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Departments of Reproductive Biology, and Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106
Address all correspondence and requests for reprints to: George I. Gorodeski, M.D., Ph.D., University MacDonald Women’s Hospital, University Hospitals of Cleveland, 11100 Euclid Avenue, Cleveland, Ohio 44106. E-mail: gig{at}po.cwru.edu
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1 nM), and the effects were inhibited
by tamoxifen. Treatment with SNP increased cGMP maximally, even in
estrogen-deficient cells. LY-83583 blocked the estrogen-induced
increase in cGMP, but neither LY-83583 nor KT-5823 had a significant
effect on the estrogen-induced increases in NO release and cellular
cGMP. The NO synthase (NOS) inhibitor
NG-nitro-L-arginine methyl ester
decreased NO release, and pretreatment of cells with
L-arginine reversed the effect. Cultured human cervical
epithelial cells express messenger RNA for the NOS isoforms endothelial
NOS (ecNOS), brain NOS, and inducible NOS. 17ß-Estradiol up-regulated
ecNOS messenger RNA, and tamoxifen blocked the effect. Based on these
results we suggest that the effect of estradiol on permeability
involves four signaling steps: 1) activation of estrogen receptors, 2)
increase in ecNOS transcription and up-regulation of NO activity, 3) NO
activation of guanylate cyclase and increase in cGMP, and 4) cGMP
activation of cGMP-dependent protein kinase. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Estrogens increase the secretion of cervical mucus in women (1) and the secretion of mucins into the cervical canal by exocytosis from the apical cell membrane of endocervical cells. The mechanisms by which estrogens regulate mucins secretion are relatively well understood (1). In contrast, relatively little is known about mechanisms of estrogen regulation of the cervical plasma. In women, cervical plasma composes 80–99% of the total weight of the cervical mucus. It originates by transudation of water, electrolytes, carbohydrates, proteins, and lipids from the blood through the endocervical and ectocervical epithelia (1). Recent studies revealed that estrogen increases the permeability of cultured human cervical epithelia (2, 3). The conclusions from these studies were that estrogen increases transudation of fluid and secretion of the cervical plasma by decreasing the resistance of the paracellular transcervical pathway to the movement of fluid and solutes from the blood into the lumen.
The molecular mechanisms by which estrogen modulates paracellular
resistance are not entirely understood. The estrogen-induced increase
in cervical permeability is transcriptionally regulated, and it
involves estrogen receptor 
(3). However, estrogen receptor does
not directly modulate permeability, and the effect is mediated by a
secondary signaling system (3). In some types of cells the effects of
estrogen are mediated by nitric oxide (NO) (4, 5, 6). NO can modulate the
permeability of endothelial cells (7, 8, 9, 10) and epithelial cells
(11, 12, 13, 14, 15), but until recently its actions in the human cervix were
unknown. NO is synthesized from L-arginine during the NO
synthase (NOS)-catalyzed conversion of L-arginine to
L-citrulline (10, 11). Previous studies have identified NO
activity in mammalian cervical cells (16) and NOS expression in human
uterine and vaginal tissues (17), but relatively little is known about
the role of NOS in human cervical epithelium. The objective of the
present study was to determine the degree to which the effects of
estrogen on paracellular permeability of cultured human cervical
epithelia are mediated by the NO system.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Changes in paracellular permeability were determined from changes in the permeability to pyranine (Ppyr) and in the transepithelial electrical conductance (GTE). The methods, including conditions for optimal determinations of Ppyr and GTE across low resistance epithelia, calibrations and controls, potential pitfalls, and the appropriate measures to prevent artifacts were described and discussed previously (19).
