| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Original Studies |
Departments of Reproductive Biology and Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106
Address 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
 |
Abstract |
|---|
 Top Abstract IntroductionPatients and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
|---|
The cervical mucus is a mixture of water, water-insoluble components (mucins), and water-soluble components (cervical plasma). The water-soluble cervical plasma constitutes 80–99% of the total weight of the cervical mucus, and it originates by transudation of fluid and solutes from the blood into the cervical canal (1). Mechanisms of secretion and regulation of cervical plasma by estrogen are not well understood. Most previous studies in the field used samples of cervical mucus (2), but studies in vivo may be inaccurate due to sampling errors and due to contamination with blood, uterine, and vaginal secretions. An important progress in the understanding of regulation of cervical plasma was made by using new systems to culture human cervical cells on filters (3, 4).
Secretory epithelia, including the cervical epithelium, are organized as a layer(s) of confluent cells, where plasma membranes of neighboring cells come into close contact and functionally occlude the intercellular space. The current theory of epithelial control of movement of fluid and solutes from the blood into the lumen is derived from the Ussing-Zerahn model of fluid transepithelial transport (5). Molecules can move across epithelia either through the cells (transcellular route) or via the intercellular space (paracellular route). Movement in the transcellular route is restricted by the plasma membranes of the cells, whereas movement via the paracellular route is determined by the resistance of the intercellular tight junctions and by the resistance of the lateral intercellular space (RLIS). Because the resistance of plasma membranes (RPM) to passive movement of molecules is higher than that of the paracellular route (6), the overall permeability properties of secretory epithelia are determined by the paracellular route.
Secretory epithelial cells form two types of barriers to the free movement of molecules in the intercellular space: the tight junctions (7, 8, 9, 10, 11) and the RLIS (12). The latter is determined by the length of the intercellular space from the tight junctions to the basal lamina and by the proximity of the plasma membranes of neighboring cells. Cells can control the volume of the intercellular space and, therefore, the resistance to flow through the space, by changing their width; changes in cell size will affect inversely the size of the intercellular space.
The cervical plasma is derived by transudation of plasma from the
capillaries that feed the cervix, through the body of the cervix and
into the cervical canal. The driving force for transudation is the
blood pressure in the capillaries that generates a transepithelial
hydrostatic gradient between the capillaries and the cervical canal
(1). Most cell types in the cervix, including stromal and smooth muscle
cells, do not significantly restrict the movement of fluid through the
cervix. In contrast, cervical epithelial cells do restrict fluid
transudation through the intercellular space. The Ussing-Zerahn model
describes net total transepithelial resistance
(RTE) in terms of the RPM
and the paracellular resistance (RPCR), in
parallel [i.e.
1/(RTE) = 1/(RPM) +
1/(RPCR)]. Because RPM is
larger than the RPCR by about
105 (6), 1/(RTE) 
1/(RPCR) and RTE
RPCR. This indicates that in most epithelia the
RPCR determines the total
RTE. RPCR, in turn, is
determined by the tight junctional resistance
(RTJ) and by the RLIS in
series, [i.e. RPCR =
RTJ + RLIS], and,
therefore, RTE RTJ +
RLIS. In cultured human cervical epithelia the
RTJ and RLIS can be
independently regulated and separately assayed (4), making these
cultures an important system to study regulation of paracellular
permeability.
After menopause, women experience a significant decrease in cervical secretions (1). Until recently, this phenomenon was attributed to hypoestrogenism that follows the menopause (13). Estrogens increase secretion of cervical mucus in women, including postmenopausal women (1), but not all postmenopausal women improve on estrogen (13). These observations raise the possibility that hypoestrogenism may not be the only factor that contributes to decreased cervical secretions in postmenopausal women. The first objective of the present study was to determine the paracellular permeability characteristics in cervical epithelial cells of postmenopausal women and to compare them with those of premenopausal women.
Previous studies in cultured human cervical epithelia showed that estrogens increase cervical permeability by decreasing the RLIS (14). These studies used cells that were obtained from premenopausal women, and until recently little was known about effects of estrogen on the RLIS in cervical epithelial cells of postmenopausal women. The second objective of the study was to determine the paracellular mechanisms that are affected by menopause and the effect of estrogen on RTJ and RLIS in cultured cervical epithelia of postmenopausal women.
The experiments used cultures of human ectocervical epithelial (hECE) cells, which are normal cells derived from minces of the ectocervix and express the estrogen receptor. These cells were used in previous studies as a model of the ectocervical epithelium (3, 14, 15). In vivo, both the endocervical epithelium and the ectocervical epithelium control transudation of fluid into the cervical canal and the vagina (1). The main difference between the endocervical and ectocervical epithelia is the secretion of mucins, which normally are contributed only by the former. However, the permeability characteristics of hECE cells are similar to the permeability of the cervical epithelium in vivo, as calculated from sampling cervical secretions in women (1, 4, 16). hECE cells grow in vivo (1) and in vitro (3, 17, 18) as a stratified epithelium. We (19) and others (20) have shown that only the basal layer of cells determines the RPCR of stratifying epithelia and that suprabasal cells contribute less than 5% to the total resistance (17). These characteristics make hECE cultures an appropriate and useful model to study estrogen- and menopause-related regulation of transcervical paracellular permeability.
