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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-1223
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 1 345-351
Copyright © 2005 by The Endocrine Society

Aging and Estrogen Effects on Transcervical-Transvaginal Epithelial Permeability

George I. Gorodeski

Departments of Reproductive Biology, Physiology and Biophysics, and Oncology, 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}cwru.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The objective of the study was to understand age-related contributions of the resistance of the intercellular tight junctions (RTJ) and the resistance of the lateral intercellular space (RLIS) to the transcervical-transvaginal permeability. The experiments used normal human ectocervical epithelial cells obtained from women aged 36–65 yr. Twenty-four hours of treatment of cells with 10 nM 17ß-estradiol decreased the RLIS, whereas longer treatments were required to decrease The RTJ. Aging had no effect on baseline or estrogen decrease in RTJ, but it blocked both baseline and the estrogen decrease in RLIS. Actin assays showed age-related decrease in the fraction of monomeric G-actin and attenuation of sodium-nitroprusside-induced increase in G-actin. These results suggest that the aging-related diminished capacity of human ectocervical epithelial cells to remodel cellular actin from polymerized actin toward monomeric G-actin confers increased cell rigidity and therefore increased RLIS. Therefore, the hypoestrogenism-related decrease in RTJ and the hypoestrogenism- and aging-related increase in RLIS could be the cellular mechanisms of decreased permeability that lead to decreased fluid transport and decreased lubrication of the lower genital tract in older postmenopausal women.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
VAGINAL DRYNESS IS a common symptom of postmenopausal women, and it is the result of decreased fluid transport from the blood through the cervical-vaginal epithelia and into the lumen. Until recently this phenomenon was attributed to hypoestrogenism, but not all postmenopausal women improve on estrogens, and responses vary among older postmenopausal women (1). These clinical data suggest that estrogen may not be the only factor that contributes to the diminished cervical-vaginal secretions after menopause. The present study tested the hypothesis that aging, independent of estrogen, also affects the permeability of the cervical-vaginal epithelia thereby controlling transepithelial fluid transport.

The experiments used cultures of normal human ectocervical-vaginal (hECE) cells on permeable support, which are a good experimental model to study transcervical-transvaginal transport (2, 3). Results of studies using filter-generated hECE cultures can be modeled by the Ussing-Zerahn model of fluid transepithelial transport (Fig. 1Go) (4). This model predicts that the overall permeability properties of secretory epithelia are determined by the intercellular (paracellular) route and that the paracellular permeability is determined by the resistance of the intercellular tight junctions (RTJ) and the lateral intercellular space (RLIS) in series. The regions of the tight junction are considered high resistive element due to the occlusion of the intercellular space by the tight junctional complexes. In contrast, RLIS is considered a low-resistive element, and it is determined by the proximity of the plasma membranes of neighboring cells and the length of the intercellular space from the tight junctions to the basal lamina (5). Hence, the transepithelial permeability can be described in terms of the inverse of the net RTJ and RLIS as



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  		FIG. 1. The Ussing-Zerahn model of transepithelial transport. Intercellular tight junctions located near the apical border of the cells occlude the paracellular pathway. The impedance to fluid and solutes movement in the paracellular route is determined by the RTJ, shown schematically as a thick resistor, and the RLIS, shown as a thin resistor, in series.

 
Recent studies validated that statement (6, 7) and have determined a novel complex pattern of estrogen modulation of both the RTJ and RLIS (8, 9).

The specific objective of the present study was to understand the effect of aging on the RTJ and RLIS. The results show an apparent lack of aging effect on cervical-vaginal RTJ but a significant aging-related increase in RLIS. Furthermore, hECE cells obtained from older women showed diminished capacity to remodel cellular actin toward monomeric G-actin. Because a shift in actin steady state toward polymerized actin confers increased cell rigidity (10, 11, 12) and therefore increased RLIS (13), the results suggest diminished deformability of the cytoskeleton of cervical-vaginal epithelial cells with age, which could be aggravated by estrogen deficiency. The net result would be reduced epithelial permeability as well as decreased fluid transport and decreased lubrication of the lower genital tract.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
The experiments used secondary-tertiary cultures of epithelial cells that were cultivated from histologically normal ectocervical tissues, using previously published methodology (2, 14, 15). Ectocervical tissues were obtained from pre- and postmenopausal women aged 35–65 yr. Menopause was defined by the treating physicians as amenorrhea for 1 yr or longer or amenorrhea of at least 6 months plus climacteric symptoms and plasma levels of FSH greater than 25 mIU/ml. Chosen for the study were samples of pre- and postmenopausal women not treated with sex steroid hormones for at least 3 months before tissue collection. All women were selected from among those who underwent a hysterectomy by their treating physician for indications that were unrelated to the present study and had 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. Tissues that were collected by the Cooperative Human Tissue Network at the University Hospitals of Cleveland and CASE (Case Western Reserve) University according to institutional review board protocol 03–90-TG. After their removal, tissues were washed with cold and sterile saline to clear mucus and blood, carefully dissected of stroma, and placed in cold saline. Ectocervical tissues were cut under the microscope to the size of about 1 mm and plated epithelial face down on culture dishes as described (2, 14, 15).