Changes in Ppyr were determined from unidirectional (luminal to subluminal) fluxes across filters mounted vertically in the modified Ussing/diffusion chamber to prevent hydrostatic gradients (19, 20). Pyranine is a trisulfonic acid with a molecular mass of 510 Da; it traverses epithelia via the paracellular pathway, and its concentration can be measured down to nanomolar levels by fluorescence techniques (18). Pyranine was added to the luminal compartment, and the amount of pyranine in the subluminal compartment was measured after 10 min. The transepithelial permeability coefficient (Ppyr) was calculated as previously described (15, 16). Cytolysis of human cervical epithelial cells that were previously incubated with 0.1 mM pyranine did not increase pyranine fluorescence significantly above the background (not shown).
Changes in GTE were determined continuously across filters mounted vertically in a modified Ussing chamber from successive measurements of the transepithelial electrical current (I) and the transepithelial potential difference (PD; lumen negative): GTE = I/PD. All reagents used for the Ussing chamber experiments were added from concentrated stocks (x300–1000) of 1% ethanol, dimethylsulfoxide, or saline to both the luminal and subluminal solutions (19).
Generation of osmotic gradients
Transepithelial hypertonic gradients of 325 to 285 mosmol/liter
in the subluminal to luminal direction across cultures on filters were
established by adding 120 µl 2 M sucrose solution to the
subluminal solution (21).
Assay of nitric oxide
Release of NO into the extracellular medium was determined as
the accumulation of nitrite
(NO2-) and nitrate
(NO3-) in the extracellular
fluid by a modified Greiss method (7). The detection limit of the assay
is 2 µM. Results were expressed as picomoles per min/mg
protein.
Determinations of cGMP
Cells on filters were homogenized in trichloroacetic acid, and
the cGMP content within the cell homogenate was assayed using a
commercially available RIA kit (Amersham Pharmacia Biotech, Arlington Heights, IL) (7). Results were expressed as
picomoles per min/mg DNA. Levels of cGMP in the extracellular fluid
bathing filters seeded with human cervical epithelial cells were
undetectable by this method (not shown).
Cellular DNA and total protein were measured as previously described (18).
Isolation of RNA (22)
Total RNA from cultured cells was isolated with the
QIAGEN kit (Chatsworth, CA), using lysis buffer plus
ß-mercaptoethanol at 350 µl/107 cells. The
final total RNA pellets were resuspended in 50 µl
diethylpyrocarbonate-water and quantitated by measuring
OD260.
RT-PCR was described previously (22). The following PCR conditions were applied: for endothelial NO synthase (ecNOS), 35 cycles of 1-min denaturation at 94 C, 1-min annealing at 62 C, and 2-min extension at 72 C; for neuronal (brain) NOS (bNOS), 35 cycles of 1 min at 94 C, 2 min at 56 C, and 2 min at 72 C; for the inducible NOS (iNOS), 35 cycles of 1 min at 94 C, 2 min at 56 C, and 2 min at 72 C; and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 30 cycles of 1 min at 94 C, 1 min at 60 C, and 1 min at 72 C. The following oligonucleotide primers were used: human ecNOS (23): 5'-forward (sense), 5'-CAG TGT CCA ACA TGC TGC TGG AAA TTG-3'; and 3'-reverse (antisense), 5'-TAA AGG TCT TCT TGG TGA TGC C-3'; human bNOS (24): 5' forward (sense), 5'-TTT CCG AAG CTT CTG GCA ACA GCG GCA ATT-3'; and 3'-reverse (antisense), 5'-GGA CTC AGA TCT AAG GCG GTT GGT CAC TTC-3'; iNOS (25): 5'-forward (sense), 5'-GCC TCG CTC TGG AAA GA-3'; and 3'-reverse (antisense), 5'-TCC ATG CAG ACA ACC TT-3'; and human GAPDH (22): 5'-forward (sense), 5'-TGA AGG TCG GAC TCA ACG GAT TTG GT-3'; and 3' reverse (antisense), 5'-GTG GTG GAC CTC ATG GCC CAC ATG-3'.
Densitometry
X-Ray films were analyzed with laser densitometer Sciscan 5000
(United States Biochemical Corp., Cleveland, OH) and
normalized relative to GAPDH RNA.