|
Patients and Methods |
|---|
|
TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
|---|
hECE cells were cultivated from minces of ectocervix of pre- and postmenopausal women 35–65 yr of age. Women were divided into three groups, based on age and on history of estrogen treatment in vivo: 1) group A: age, 35–45 yr; premenopausal women in the estrogenic phase of their menstrual cycle; 2) group B: age, 55–65 yr; postmenopausal women not treated with hormone replacement (estrogen or progestin) for at least 1 yr prior to the study; and 3) group C: age, 55–65 yr; postmenopausal women who received estrogen replacement for at least 6 months. Menopause, as defined by the treating physicians, was considered as amenorrhea for 1 yr or more, or amenorrhea of at least 6 months plus climacteric symptoms and plasma levels of FSH more than 25 mIU/mL. All women were selected among those who underwent a hysterectomy by their treating physician, for indications that were unrelated to the present study, and had a histologically normal cervix. Indications for hysterectomy included uterine fibroids, adnexal mass, abnormal uterine bleeding, and prolapsed uterus, and the ectocervical tissues were defined as discarded tissues. The study was carried out according to institutional regulations and had been approved by the hospital’s Institutional Review Board. After their removal, ectocervical tissues were washed with cold and sterile saline to clear mucus, carefully dissected of stroma, and placed in cold saline. Ectocervical tissues were minced under the microscope to the size of 1 mm and plated epithelial face down on culture dishes, as described (3).
Cell cultures
Primary to tertiary cultures of hECE cells were generated from minces of ectocervix, as described (3). The cells were grown and maintained in a culture dish at 37 C in DMEM/Ham’s F12 (3:1) supplemented with nonessential amino acids, 1.8–10-4 M adenine, 100 U/mL penicillin, 100 mg/mL streptomycin, 50 ng/mL gentamicin, 2 mM L-glutamine, 5 µg/mL insulin, 1–10-6 M hydrocortisone, 5 µg/mL transferrin, 2–10-9 M triiodothyronine, 10 ng/mL epidermal growth factor, and 8% FCS (Sigma, St. Louis, MO) in a 91% O2/9% CO2 humidified incubator and plated on filters for experiments (3). The cells were routinely tested for mycoplasma. For experiments with estrogen, cells on filters were shifted to steroid-free medium for 3–5 days (14). This medium was composed of phenol-red-deficient DMEM/Ham’s F12 or RPMI 1640 (Sigma) containing 8% heat-inactivated FBS that was previously treated with charcoal to remove steroids. Preparation of charcoal-treated serum was described (14); briefly, dextran-coated charcoal (Sigma) was dissolved at 8% in 0.15 M NaCl, autoclaved, mixed by stirring, spun, and the pellet was resuspended as 1 g/1.25 mL in H2O. FBS (HyClone Laboratories, Inc., Logan, UT) was mixed with the activated charcoal-dextran at 20:1 (vol:vol) and incubated for 45 min at 55 C. At the completion of incubation the mixture was spun twice at 800 x g for 20 min and the supernatant (serum) was decanted and collected.
Changes in paracellular permeability were determined in terms of changes in the permeability to cell-impermeant tracers (1, 19) and in terms of changes in the RTE. Because permeability relates reciprocally to resistance, RTE was expressed in terms of the transepithelial electrical conductance (GTE). Before experiments, filters containing cells were washed three times and preincubated for 15 min at 37 C in a modified Ringer buffer composed of 120 mM NaCl, 5 mM KCl, 10 mM NaHCO3 (before saturating with 95% O2/5% CO2), 1.2 mM CaCl2, 1 mM MgSO4, 5 mM glucose, 10 mMn-(2-hydroxyethyl) piperazine-N'-2-ethanesulphonic acid (pH 7.4), and 0.1% BSA in volumes of 4.7–5.2 mL in the luminal and subluminal compartments.
Flux experiments
Changes in epithelial permeability to cell-impermeant molecules were determined from measurements of unidirectional (luminal to subluminal) fluxes of cell-impermeant tracers across filters mounted vertically in a modified Ussing/diffusion chamber (to prevent hydrostatic gradients), as described (3, 19). Most experiments used pyranine, which is a trisulfonic acid with a mol wt of 510 that traverses epithelia via the paracellular pathway, and its concentration can be measured down to nanomolar levels by fluorescence techniques (3, 19). Cytolysis of hECE cells that were previously incubated with 0.1 mM pyranine did not increase pyranine fluorescence significantly above background (data not shown). Pyranine (Ppyr) was determined from unidirectional (luminal to subluminal) fluxes: 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 from the area-normalized unidirectional pyranine flux as Ppyr = -JP/[Pyr]cis, where JP is the unidirectional pyranine flux and [Pyr]cis is the concentration of pyranine in the cis (luminal) compartment. Pyranine fluxes were calculated by JP = ([Pyr]cis) x ([Ftrans])/([Fcis] x S x time), where F is the background-corrected pyranine-fluorescence in the cis and trans compartments and S is the surface of the filter insert (0.6 cm2). Some experiments also used other tracers at the following concentrations or specific activities: 10 µCi/mL [14C]mannitol (mol wt, 180), 12 µCi/mL [14C]sucrose (mol wt, 320), and 10-4 M polydextrans (mol wt of 3, 10, 40, and 70 kDa). The specific activity of the tracers was determined by measuring either radioactivity ([14C]mannitol and [14C]sucrose) in a Beckman Coulter, Inc. (Boston, MA) LS 3801 ß counter, or fluorescence (Pyranine and polydextrans) in an Amino Bowman Spectrophotofluorimeter (American Instrument Company, Inc., Silver Spring, MD). The excitation/emission wavelengths (nm) for the latter were: pyranine, 390/520; polydextrans 3K and 70K (rhodamine), 540/560; polydextran 10K (cascade blue), 360/445; and polydextran 40K (fluresceine), 490/520.
