNOS expression.
Data were analyzed by two-way ANOVA, followed by Student’ s t test when appropriate.
Data are presented as the mean + standard deviation. P <0.05 was considered significant.
Page 9 of 26
Uterus and body weight
Mean body weights were similar in all groups (E2-treated mice 23.3 ± 1.0 g, ovariectomized
22.9 ± 0.9 g sham-operated 23.1 ± 1.1 g, respectively, N.S.).
As compared to sham-operated mice (102 ± 12 mg), mean uterus weights were similar in E2-
treated mice (117 ± 22 mg, n.s.) and drastically decreased in ovariectomized mice (23 ± 3 mg,
At the time of sacrifice (16 weeks of age), 17ß-estradiol serum levels were undetectable in
ovariectomized animals, while a four-fold increase was observed in E2-treated animals as
compared to sham operated animals (80±9 vs. 19±3 pg/ml, p<0.001, Table I). Similar results
were observed at 12 weeks of age (data not shown).
E2-treated mice exhibited a lower frequency of micturition than ovariectomized and sham-
operated animals (1.2 ± 0.8 micturition/8h vs. 4.0 ± 1.7 and 3.6 ± 1.5, respectively, P =
0.008). At the end of the experiment, the residual urine volume was determined by bladder
catheterization. E2-treated mice exhibited a 30-fold increase in residual volume compared to
ovariectomized or sham-operated animals, which were almost devoid of residual (0.32 ± 0.11
ml vs. 0.01 ± 0.01 ml and 0.01 ± 0.01 ml, respectively, P = 0.008).
The role of nNOS derived NO - was evaluated by subjecting the animals to pharmacological
inhibition by 7-nitroindazole. Acute inhibition of nNOS resulted in a significant decrease in
the frequency of micturition in ovariectomized and sham-operated animals paralleled by a
significant increase in residual volume (Table II), giving evidence of the tonic inhibitory
effect of nNOS in micturition. In contrast, 7 NI did not significantly influence the micturition
pattern of E2 animals (Table II). nNOS inhibition significantly reduced urine output in
Page 10 of 26
ovariectomized animals (190 vs. 347 µl, P = 0.016) but not in sham-operated (352 vs. 389 µl,
P = 0.45) and in E2-treated animals (261 vs. 474 µl, P = 0.11).
Leak Point Pressure
E2-treated mice exhibited a two-fold increase in LPP, compared to ovariectomized and sham-
operated animals (8.8 ± 2.3 cmH2O, vs. 4.3 ± 0.7 and 4.9 ± 0.9 cm H2O respectively,
In addition, acute nNOS inhibition significantly increased urethral resistances in sham-
operated (7.60 ± 1.55 vs. 4.90 ± 0.89 cm H2O, p=0.03) and ovariectomized animals (9.90 ±
5.13 vs. 4.25 ± 0.66 cm H2O, P=0.03) but not in E2 animals (10.67 ± 6.12 vs. 8.75 ± 2.29 cm
H2O, n.s., P=0.70)) highlighting both the relaxing role of nNOS in sham-operated and
ovariectomized animals and its impairement by supraestrus levels of E2.
Histology and histomorphometry (Figure 1 and 2)
As expected in mice, urethral sections were devoid of striated muscle. E2-induced increase in
urethral resistances was associated with thicker muscularis layers in ureter and bladder and by
a parallel increase in bladder weight (31.2 ± 7.8 mg vs 21.0 ± 1.8 vs 22.1 ± 2.2 in E2-treated
mice vs sham-operated and ovariectomized mice, respectively, P < 0.01). No differences were
observed in various urethral layers, whatever the hormonal status.
nNOS urethral expression (Figure 3)
E2 strongly impacted on nNOS urethral expression as supraestrus E2 resulted in a significant
62 % decrease whereas ovariectomy led to a 69 % increase in expression, as compared to
Page 11 of 26
In the present work, we confirmed the influence of E2 on urethral tone and asserted whether it
was mediated through local nNOS activity and expression as reported in other target organs
(summarized in figure 4). Contrary to sham-operated and ovariectomized animals where acute
nNOS inhibition increased urethral resistances, nNOS inhibition had no impact on the
chronically elevated resistances observed in E2-treated animals.
As shown by increased residual volume and fewer micturitions in E2-treated animals,
estradiol status profoundly influenced the micturition pattern which raises the concern that
supraestrus levels of estradiol could lead to overflow incontinence by urinary retention.