Statistical analysis of the data
Data are presented as the mean (±SD), and
significance of differences among means was estimated by ANOVA. Trends
were calculated using GB-STAT V5.3 (Dynamic Microsystems, Inc., Silver
Spring, MD) and analyzed with ANOVA. Best fit of regression equations
(least squares criterion) was achieved with SlideWrite Plus (Advanced
Graphics Software, Inc., Carlsbad, CA), which uses the
Levenberg-Marquardt algorithm, and was analyzed using ANOVA.
Chemicals and supplies
Anocell (Anocell-10) filters were obtained from Anotec (Oxon,
UK). Fluorescent microspheres (FluoresBrite beads, calibration grade)
were obtained from Polysciences, Inc. (Warrington, PA). All other
chemicals were obtained from Sigma (St. Louis, MO).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/cm-2). Treatment with 17ß-estradiol
increased GTE 2-fold in both hECE and CaSki
cultures in a time- and dose-related manner (Fig. 1
, A and B), confirming our previous
results (2). Increases in GTE were observed after
3 h and reached a plateau after 6 h of treatment with the
hormone (Fig. 1A). Maximal increases in GTE were
induced by doses of 10 nM or more, with an
EC50 of 17ß-estradiol of 1.1 ± 0.2
nM (Fig. 1B). Estradiol also increased the permeability to
pyranine (Fig. 1A, inset). These results indicate that
treatment with physiological concentrations of 17ß-estradiol
increases the permeability of cultured human cervical epithelia.
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). A similar effect (not shown) was
obtained with the NO donor molisidomine
[N-[ethoxycarbonyl]-3-[4-morpholinyl]sydnoneimine
(SIN-I)] in the presence of superoxide dismutase (to degrade
superoxide anions). These results indicate that the increases in
GTE in response to SNP or SIN-I are not the
result of toxic effects on the cells, but are mediated by NO or a
related metabolite.
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). Combined treatment with 1 mM SNP and 50
µM 8-Br-cGMP also increased GTE,
but the effect was similar to that produced by SNP alone (not shown).
Collectively, the results shown in Fig. 2 suggest that NO and cGMP
increase the permeability of cultured human cervical epithelia.
Effects on GTE of estradiol plus SNP or
estradiol plus 8-Br-cGMP do not summate
The next experiment tested the effect of combined treatments of
estradiol plus SNP and estradiol plus 8-Br-cGMP on
GTE. The null hypothesis was that the effect of
estrogen is not mediated by NO and/or cGMP; subsequently, the addition
of SNP or of 8-Br-cGMP to estradiol-treated cells should increase
GTE to the same degree as in cells not treated
with the estrogen and more than in cells treated only with estrogen. In
the present experiment cells were pretreated with 1 nM
17ß-estradiol, a concentration that induces a half-maximal increase
in GTE (Fig. 1B).
In estradiol-treated cells 1 mM SNP increased
GTE, but this increase in
GTE was not different from the increase seen with
SNP alone (i.e. in cells not treated with the estrogen; Fig. 3). Similarly, in estradiol-treated cells
50 µM 8-Br-cGMP increased
GTE, but this increase in
GTE was not different from the increase produced
by 8-Br-cGMP alone (i.e. in cells not treated with the
estrogen; Fig. 3). These results indicate that the effects of estradiol
plus SNP or estradiol plus 8-Br-cGMP on GTE do
not summate.
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). In the present study a hypertonic gradient of 325 to 285
mosmol/liter in the subluminal to luminal direction applied across
cells treated with 1 nM 17ß-estradiol significantly
increased GTE (Fig. 3). In cells treated with 1
nM 17ß-estradiol plus 1 mM SNP or with 1
nM 17ß-estradiol plus 50 µM 8-Br-cGMP,
hypertonic gradients also increased GTE
significantly, but to a similar degree as in cells treated only with
estrogen (Fig. 3). Therefore, the lack of an additive effect on
GTE in response to estradiol plus SNP or
estradiol plus 8-Br-cGMP cannot be explained by the fact that 1
nM 17ß-estradiol stimulates a near-maximal increase in
GTE. Based on these results the null hypothesis
was rejected; another possible explanation is that the effects of
estrogen, NO, and cGMP on GTE are interrelated,
and that NO and cGMP possibly mediate the estrogen-induced increase in
permeability.