Determinations of GTE
GTE was chosen as an end point to assess
paracellular permeability because this method is sensitive to small
changes in transepithelial permeability and it allows for real-time
measurements of changes in transepithelial conductance. The
electrophysiological methods, including appropriate measures to prevent
artifacts were previously discussed by us (21) and by others (6).
Changes in GTE were determined continuously
across filters mounted vertically in a modified Ussing chamber from
successive measurements of the transepithelial electrical current
(
I, obtained by measuring the current necessary to clamp the offset
potential to zero and normalized to the 0.6-cm2
surface area of the filter) and the PD (lumen negative), switching
between I (pulses of 200-1400 µAmp/cm2) and PD at a
rate of 20 Hz: GTE = I/ PD (21). The
volumes and content of the buffer solutions used in the Ussing chamber
experiments were kept constant. The temperature in the chamber was
maintained at 37 C, and the medium in each compartment was continuously
bubbled with an airlift of 95% O2/5%
CO2 that flows parallel to the surface of the
culture. The output of the voltage clamp was recorded in parallel on a
strip-chart recorder (Linseis L6514; Linseis, Cleveland, OH) and
on an IBM-PC 386–30 equipped with a DI-220 A/D converter board, a
AT/MCA CODAS hardware and software (DATA Q, Akron, OH), and a Bernoulli
90 megabyte hard disk. The CODAS board allows acquisition of up to
50,000 samples/sec with gap-free display. AT-playback provides for data
analysis.
The experimental design of the electrophysiological measurements,
including calibrations and controls, the significance of the PD and
I, and the conditions for optimal determinations of
GTE across low-resistance epithelia
(e.g. hECE cells), were described and discussed (21, 22). In
hECE cultures the electrical resistance of the filter itself as
measured in the Ussing chamber is about 20
/cm2, which is approximately 1.5–2-fold
higher than the resistance generated by the cells. To determine the
epithelium-generated resistance, the following steps were taken: 1)
every experiment included additional controls, such as blank filters
without cells, and filters containing cells grown in regular medium
with known GTE, based on previous studies (21, 22); 2) each experiment began by adjusting the potential difference
across a blank filter without cells to zero, to allow for subtraction
of the filter-generated resistance; 3) at the conclusion of some
experiments, calcium in the medium was lowered to zero by adding 2
mM EGTA to disrupt intercellular connections,
including the tight junctions (23, 24, 25), and to abolish the
RPCR. Under these conditions, the
GTE was similar to the conductance of the filter
itself.
Determinations of the dilution potential (Vdil) were performed in the
Ussing chamber as described (21). Transepithelial dilution potentials
(Vdil) were determined by measuring the effect of lowering NaCl in the
luminal solution on changes in voltage generated across the epithelial
culture. This was done by replacing the Ringer’s buffer in the luminal
compartment (130 mM NaCl) with low (10 mM) NaCl
solution. The latter buffer was similar to the Ringer’s solution,
except that it lacked 120 mM NaCl and was supplemented with
240 mM sucrose to compensate for osmolarity. The methods of
electrophysiological data evaluation were previously described and
discussed (21). Vdil was the measured potential difference
(VoltageSL-VoltageL) after
lowering NaCl in the luminal solution, corrected for the
potential-electrodes asymmetry, where the subscripts
SL and L are the subluminal
and luminal solutions, respectively. The Henderson diffusion equation
for monocations and monoanions was used to interpret the
transepithelial dilution potential in terms of ionic permeabilities
(26). With the assumption that Na+ and
Cl- are the major permeant ionic species, the
relative mobilities of Na+ and
Cl- in the intercellular space
uCl and uNa can be
determined as ß= uCl/uNa = (K +
||Vdil||)/(K - ||Vdil||), where K (R x T/F)
x ln(NaSL/NaL) = 68.5 mV
at the given [Na+SL] = 130
mM, and
[Na+L] = 10
mM (27).
Generation of hydrostatic and hypertonic gradients
Aliquots of buffer were added to the subluminal compartment to establish hydrostatic gradients of 5–20 mm H2O in the subluminal to luminal direction (4). Extracellular osmolarity in the subluminal side was increased from 285 mosmol/L to 325 mosmol/L by adding aliquots (about 120 µL) of 2 M sucrose solution to the subluminal solution, thus establishing a subluminal to luminal hypertonic gradient (4). To compensate for volume changes, a similar amount of Ringer’s buffer was added to the luminal solution.
For all experiments using cells on filters, reagents were added from concentrated stocks (x300–x1000) of either 1% ethanol or saline to both the luminal and subluminal solution.
Determinations of free calcium
Levels of calcium in the extracellular buffer were manipulated using the calcium chelator EGTA. Concentrations of free calcium were calculated as described (24, 28).