The epithelial cells outgrown from the peripheral rim of the ectocervix are identical with ectocervical-vaginal epithelial cells (2, 14, 15). The cells, designated hECE cells, grow and differentiate on filters as multilayered squamous stratifying epithelium and retain phenotypical and biological characteristics of the native ectocervical-vaginal epithelium (2, 14, 15). This makes the filter-generated cultures a suitable experimental model for the study of transcervical-transvaginal transport phenomena.

The experimental methods of cell culture techniques (2, 14, 15), and depletion of intracellular estrogens (13) were described. Changes in paracellular permeability were determined in terms of changes in the permeability to the cell-impermeant pyranine (Ppyr) (2) and changes in the transepithelial electrical resistance (RTE) (7). Changes in the RTJ were determined in terms of changes in the relative mobilities of Na+ and Cl– in the intercellular space (UCl/UNa) (6, 7, 16).

Transepithelial hydrostatic gradients were used to manipulate and assay changes in the RLIS. Transepithelial hydrostatic gradients were generated by adding aliquots of buffer to the subluminal compartment to establish hydrostatic gradients of 20 mm H2O in the subluminal to luminal direction (6, 7). This perturbation is similar in magnitude and equal in direction to the effect of blood pressure in the capillaries in vivo (17). Capillary blood flow exerts pressure in the subluminal to luminal direction, which dilates the intercellular space at basolateral aspects of the plasma membrane (18), thereby increasing the volume of the lateral intercellular space and reducing the RLIS (3, 6, 13).

Levels of calcium in the extracellular buffer were lowered by adding aliquots from 0.3 M EGTA. Concentrations of free calcium were calculated as described (6, 7). All other reagents were also added from concentrated stocks (x 300 to x 1000) of either 1% ethanol or saline to both the luminal and subluminal solutions. Cellular DNA and total protein content were determined as described (2). Cellular G-actin content was determined using the DNase-I inhibition assay (13).

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. Anocell (Anocell-10) filters were obtained from Anotec (Oxon, UK). All other chemicals were obtained from Sigma Chemicals (St. Louis, MO).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Age-related changes in transcervical-transvaginal permeability

Ectocervical tissues were collected from a total of 26 women and were grouped by age as follows: 36–40 yr (n = 5), 41–45 yr (n = 4), 46–50 yr (n = 5), 51–55 yr (n = 5), 56–60 yr (n = 3), and 60–65 yr (n = 4). One woman aged 49 yr, three women aged 51, 53, and 54 yr, and all women aged 55 yr and above were postmenopausal. Cultures of hECE cells were generated from those tissues and used to determine age-related changes in paracellular resistance. As shown in Fig. 2Go, levels of Ppyr decreased from a mean (± SD) of 22 ± 2 cm/sec·10–6 at ages 36–40 yr to 6 ± 2 cm/sec·10–6 at ages 61–65 yr, whereas levels of RTE increased from 15 ± 3 {Omega}/cm2 at ages 36–40 yr to 39 ± 4 {Omega}/cm2 at ages 61–65 yr. Because Ppyr relates directly to, and RTE inversely to, paracellular permeability (3), the data in Fig. 2Go indicate age-dependent decrease in transcervical-transvaginal paracellular permeability.



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  		FIG. 2. Age-related changes in permeability across cultured hECE cells. Cells generated from tissues of premenopausal women were maintained on filters in regular medium for 3 d after confluence. Determinations of Ppyr and RTE were made in the Ussing chamber on the same filter insert. Shown are means (± SD) of three to five specimens at each age group with two to four repeats per subject. The age-related changes for both Ppyr and RTE were significant (P < 0.01 for both).