However, the NIH consensus definition of overflow incontinence is that of frequent
micturitions of low volumes
(http://kidney.niddk.nih.gov/kudiseases/pubs/uiwomen/index.htm) while on the contrary we
observed in supraestrus animals a reduction in frequency associated with an increase in
volume, that failed this definition and suggested a physiological benefit of estradiol
One limitation of the present study is that it did not address any direct impact on bladder
contractility. While nNOS was shown to be the major NOS isoform in the lower urinary tract
(Burnett et al. 1997) its gene invalidation did not impact on bladder strip contractility or
relaxation after chemical and electrical stimulation (Sutherland et al. 1997), suggesting that
the reported impact on urodynamics and micturition were secondary to alterations of urethral
resistances (Burnett et al. 1997; Mamas et al. 2003; Sutherland et al. 1997). We therefore
focused on the dynamic control of urethral resistances by estradiol through the nNOS
Page 12 of 26
As urethral resistances may reflect any combination of dynamic obstructions resulting from
smooth muscle tonus and static tissue resistances, we investigated their respective
contributions depending on E2 status and nNOS activity by measuring the bladder pressure at
urine leakage - similar to the leak point pressure (LPP) in man - and searched for structural
urethral alterations. In contrast to the sham-operated and ovariectomized controls, E2-treated
mice exhibited a two-fold increase in urethral resistances that was not further modified by
nNOS inhibition and the structure of the bladder as assessed by image analysis and weight
was significantly altered. Of note, no differences between groups were observed in the
urethral layers suggesting that the E2-induced urethral resistances were not related to
structural changes of the outlet. In ovariectomized and sham-operated controls, acute
inhibition of nNOS increased the bladder pressure at urine leakage, confirming in the female
urinary tract the relaxing effect of NO on urethral tone (Sutherland et al. 1997). However,
such inhibition had no influences on the increased urethral resistances observed in E2-treated
We therefore assessed nNOS expression and for the first time in urethra highlighted the
significant decrease of nNOS-expression in E2-treated animals mirrored by its sharp increase
in ovariectomized animals.
Ten years ago, Takahashi first reported that short-term high-dose estrogen treatment reduced
NOS activity and inhibited the nitrergic-nerve-stimulation-induced relaxation of rabbit
urethral smooth muscle (Takahashi et al. 1997). Al Hijji showed that such E2 reduced NOS
activity was observed in all segments of the lower urinary tract (Al-Hijji and Batra 1999).
Both experiments studied the influence of short-term stimulation (2 and 1 weeks,
respectively) with high doses of estradiol (5mg.kg-1.wk-1 and 1 mg. kg-1.wk-1, respectively)
on NOS activity and did not assess selective implication of specific NOS subtypes. To our
knowledge, the present report is the first evidence of long-term hormonal modulation of
Page 13 of 26
nNOS expression in the female lower urinary tract by supraestrus levels of estradiol, a key
finding in view of the function of NO as a major mediator of smooth muscle relaxation in the
lower urinary tract.
Sutherland and Burnett previously emphasized the key role of the nNOS subtype on lower
urinary function. Sutherland showed that gene inactivation of nNOS in female mice resulted
in profound decrease of overall NOS activity in bladder and urethra, indicating that it
accounts for most of the NOS activity in the lower urinary tract. Increased bladder weight
and maximal bladder pressure at leakage suggested that nNOS disruption resulted in increased
uretral resistances which did not however translate into measurable alterations of the voiding
patterns (Sutherland et al. 1997). In male animals however, targeted disruption of the nNOS
gene resulted in dysfunctional bladder outlet (Burnett et al. 1997). Interestingly, eNOS
immunoreactivity was observed in endothelium of submucosa blood vessels and in
urothelium of the urethra, whereas nNOS expression was restricted to nerve fibers throughout
the inner lamina propria and muscular layer thereby supporting an exclusive control of
urethral muscle relaxation by nNOS (Burnett et al. 1997). Our present findings in female
sham-operated animals where 7NI-selective nNOS inhibition increased urethral tone are
consistent with these two studies.
We show here that supraestrus E2 decreased urethral nNOS expression resulting in profound
modifications of bladder function and structure. Beside direct regulation of nNOS expression,
E2 can also indirectly impair NOS activity by generating urethral production of endogenous
NOS inhibitors such as NG-monomethyl-L-arginine (L-NMMA) and asymmetrical NG,NG-
dimethyl-L-arginine (ADMA) through decreasing dimethylarginine dimethylaminohydrolase
(DDAH) activity (Okuno et al. 2004). In human, endogenous NOS inhibitors have proven to
be of pathophysiological relevance in pathological conditions such as peripheral and coronary
Page 14 of 26
arterial diseases (Boger et al. 1997; Valkonen et al. 2001). Beside modulating the urethral
outlet function or as its consequence, E2 has strong impact on the bladder reservoir. Indeed, it
can induce detrusor hypertrophy (Lin et al. 2006) or reduce the density of Muscarinic
receptors (Shapiro 1986) in keeping with the significant improvement of overactive bladder
symptoms in postmenopausal women (Cardozo et al. 2004).
As a whole, by decreasing the expression and the activity of nNOS and by enhancing the local
concentration of NOS inhibitors, supraestrus E2 can locally disrupt the NO regulation leading
to an increase in the urethral tone. This could account for the reported gradual increase in
urethral tone and relative lack of stress urinary incontinence during pregnancy (Iosif et al.
1980) and suggest a beneficial effect of estrogens in postmenopausal incontinence.
This effect was not observed with physiological estradiol serum levels in line with the
reported lack of efficacy of estrogen supplementation within the estrus range on urinary
incontinence (Hendrix et al. 2005; Robinson and Cardozo 2003). This apparent discrepancy
between physiological and supraestrus levels of E2 was previously reported by our group in
other extrareproductive effects such as atherosclerosis prevention (Elhage et al. 1997) and
modulation of invariant Natural Killer T cells (Gourdy et al. 2005).
The present study also confirmed the profound impact of high doses of estradiol on kidney
function and nNOS renal expression. Indeed, in E2-treated animals urine output - as
represented by the sum of the volume urinated and the residual volume – increased
reminiscent of the report by Carlberg that high-dose supplementation with estrogens increased
urine volume by decreasing osmolality (Carlberg et al. 1984). In addition, this was exquisitely
influenced by nNOS inhibition confirming the observation of Alexander in the pregnant rat
Page 15 of 26