Statistical analysis of the data
Data are presented as means (±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 analyzed using ANOVA.
Chemicals and supplies
Anocell (Anocell-10) filters were obtained from Anotec (Oxon, UK). All other chemicals were obtained from Sigma.
|
Results |
|---|
|
TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
|---|
The first objective of the study was to determine the paracellular
permeability characteristics of cultures of hECE cells obtained from
postmenopausal women and to compare the results to hECE cultures from
premenopausal women. Ectocervical tissues were collected from a total
of 24 women. Group A included nine premenopausal women who underwent
hysterectomy 8–11 days after a menstrual cycle and were diagnosed as
being in the estrogenic phase of their menstrual cycle as confirmed by
pathology reports of the endometrium. Group B included seven
postmenopausal women who have not used hormone (estrogen or progestin)
replacement for at least 1 year prior to surgery. Group C included
eight postmenopausal women who were treated with estradiol orally or
transdermally, or with conjugated estrogens, for more than 6 months
prior to surgery. All women in group C also used add-on progestins, but
all underwent hysterectomy 1–2 weeks after stopping the progestin.
Primary hECE cultures were successfully generated from all patients and
sufficed for the experiments shown in Fig. 1
, A and B. Secondary and
tertiary cultures were generated from some patients and were use for
subsequent experiments.
|
shows the results of GTE (Fig. 1A) and
Ppyr determinations (Fig. 1B) in cultures of hECE cells obtained from
women in groups A–C. In premenopausal women (group A), GTE
was 76 ± 3 mS/cm2 and significantly higher than in
postmenopausal women not treated with estrogen (group B, 37 ± 2
mS/cm2, P < 0.01). In postmenopausal women
treated with estrogen (group C), GTE was 57
± 2 mS/cm2; this level was significantly higher than
in group B (P < 0.01), but significantly lower than in
group A (P < 0.01, Fig. 1A). Similar trends were
obtained using Ppyr as the end point for paracellular permeability. In
group A, Ppyr was 13.5 ± 3.1
cm/sec1·10-6
and significantly higher than in group B (7.1 ± 1.9
cm/sec1·10-6, P
< 0.01). In group C, Ppyr was 9.0 ± 0.9
cm/sec1·10-6; these levels were
significantly higher than in group B (P < 0.01), but
significantly lower than in group A (P < 0.01, Fig. 1B). These results indicate: 1) hECE cells of postmenopausal women form
epithelia with lower paracellular permeability than hECE cells of
premenopausal women; and 2) treatment of postmenopausal women with
estrogen in vivo increases paracellular permeability of hECE
cultures, but levels remain lower compared with premenopausal
women. Effects of estrogen on paracellular permeability
The results shown in Fig. 1, A and B, suggest that treatment with
estrogen in vivo increases the paracellular permeability
across cultured hECE cells of postmenopausal women. These experiments
used cells that were outgrown from cervical minces in regular culture
medium containing phenol red and enriched with FCS. It is, therefore,
possible that cells were exposed to estrogen in vitro, and
the results in Fig. 1 in groups A–C do not accurately reflect the
effects of menopause and estrogen in vivo on paracellular
permeability. The experiments in Fig. 2, A and B, were designed to answer this question. Cells were obtained
from premenopausal women (group A) and from postmenopausal women not
treated with estrogen (group B). Cells plated on filters were grown in
steroid-free medium and supplemented with 10 nM
17ß-estradiol (or the vehicle) for 2 days before determinations of
GTE and Ppyr. The objective was to deplete cells
of estrogen, or alternatively to control the exposure to estrogen,
using well described methods (14, 15).
|
, 
) and significantly lower than in
cells of group A that were maintained in regular medium (Fig. 1A, P < 0.01). Treatment with estradiol in
vitro increased GTE to 96 ± 10
mS/cm2 (Fig. 2A, 
), confirming previous results in cell
of premenopausal women (14, 15). In cultures of hECE cells from
postmenopausal women not treated with estrogen (group B) incubation in
steroid-free medium lowered GTE to 32 ± 3
mS/cm2 (Fig. 2A, ). This level was lower than in cells
of group B, which were maintained in regular medium (Fig. 1A, P < 0.01). Treatment with estradiol in
vitro increased GTE to 65 ± 4 mS/cm2
(Fig. 2A, ). This level was higher than in cells of postmenopausal
women not treated with estrogen in vivo (group B, Fig. 1A, P < 0.01), but similar to that in cells of
postmenopausal women who were treated with estrogen in vivo
(group C, Fig. 1A, P > 0.1). Similar trends were
observed using Ppyr as the end point for paracellular permeability
(Fig. 2B). Collectively, the results shown in Figs. 1 and 2 indicate
similar trends—that lack of estrogen decreases the permeability and
treatment with estrogen in vivo or in vitro
reverses the effect and increases the permeability.