Tamoxifen, LY-83583, and KT-5823 modulate the effects of SNP and
8-Br-cGMP on GTE
To better understand the mechanisms of action of SNP and
8-Br-cGMP, two experiments were performed. In the first
experiment, cells were treated with tamoxifen and then exposed to SNP
and 8-Br-cGMP. Tamoxifen inhibits the estrogen-induced increase in
cervical permeability (2), and the objective was to determine whether
treatment with tamoxifen also blocks increases in
GTE induced by SNP or 8-Br-cGMP. When
administered alone, tamoxifen had no significant effect on permeability
(Fig. 4). Tamoxifen also did not modulate
the increases in GTE induced by SNP or 8-Br-cGMP
(Fig. 4), suggesting that the effects of SNP and 8-Br-cGMP on
permeability do not involve activation of estrogen receptor(s).
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); however, both agents
blocked the increase in GTE induced by SNP, and
KT-5823 also blocked the increase in GTE induced
by 8-Br-GMP (Fig. 4). Collectively, these results indicate that 1) SNP-
and 8-Br-cGMP-induced increases in GTE are
independent of estrogen action; 2) LY-83583 blocks a necessary
signaling step for both SNP and 8-Br-cGMP; and 3) KT-5823 blocks a
necessary signaling step only for 8-Br-cGMP.
Estrogen increases NO release and up-regulates cellular cGMP
To test the hypothesis that the effect of estrogen on cervical
permeability is mediated by NO and cGMP, experiments were conducted to
determine the effects of estrogen on NO activity and cellular cGMP. NO
activity was determined in terms of NO release into the extracellular
fluid as the accumulation of nitrite
(NO2-) and nitrate
(NO3-) in the bathing medium. NO
is a volatile gas that can permeate cell membranes (13, 14), and in
biological systems such as cultured cells on filters it is in
equilibrium between the intracellular and extracellular fluids. Levels
of cGMP were measured directly by RIA.
Cultured human cervical epithelial cells on filters release NO
constitutively into the extracellular fluid (Fig. 5A). Treatment with 17ß-estradiol
increased NO release in a dose-related manner: effects began at
concentrations of 0.1 nM or more and reached saturation at
10 nM estradiol or more. The dose-response curve was
sigmoidal, with an EC50 of estradiol of 0.9
± 0.1 nM (Fig. 5A). Treatment with 17ß-estradiol also
increased cellular cGMP, reaching saturation at 10 nM or
more, with an EC50 of estradiol of 1.2 ±
0.1 nM (Fig. 5B). The dose-response curves of NO release
vs. estradiol (Fig. 5A) and cGMP vs. estradiol
(Fig. 5B) were similar. Also, both curves were similar to that of
GTE vs. estradiol (Fig. 1B),
suggesting that the effects of estradiol on NO release, cGMP
accumulation, and permeability involve a common signaling pathway.
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). In cells
treated with 10 nM estradiol or more, levels of cGMP were
maximal, and SNP had no additional effect (Fig. 5B). These results
suggest that SNP up-regulates cellular cGMP maximally, and that
estradiol has no additive effect. Treatment of cells with estradiol
plus 8-Br-cGMP did not produce an additional increase in NO release
compared with the effect of estradiol only (Fig. 5A).
Experiments were also performed to determine the effects of tamoxifen,
LY-83583, and KT-5823 on the estrogen-dependent increases in NO
release and cellular cGMP. In cells not treated with estradiol, none of
the three drugs had a significant effect on NO release or levels of
cellular cGMP (Fig. 6). In cells treated
with 10 nM 17ß-estradiol, tamoxifen blocked the
estrogen-induced increase in NO release and attenuated the
estrogen-induced increase in cellular cGMP (Fig. 6). In
estradiol-treated cells, LY-83583 had no significant effect on NO
release, but it attenuated the increase in cGMP (Fig. 6). In
estradiol-treated cells KT-5823 had no effect on NO release or cGMP
(Fig. 6).