 
Uncoupling RTJ and RLIS

To better understand the effects of age and estrogen on permeability, it was necessary to uncouple the contributions of the RTJ and RLIS to the total RTE because estrogen modulates both the RTJ (19) and RLIS (13). To modulate the RTJ, cells were treated with sn-1,2-dioctanoyl diglyceride (diC8), a cell-permeable analog of diacylglycerol that increases the RTE across hECE cells within minutes (Fig. 3AGo) by increasing the RTJ (3). Under these conditions the increase in RTE is near maximal (3), hence designated maximal RTE (Fig. 3Go). The RLIS was perturbed by using a hydrostatic gradient of 20 mm H2O in the subluminal to luminal direction (Fig. 3AGo). This perturbation produces near maximal decrease in RLIS (3), hence designated maximal RLIS (Fig. 3Go).



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  		FIG. 3. Uncoupling the RTJ and RLIS. Shown are experiments using hECE cells from premenopausal women. A, RTE was modulated by adding 10 µM diC8 followed by exposing cells to a hydrostatic gradient (HSG) of 20 mmH2O in the subluminal to luminal direction. B, RTE was modulated by diC8 followed by adding 0.8 mM EGTA. Results of 24 experiments are summarized in Table 1Go.

 
This experimental design was used to determine the contributions of RTJ and RLIS to the maximal RTE. After treatment with diC8, when RTE reached plateau, i.e. maximal RTE (Fig. 3AGo), cells were exposed to a hydrostatic gradient of 20 mm H2O in the subluminal to luminal direction. The decrease in RTE represents the maximal RLIS (Fig. 3AGo). Subsequently and based on the Ussing-Zerahn model (4), the differences between the diC8-induced increase in RTE and the hydrostatic gradient-induced decrease in RLIS represent the maximal RTJ (Fig. 3AGo). The validity of these determinations was tested by using EGTA to lower extracellular Ca2+. Calcium is required for gating the tight junctions (20, 21, 22), and lowering extracellular calcium across hECE cultures abrogates the RTJ (22). Lowering extracellular calcium decreased within minutes the RTE by abrogation of the RTJ, and the residual measured resistance is contributed mainly by the RLIS (Fig. 3BGo). Table 1Go summarizes results of 24 experiments in cultured hECE cells of premenopausal and postmenopausal women using the above experimental designs. The data show that baseline RTE as well as the responses to diC8, hydrostatic gradients, and EGTA depended on patient age and menopause, i.e. estrogen status. However, the calculated maximal RTJ and maximal RLIS were similarly irrespective whether cells were treated with diC8/hydrostatic gradient or diC8/EGTA.


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  		TABLE 1. Uncoupling modulation of RTJ and RLIS

 
Estrogen modulation of RTJ and RLIS

To better understand the effect of estrogen on permeability, cells were grown in a medium lacking estrogen, and the effect of estrogen was determined at successive time intervals after adding the hormone. Shifting hECE cells to steroid-free medium increased RTE to about 40 {Omega}/cm2 (Fig. 4Go). This increase was less than the maximal attainable RTE in hECE cells (about 51 {Omega}/cm2), suggesting that postmenopausal estrogen deficiency per se cannot explain the age-related increase RTE shown in Fig. 2Go. The data in Fig. 4Go also show that treatment with 17ß-estradiol at the physiological concentration of 10 nM stimulates a biphasic decrease in RTE: an early decrease in RTE that is induced by a decrease in RLIS and a slower additional decrease in RTE that was associated with an increase in UCl/UNa and is therefore the result of a decrease in RTJ.



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  		FIG. 4. Time course of estrogen effect on RTE (•) across hECE cultures and the effects on the ratio of monoion mobilities ({circ}, UCl/UNa). Cultures were generated from five postmenopausal women aged 53–57 yr. Cells were plated on filters, and after 2 d cells were shifted to steroid-free medium and treated with 10 nM 17ß-estradiol. At the indicated times after treatment with estrogen, filters were mounted in the Ussing chamber under sterile conditions for RTE and UCl/UNa determinations and returned to the incubator; measurements were then repeated on the same filter. Repeated measurements were done on 10 of 17 filters. The remaining filters became unsuitable for Ussing chamber experiments because of loss of confluence or contamination.