Treatment with 10 nM 17ß-estradiol in vitro
increased GTE and Ppyr in cells both of
postmenopausal and premenopausal women, but levels in the former
remained lower than in the latter (Fig. 2). In cells of premenopausal
women 10 nM 17ß-estradiol exerts a maximal
effect on permeability (14). A possible explanation to the finding that
the permeability in cells from postmenopausal women did not increase to
a similar level as in cells from premenopausal women is that the
concentration of 10 nM 17ß-estradiol did not
exert a maximal effect on permeability. To determine the concentration
of 17ß-estradiol that exerts maximal effect on
GTE in cells of postmenopausal women, the
response to different doses of estradiol was studied. As is shown in
Fig. 3, in hECE cultures obtained from
postmenopausal women GTE increased already with
0.1 nM, and saturation was achieved with 10
nM 17ß-estradiol. The
EC50 of estradiol was 1.2
nM, and the dose-response curve could be fitted
into a modified Hill equation with a Hill coefficient number of
1.1 (Fig. 3), suggesting interaction of estradiol with single-class
binding sites. These responses are similar to the effects of
17ß-estradiol in cells of premenopausal women (14, 15). Therefore,
the increase in permeability in cells from postmenopausal women in
response to 10 nM 17ß-estradiol is a maximal
effect. Collectively, the results shown in
Figs. 1–3 indicate that
steroid deprivation decreases paracellular permeability across cultured
hECE cells and 17ß-estradiol, at concentrations that are
physiological in premenopausal women, can increase the permeability
regardless if cells were obtained from premenopausal or postmenopausal
women. However, the permeability across cultures from postmenopausal
women remained low compared with premenopausal women, even after
treatment with estrogen.
|
The experiment was done using cells from groups A and B that were
cultured in steroid-free medium, or were treated with 17ß-estradiol,
similar to the design in Fig. 2. As is shown in Fig. 4, in all four experimental groups of
cells the paracellular permeability decreased exponentially with the
mol wt of the probe. In cells of group B (postmenopausal women) the
permeability for large molecules decreased to a greater degree than in
cells of group A (premenopausal women). In both groups treatment with
estradiol increased the permeability, and in both groups the
permeability for large molecules increased to a greater degree than the
permeability for smaller molecules (Fig. 4). These results indicate
that the differences in permeability among hECE cultures of
premenopausal and postmenopausal women involve differences in the
paracellular permeability and that in both types of cells the
estrogen-induced increase in permeability involves increases in
paracellular permeability.
|
The second objective of the study was to understand how the menopause transition affects paracellular mechanisms that control cervical permeability. In epithelia, including cultured hECE cells, the paracellular permeability is determined by the RTJ and by the RLIS (5, 21). The specific objective of the next experiments was to determine the degree to which menopause and estrogen modulate RLIS and RTJ.
To determine the degree to which menopause affects
RTJ, cells were exposed to conditions that are
known to modulate tight junctions in cultured human cervical epithelia.
These conditions include lowering extracellular calcium, treatment with
cytochalasin-D, and activation of protein kinase-C by sn-1,2-dioctanoyl
diglyceride (diC8). Lowering extracellular calcium decreases
RTJ by changing the gating properties of tight
junctional proteins (24). Calcium in the extracellular medium was
lowered from the normal level of 1.2 mM to 1.0–0.4
mM by adding aliquots of the calcium chelator EGTA.
Cytochalasin-D perturbs F-actin (29, 30) and decreases
RTJ by disrupting the cytoskeleton (14). DiC8 is
a cell-permeable stable analog of diacylglycerol, and it increases
RTJ by a mechanism that involves activation of
calcium-dependent protein kinase-C (28). The experiments were done on
cells from groups A and B that were cultured in steroid-free medium, or
were treated with 17ß-estradiol, similar to the design in Fig. 2.
Lowering extracellular calcium increased GTE in a
dose-related manner (Fig. 5A). In cells
of premenopausal women GTE increased to higher
levels than in cells of postmenopausal women, but the net increases in
the latter were bigger than in the former. For instance, in cells of
postmenopausal women lowering extracellular calcium to 0.6
mM increased GTE 10-fold, compared
with a 6-fold increase in cells of premenopausal women (Fig. 5A).
Estrogen had no significant effect on the responses in cells bathed in
1.0 and 0.8 mM calcium, and the differences in permeability
that prevailed at baseline conditions remained also after lowering
extracellular calcium (Fig. 5A). However, pretreatment with estrogen
augmented the responses in cells bathed in 0.6 mM
calcium.
|
. In these
experiments, cytochalasin-D was used at concentrations of 0.1–10
µg/mL for 30 min, which produce submaximal changes in
GTE (16). Cytochalasin-D increased
GTE in a dose-related manner (Fig. 5B). In cells
of postmenopausal women, 0.1 and 1 µg/mL cytochalasin-D increased
GTE 8-fold, compared with a 4-fold increase in
cells of premenopausal women (Fig. 5B). These trends were similar to
those induced by lowering extracellular calcium, but they did not reach
statistical significance. At 10 µg/mL, cytochalasin-D increased the
permeability to about 600 mS/cm2 (Fig. 5B); above this
level, quantification of changes in GTE is
nonlinear and unsatisfactory (21, 24). Estrogen had no significant
effect on the responses to cytochalasin-D, and the differences in
permeability that prevailed at baseline conditions remained after
treatments with 0.1 or 1 µg/mL cytochalasin-D (Fig. 5B).
Collectively, the results in Fig. 5, A and B, indicate: 1) menopause
shifted the dose-response curves of [GTE]
vs. [extracellular calcium] and
[GTE] vs. [cytochalasin-D] to the
right; 2) net increases in GTE were bigger in
cells of postmenopausal women than in cells of premenopausal women; and
3) the estrogen-induced increases in GTE were
additive to those induced by mild perturbations of extracellular
calcium and by 0.1 and 1 µg/mL cytochalasin-D.