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indicate that 1)
increases in NO and cGMP are sufficient to increase
GTE; 2) increases in NO and cGMP are necessary
for estrogen to exert an increase in GTE; and 3)
an increase in cGMP is necessary for NO to exert an increase in
GTE. These results therefore suggest that NO
precedes cGMP in the estrogen signaling cascade.
Mechanism of estrogen-induced increase in NO release
In eukaryotic cells NO is often synthesized from
L-arginine during the NOS-catalyzed conversion of
L-arginine to L-citrulline (13, 14). Three
broad categories of NOS isoenzymes have been characterized, all
products of different genes: ecNOS, bNOS, and iNOS (13, 14). The
objective of the next two experiments was to determine the degree to
which the estrogen-induced increase in NO release involves
up-regulation of NOS(s).
The first experiment tested the effect of the NOS inhibitor
NG-nitro-L-arginine
methyl ester (L-NAME) on NO release. In estrogen-deprived cells L-NAME
decreased NO release, but the effect did not reach statistical
significance (Fig. 7). In
estrogen-treated cells L-NAME decreased NO release significantly, to a
level not different from that observed in estrogen-deprived cells (Fig. 7). In most biological systems the inhibitory effects of L-NAME on NOS
can be attenuated by pretreatment with L-arginine
(13, 14). As is shown in Fig. 7, L-arginine alone
did not have a significant effect on NO release, but it abrogated the
L-NAME-induced inhibition of NO release in estrogen-treated cells.
These results indicate that the estrogen-induced increase in NO release
involves up-regulation of the L-NAME-sensitive mechanism.
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). The cDNA
fragments were isolated, amplified, and purified, and the products were
sequenced by the dideoxy chain termination method. Sequence analysis of
the cloned segments revealed homologies of 98–99% (sense and
antisense) with the human ecNOS, bNOS, and iNOS (the differences were
sequence errors). These results indicate that cultured human cervical
epithelial cells express mRNA for ecNOS, bNOS, and iNOS.
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). Estradiol also had no significant
effect on the expression of bNOS or iNOS mRNA (Fig. 8C). In contrast,
treatment with estradiol increased the expression of ecNOS mRNA (Fig. 8, B and C). To ascertain that the RT-PCR technique is sensitive in
measuring changes in the expression of ecNOS mRNA, the experiment was
repeated using different amounts of cDNA for PCR amplification. As
shown in Fig. 8B, the quantity of the amplified products of both the
GAPDH and ecNOS cDNA was dependent on the amount of cDNA used for the
amplification. Treatment with estradiol had no effect on GAPDH RNA, but
it increased ecNOS RNA (Fig. 8B). Subsequently and regardless of the
initial amount cDNA used for the PCR, the ratio of ecNOS RNA relative
to GAPDH RNA was greater in estradiol-treated cells than in
estrogen-deprived (control) cells. Densitometry analysis of three
experiments in hECE cells and three experiments in CaSki cells revealed
that estradiol increased the ratio of ecNOS/GAPDH mRNA 12- to
25-fold.
To ascertain that the RT-PCR technique can yield interpretable
semiquantitative results, the effect of number of PCR cycles on the
expression of ecNOS and GAPDH mRNA was determined. As shown in Fig. 8C, the quantity of amplified products of both ecNOS and GAPDH was
dependent on the number of PCR cycles. Individual analysis of ecNOS and
GAPDH results showed that using 35 and 30 cycles, respectively,
resulted in a synthesis reaction that did not reach a plateau. This
indicates that using 35 and 30 cycles, respectively, for ecNOS and
GAPDH provide amplification conditions for log phase synthesis.