Treatment of cells with diC8 decreased GTE in all
four groups of cells (Fig. 6), but net
decreases in GTE in cells of postmenopausal women
were significantly greater than in cells of premenopausal women.
Estrogen had no significant effect on the responses to diC8
(inset in Fig. 6). Collectively, the results shown in Figs. 5 and 6 indicate that lowered extracellular calcium, cytochalasin-D,
and diC8 modulate GTE significantly more in cells
of postmenopausal women than in cells of premenopausal women.
|
Hydrostatic gradients (Fig. 7A) and
hypertonic gradients (Fig. 7B) increased GTE in
cultures of cells from premenopausal and postmenopausal women. In cells
maintained in steroid-free medium, a hydrostatic gradient of 20 mm
H2O and a hypertonic gradient of 40 mosmol/L
increased GTE by about 30 mS/cm2,
regardless if cells were obtained from premenopausal or from
postmenopausal women (insets in Fig. 7). In cells treated
with estradiol GTE increased by about 70
mS/cm2 both in cells that were obtained from premenopausal
women and from postmenopausal women (insets in Fig. 7, P < 0.01 for both hydrostatic and hypertonic
gradients). These results indicate that hydrostatic and hypertonic
gradients in the subluminal to luminal direction increase
GTE more in estrogen-treated cells than in cells
grown and maintained in steroid-free medium, and the effect is similar
in cultures of cells from premenopausal and postmenopausal women.
|
The experiments shown in
Figs. 5–7 suggest that the low
permeability across cultures of cells obtained from postmenopausal
women and maintained in estrogen-deficient medium is determined by high
RTJ. The objective of the next experiment was to
confirm this finding, using measurements of the dilution potential
(Vdil) and determinations of the ratio of mobilities of
Cl- to Na+ across the
epithelium (uCl/uNa). These
parameters were chosen because tight junctions influence the mobilities
of monoions, and the cation selectivity reflects the degree of
occlusion of the paracellular space by the tight junctions (12). The
positive control in this experiment was the effect of treatment with 1
µg/mL cytochalasin-D for 30 min; as is shown in Table 1, cytochalasin-D increased
GTE across cultures both of postmenopausal women
and of premenopausal women. The changes in GTE
were associated with a decrease in Vdil and an increase in
uCl/uNa (Table 1),
supporting previous studies that cytochalasin-D increases permeability
by abrogating the RTJ (16).
|
). This result supports the hypothesis that the lower
permeability across cultures of postmenopausal women is associated with
higher RTJ. Treatment of cells in
vitro with 17ß-estradiol increased GTE in
both types of cells, but had little effect on Vdil and on
uCl/uNa (Table 1). This
result supports the hypothesis that the estrogen-increase in
permeability is not associated with changes in
RTJ. Contributions of RTJ and RLIS to the total RPCR
The objective of the next experiment was to estimate the relative
contributions of RLIS and
RTJ to the total RPCR in
hECE cultures of cells obtained from postmenopausal women. Cells
maintained in steroid-free medium were shifted to low calcium medium to
increase GTE submaximally to about 600
mS/cm2 and then exposed to hydrostatic gradients in the
subluminal to luminal direction. In these experiments, we used
hydrostatic gradients that were generated by low hydrostatic pressures
of 5–20 mm H2O, which prevail in the capillary
bed in vivo (32). As is shown in Table 2, hydrostatic gradients increased
GTE in a dose-response manner in cells of both
premenopausal and postmenopausal women, confirming our previous studies
in cultured human cervical epithelia (4, 33).
|
). By extrapolation to the submaximal
GTE of 600 mS/cm2, the hydrostatic
gradient-dependent increase in GTE
contributed 100/600, or about 16%, to the total paracellular
permeability. Because the RPCR is determined by
RLIS and RTJ in series (5),
this result suggests that in cultures of premenopausal women the
RLIS contributes 16% and the
RTJ about 84% to the total
RPCR.
To obtain submaximal increase in GTE in cells of
postmenopausal women, it was necessary to lower extracellular calcium
to 0.4 mM. In cells of postmenopausal women hydrostatic
gradients also had an additive effect on GTE to
that of low extracellular calcium (Table 2). At 0.4 mM
calcium a hydrostatic gradient of 20 mm H2O
increased GTE by about 20 mS/cm2,
regardless of the calcium level in the extracellular medium (Table 2).
By extrapolation to a submaximal GTE of 600
mS/cm2, the hydrostatic gradient-dependent increase in
GTE contributed 20/600, or about 3%, to the
total paracellular permeability. This suggests that in hECE cultures of
postmenopausal women the RLIS contributes 3%,
and the RTJ about 97% to the total
RPCR.
|
Discussion |
|---|
|
TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
|---|
The statement that some of the increase in RPCR results from an increase in RTJ is supported by the following experimental findings: 1) hECE cells obtained from postmenopausal women formed confluent cultures on filters that restricted the free movement of cell-impermeant molecules and electrical charge across the culture; 2) the paracellular permeability for a given molecule decreased exponentially with the molecular weight of that molecule, thus supporting the hypothesis that tight junctions of cultured human cervical epithelia can be modeled as a series of parallel narrow tubes, as we have previously proposed (24). According to this model, the movement of fluid through the tight junctional spaces can be predicated in terms of laminar flow by Poiselle’s law (34); 3) the paracellular permeability across hECE cultures of postmenopausal women could be increased by lowering extracellular calcium and by treatment with cytochalasin-D, conditions that modulate RTJ (16, 24). Likewise, the paracellular permeability could be decreased by diC8, which increases RTJ in cultured human cervical epithelia (4, 35); and 4) cultures of postmenopausal women expressed greater transepithelial, cytochalasin-D-sensitive cation selectivity than cultures of premenopausal women. Because cation selectivity is a property determined by tight junctions (12), the present results suggest that the tight junctions expressed by hECE cells of postmenopausal women impede effectively the movement of fluid and solutes through the intercellular space.
The RTJ modulators’ low extracellular calcium, cytochalasin-D, and diC8 resulted in greater net changes in GTE in cells of postmenopausal women than in cells of premenopausal women. These results can be explained according to hydrodynamic principles of Poiselle’s law (34): for a given increase in the diameter of a tube, such as that induced by lowered extracellular calcium or by cytochalasin-D, the net decrease in RPCR will be greater for smaller tubes. The present results support this model in hECE cultures because in cultures from postmenopausal women, which have higher baseline resistance than cultures from premenopausal women (and presumably narrower tight-junctional space), low extracellular calcium and cytochalasin-D increased GTE to a greater degree than in cultures of premenopausal women. Poiselle’s law also predicts that for a given decrease in the diameter of a tube, such as that induced by diC8, the net increase in RPCR will be greater for smaller tubes. The results with diC8 support this statement as well. These results support the modeling of tight junctions of cultured human cervical epithelia as a series of parallel narrow tubes, as we have previously proposed (24).
At present, little it is known about what molecular mechanisms regulate the increase in RTJ after menopause. The tight junctional apparatus is a complex of strands and grooves formed by plasma membranes of neighboring cells. It involves proteins that face the extracellular space, as well as intracellular proteins that are associated with cytoskeletal proteins (36, 37). The strands and grooves narrow the intercellular space at the region of the tight junction and form spaces that are modeled functionally as a series of parallel tubes (24). The tight junctional apparatus can be regulated (10, 11, 21), and the present results indicate that the menopause transition in women can affect the RTJ, but the effect is unrelated to lack of estrogen. We are currently studying what aging-related mechanisms may be involved in the increase in RTJ in hECE cells of postmenopausal women.
Some of the decrease in paracellular permeability in hECE cultures from
postmenopausal women was the result of an increase in the
RLIS. This conclusion is supported by the
findings related to treatment with estrogen. In human cervical
epithelial cells estrogen increases paracellular permeability. The
signaling and mechanism of estrogen action have been recently
characterized (15, 38, 39). They involve
estrogen-receptor-
-dependent increase in nitric oxide and in cGMP;
and cGMP-dependent protein kinase stimulation of ADP-ribosylation of
monomeric G-actin and fragmentation of the cytoskeleton (14). Cells
become more deformable and tend to decrease their size more readily in
response to stimuli that modulate the cytoskeleton. Decreases in cell
size cause reciprocal increase in the volume of the intercellular space
and decrease the RLIS (4).
In cells from postmenopausal women that were treated with estrogen in vivo the permeability was higher compared with cells from women not treated with estrogen, but lower compared with cells from premenopausal women. This finding was confirmed in experiments using estrogen treatment in vitro. In the latter experiments, cells were shifted to steroid-free medium to abrogate the effect of phenol red and to remove the estrogen that may have been present in the culture medium (14). The permeabilities of cultures from postmenopausal and from premenopausal women were lower after shifting cells to steroid-free medium. Treatment with 10 nM 17ß-estradiol, a concentration that is in the physiological range for premenopausal women, increased the permeability to levels that prevailed in primary cultures grown in regular culture medium. This result indicates that the effect of estrogen-deprivation on permeability was reversible on adding estradiol, suggesting that hECE cells of postmenopausal women express the estrogen receptor and maintain the signaling machinery that leads to decreased RLIS. It also suggests that the effect of estrogen on RLIS does not depend on the status of menopause or on the woman’s age.
Treatment with 10 nM 17ß-estradiol increased permeability to a similar degree in cultures from cells of premenopausal and postmenopausal women. The estrogen-induced increase in permeability was less than that induced by modulators of RTJ, indicating that changes in RLIS have a limited capacity to modulate the total RPCR. This conclusion is also supported by the experiments with the RLIS modulators’ hydrostatic and hypertonic gradients. Similar to previous studies in other types of cultured human cervical epithelia (4, 14), hydrostatic and hypertonic gradients in the subluminal to luminal direction increased the permeability also in hECE cells from postmenopausal women, and the effects were independent and additive to those induced by modulators of the RTJ. Furthermore, in cells from postmenopausal women the net increase in permeability that was induced by estrogen (modulator of the RLIS) (14) was similar to that induced by hydrostatic and hypertonic gradients. One of the conclusions from these results is that lack of estrogen contributes to some of the increase in RPCR in hECE cells of postmenopausal women.