In estrogen-deprived cells tamoxifen did not have a significant effect
on ecNOS or GAPDH mRNA. However, tamoxifen blocked the
estradiol-induced increase in ecNOS/GAPDH mRNA (Fig. 8D). Tamoxifen did
not have a significant effect on bNOS/GAPDH mRNA or iNOS/GAPDH mRNA
(Fig. 8D). These results indicate that 17ß-estradiol up-regulates
ecNOS mRNA and suggest that estrogen increases NO release by
up-regulation of ecNOS.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present experiments used two types of human cervical epithelial
cells: hECE and CaSki. HECE are normal cells derived from minces of the
ectocervix and are an adequate model for the ectocervical epithelium;
CaSki are cells are a stable line of transformed cervical epithelial
cells that express phenotypic markers of the endocervix and are a
useful model for the endocervical epithelium (2, 3, 18, 19, 20, 21, 22). Both cell
types express estrogen-inducible estrogen receptors, including and
ß isoforms (3), and in both cell types the estrogen receptor
antagonist tamoxifen blocks the estrogen-induced increase in
permeability (2, 3).
The main difference between the endocervical and ectocervical epithelia
is the secretion of mucins, which normally are contributed only by the
former. In vivo, both the endocervical epithelium and the
ectocervical epithelium control transudation of fluids into the lumen
of the lower genital tract, including the cervix (1). The permeability
characteristics of hECE and CaSki cultures on filters are similar to
the permeability of the cervical epithelium in vivo, as
calculated from sampling cervical secretions in women (21, 22). Both
epithelia are characterized by relatively high transepithelial
electrical conductance (5–50 /cm-2) and a
relatively high degree of permeability to molecules that traverse the
epithelium via the intercellular space (6–28 x
10-6 cm/sec) (18). Thus, hECE and CaSki
cultures are good models to study estrogen regulation of transcervical
permeability.
The strategy used in the present study was to determine the degree to
which changes in NO activity and cellular cGMP are sufficient to
increase cervical permeability and necessary for estrogen to exert an
increase in permeability. The statement that NO mediates the effect of
estrogen is supported by the following experimental findings. 1)
17ß-Estradiol increased ecNOS mRNA. Previous studies have shown that
nerves ending in human uterine and vaginal tissues express NOS (17),
but the present results show for the first time that human
cervical epithelial cells express mRNA for all three isoforms: ecNOS,
iNOS, and bNOS. 2) Tamoxifen blocked the 17ß-estradiol-induced
increase in ecNOS mRNA. As the effect of estrogen on cervical
permeability involves up-regulation of tamoxifen-sensitive, estrogen
receptor -dependent transcription of a secondary signaling system
(3), the present results suggest that the secondary signaling system is
ecNOS. 3) 17ß-Estradiol increased NO release; L-NAME blocked the
17ß-estradiol-induced increase in NO release, and the effect of
L-NAME could be reversed by pretreatment with the NOS substrate
L-arginine. 4) The NO donors SNP and SIN-I increased
GTE. In estrogen-treated cells SNP increased
GTE to a lesser degree than in estrogen-deprived
cells, suggesting that estrogen and NO activate a common signaling
pathway. 5) Tamoxifen had no effect on the SNP-induced increase in
GTE; in contrast, LY-83583 and KT-5823 blocked
the effect, suggesting that the effect of NO on permeability does not
involve activation of the estrogen receptor(s). A possible mechanism is
activation of guanylate cyclase (14, 26, 27, 29, 30).