Unlike the results with RTJ modulators, the absolute changes in permeability induced by hydrostatic or hypertonic gradients were similar in cultures of hECE cells from premenopausal and from postmenopausal women. Because baseline permeabilities differed between these two types of cells, Poiselle’s law cannot provide an explanation to the similar responses; namely, the increase in RLIS in response to hydrostatic or hypertonic gradients cannot be explained by simple tubular expansion of the lateral intercellular space. Another explanation is that hydrostatic and hypertonic gradients increase the volume of the lateral intercellular space by changing its shape (31). This causes asymmetrical expansion of the intercellular space that cannot be described in terms of Poiselle’s law.
By probing the RTJ with low extracellular calcium and the RLIS with hydrostatic gradients, it was possible to estimate the relative contributions of RTJ and RLIS to the total RPCR. The results suggest that in hECE cultures from postmenopausal women RTJ contributes 97% to the RPCR, in contrast to 84% in hECE cultures from premenopausal women. Because RTJ is a high-resistive element that restricts movement of fluid and solutes to a greater degree than the low-resistive RLIS (12), this model explains the lower transepithelial permeability across cultured hECE cells from postmenopausal women than across cultures of premenopausal women.
The present results provide novel mechanistic data that may help us understand regulation of cervical secretions in premenopausal and postmenopausal women. Human cervical epithelial cells form epithelia with low RPCR (present results and Refs. 3, 4). In "leaky" epithelia the main driving force for fluid transudation is the pressure difference between blood and lumen. Estrogen can increase the permeability by decreasing the RLIS (Refs. 14, 15 and present results). Changes in paracellular permeability also regulate fluxes of solutes and filter molecules by mol wt and electrical charge (19). Thus, the estrogen decrease in RLIS can explain how estrogen increases cervical secretions in premenopausal women and affects the composition of the cervical mucus during the preovulatory phase of the cycle. After menopause, women produce less estrogen. Lack of estrogen can increase the RLIS (present results) and decrease blood flow in the cervix (1). Both effects tend to decrease transudation of fluid from the blood into the cervical canal and decrease cervical secretions. More importantly, after menopause RTJ increases (present results), so that the cervical epithelium is transformed into a relatively "tight epithelium." In epithelia with high RPCR, the tight junctions significantly restrict movement of fluid through the intercellular space and the blood pressure-induced hydrostatic gradient is insufficient to drive the fluid from the blood into the lumen. In these epithelia, water moves from the blood into the lumen secondary to activation of ion transporters and secretion of ions to the extracellular milieu, to compensate for osmolarity (12). hECE cells express Na+ and Cl- transporters that can be activated by neurotransmitters and agents that participate in the inflammatory/infectious response (40), suggesting that hECE cells of postmenopausal women have the potential to increase water transport indirectly. Examples are sexual intercourse (secondary to activation of local neurotransmitters) or infections. However, in contrast to the situation in leaky epithelia, water transudation in tight epithelia is more restricted (6, 10, 11). These changes can explain the diminished secretion of fluid in postmenopausal women that lead to cervical and vaginal dryness.
In the present study, treatment with estrogen could decrease RLIS and increase the permeability, but the permeability remained lower compared with that in cultures from premenopausal women. This experimental result may have clinical significance and explain why older postmenopausal women respond less favorably to estrogen relative to genital dryness than younger women. The present results and our previous studies in the field (14, 15, 38, 39) suggest that it may be possible to augment fluid transudation into the genital canal through increasing paracellular permeability, by targeting mechanisms distal to the site of estrogen action. However, more studies are needed to explore this possibility.
|
Footnotes |
|---|
Received February 21, 2000.
Revised March 31, 2000.
Accepted April 13, 2000.
|
References |
|---|
|
TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
|---|
and ß in the regulation of cervical permeability.
Am J Physiol. 278:C689–C696.
This article has been cited by other articles:
 |
|
 |
|
  G. I. Gorodeski Estrogen Decrease in Tight Junctional Resistance Involves Matrix-Metalloproteinase-7-Mediated Remodeling of Occludin Endocrinology, January 1, 2007; 148(1): 218 - 231. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Li, L. Zhou, and G. I. Gorodeski Estrogen Regulates Epithelial Cell Deformability by Modulation of Cortical Actomyosin through Phosphorylation of Nonmuscle Myosin Heavy-Chain II-B Filaments Endocrinology, November 1, 2006; 147(11): 5236 - 5248. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. I. Gorodeski Aging and Estrogen Effects on Transcervical-Transvaginal Epithelial Permeability J. Clin. Endocrinol. Metab., January 1, 2005; 90(1): 345 - 351. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. H. Wasserman Vitamin D and the Dual Processes of Intestinal Calcium Absorption J. Nutr., November 1, 2004; 134(11): 3137 - 3139. [Full Text] [PDF]
|
|
|
|
 |
|
 M. Espinosa, G. Noe, C. Troncoso, S.B. Ho, and M. Villalon Acidic pH and increasing [Ca2+] reduce the swelling of mucins in primary cultures of human cervical cells Hum. Reprod., August 1, 2002; 17(8): 1964 - 1972. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. I. Gorodeski Regulation of transcervical permeability by two distinct P2 purinergic receptor mechanisms Am J Physiol Cell Physiol, January 1, 2002; 282(1): C75 - C83. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 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 |