In some types of cells the signaling pathways of NO and cGMP converge so that NO activates guanylate cyclase and stimulates an increase in cGMP (13, 14, 26, 27). The speculation that the estrogen-induced increase in cervical permeability involves NO activation of guanylate cyclase, up-regulation of cGMP, and activation of cGMP-dependent protein kinase is supported by the following experimental findings. 1) 17ß-Estradiol increased cellular levels of cGMP, and the effect had a similar time course and dose dependency as the estrogen-induced increases in GTE and NO release. 2) Tamoxifen and LY-83583 blocked the 17ß-estradiol-induced increase in cGMP. 3) SNP increased levels of cellular cGMP, even in estrogen-deficient cells. 4) The cGMP analog 8-Br-cGMP increased GTE. In estrogen-treated cells 8-Br-cGMP increased GTE to a lesser degree than in estrogen-deprived cells, suggesting that estrogen and cGMP (as well as NO) activate a common signaling pathway. 5) KT-5823 blocked the 8-Br-cGMP-induced increase in GTE. Collectively, these results suggest that the effects of estrogen and NO on permeability are mediated by a cGMP-dependent mechanism.
Human cervical epithelial cells produce NO constitutively (present
results). As NO increases permeability, it is suggested that cervical
cells autoregulate permeability and maintain an increased state of
paracellular permeability by continuously secreting NO. This may be a
mechanism in vivo for maintaining lubrication of the
cervical canal (1). Equally important are the present findings that
estrogen can increase cervical permeability by up-regulation of NO
activity. Based on the present results as well as on our previous
results (3), it is proposed that the effect of estrogen on cervical
permeability involves four signaling steps: 1) activation of estrogen
receptor , 2) increase in ecNOS transcription and up-regulation
of NO activity, 3) NO activation of guanylate cyclase and increase in
cGMP, and 4) cGMP activation of cGMP-dependent protein kinase.
The present findings may be important for understanding cervical mucus secretion and improving estrogen treatment in women. Until recently, relatively little was known about the regulation of cervical permeability in women, and most cases of abnormal cervical mucus were attributed to defective estrogen production (1). The present results as well as our previous studies (2, 3) show that estrogen-dependent modulation of cervical permeability involves a number of regulatory sites distal to the estrogen receptor. For instance, agonists that elevate cytosolic calcium can stimulate the release of NO by the calcium-dependent ecNOS (13, 14). Secretagogues, such as ATP and histamine, can increase cytosolic calcium in human cervical epithelial cells (20); elevated intracellular calcium can stimulate NO production (13, 14) and increase the permeability (present results). The present results provide information that can be used to design clinical studies to test the degree to which pharmacological agents that act distal to the estrogen receptor can modulate cervical mucus secretion.
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Footnotes |
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Received November 4, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß in the regulation of cervical permeability.
Am J Physiol, in press
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 G. I. Gorodeski Expression, regulation, and function of P2X4 purinergic receptor in human cervical epithelial cells Am J Physiol Cell Physiol, January 1, 2002; 282(1): C84 - C93. [Abstract] [Full Text] [PDF]
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 B. Li, J. Yao, T. Morioka, and T. Oite Nitric Oxide Increases Albumin Permeability of Isolated Rat Glomeruli via a Phosphorylation-Dependent Mechanism J. Am. Soc. Nephrol., December 1, 2001; 12(12): 2616 - 2624. [Abstract] [Full Text] [PDF]
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G. I. Gorodeski Estrogen Biphasic Regulation of Paracellular Permeability of Cultured Human Vaginal- Cervical Epithelia J. Clin. Endocrinol. Metab., September 1, 2001; 86(9): 4233 - 4243. [Abstract] [Full Text] [PDF]
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 J. S. Krumenacker, S. M. Hyder, and F. Murad Estradiol rapidly inhibits soluble guanylyl cyclase expression in rat uterus PNAS, January 16, 2001; 98(2): 717 - 722. [Abstract] [Full Text] [PDF]
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G. I. Gorodeski Calcium regulates estrogen increase in permeability of cultured CaSki epithelium by eNOS-dependent mechanism Am J Physiol Cell Physiol, November 1, 2000; 279(5): C1495 - C1505. [Abstract] [Full Text] [PDF]
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G. I. Gorodeski Effects of Menopause and Estrogen on Cervical Epithelial Permeability J. Clin. Endocrinol. Metab., July 1, 2000; 85(7): 2584 - 2595. [Abstract] [Full Text]
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