 
Aging- and estrogen-related modulation of RTJ and RLIS

To better understand the significance of the age-related decrease in cervical-vaginal permeability shown in Fig. 2Go, hECE cells cultured from women of different ages were grown in estrogen-free conditions for 72 h in the absence or presence of 10 nM 17ß-estradiol. At the completion of treatments, filters containing cells were mounted in the Ussing chamber, and changes in RTE were determined in response to 10 µM diC8 (maximal RTE), followed by perturbation with a hydrostatic gradient in the subluminal to luminal direction (to determine maximal RLIS and maximal RTJ as in Fig. 3AGo). Under conditions of estrogen deprivation, maximal RTE was approximately 40 {Omega}/cm2 in cells obtained from women aged 36–40 yr, and it increased to about 50 {Omega}/cm2 in cells obtained from women aged 61–65 yr (Fig. 5AGo). The age-related increase in maximal RTE was due mainly to an increase in the maximal RLIS (Fig. 5AGo). A similar trend was seen using experiments with diC8/low extracellular Ca2+ (Fig. 5BGo), and both methods determined relatively stable, age-independent maximal RTJ of about 33 {Omega}/cm2 (Fig. 5Go).



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  		FIG. 5. Age-related changes in permeability across cultured hECE cells deprived of estrogen. Cultures were generated from women aged 35–65 yr. Cells were plated on filters, and after 2 d cells were shifted to steroid-free medium for an additional 3 d. At the completion of incubation, filters were mounted in the Ussing chamber, and after stabilization cells were subjected to one of the following treatments: cells were treated with 10 µM diC8 followed by perturbation with a hydrostatic gradient of 20 mm H2O in the subluminal to luminal direction (A); cells were treated with 10 µM diC8 followed by 0.8 mM EGTA (B). Shown are means (± SD) of three to four specimens (i.e. women) at each age group with three to four filter repeats per subject. Data about maximal RTE (•), maximal RLIS ({circ}), and maximal RTJ ({blacktriangledown}) were determined as described in Fig. 3Go, A and B, respectively. In both A and B, the age-related trends for maximal RTE and maximal RLIS were significant (P < 0.01).

 
Treatment with 10 nM 17ß-estradiol for 72 h decreased maximal RTE across hECE cultures generated from tissues of women aged 36–55 yr; in cells obtained from women aged 56–65 yr, the effect of estrogen was blocked (Fig. 6Go, A and B). The estrogen-induced decrease in maximal RTE involved decreases both in maximal RTJ and maximal RLIS (Fig. 6Go, A and B; compare with Fig. 5Go, A and B). In estrogen-treated cells, maximal RTJ was approximately 21 {Omega}/cm2 and significantly lower than that determined across estrogen-deficient cells from women of the same age (approximately 33 {Omega}/cm2). Similar to observations made in cells maintained in steroid-free conditions (Fig. 5Go), aging had relatively little effect on the RTJ responses to estrogen (Fig. 6Go).



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  		FIG. 6. Age-related changes in permeability across cultured hECE cells treated with estrogen. Cultures were generated from women aged 35–65 yr as described in Materials and Methods. Cells were plated on filters, and after 2 d cells were shifted to steroid-free medium and treated with 10 nM 17ß-estradiol for an additional 3 d. At the completion of treatment filters were mounted in the Ussing chamber. A, Effects of diC8 and hydrostatic gradient (HSG), as in Fig. 5AGo. B, Effects of diC8 and EGTA, as in Fig. 5BGo. Data about maximal RTE (•), maximal RLIS ({circ}), and maximal RTJ ({blacktriangledown}) were determined as described in Fig. 3Go, A and B, respectively. Shown are means (± SD) of three to four specimens (i.e. women) at each age group with two to five filter repeats per subject. In both A and B, the age-related trends for maximal RTE and maximal RLIS were significant (P < 0.01).

 
In contrast, aging blocked the RLIS responses to estrogen (Fig. 6Go). Treatment with 10 nM 17ß-estradiol for 72 h decreased maximal RLIS across cultures generated from tissues of women aged 36–55 yr from 8.5 to about 4 {Omega}/cm2 (Figs. 5Go and 6Go). However, in cells obtained from women aged 61–65 yr, maximal RLIS was approximately 16 {Omega}/cm2 (Fig. 6Go), and it was similar to that across cultures of comparable women’s ages that were maintained in steroid-free medium (Fig. 5Go). Similar trends were obtained when assays used the diC8/low extracellular Ca2+ method (Figs. 5BGo and 6BGo). Collectively, these data suggest that aging negates the estrogen decrease in cervical-vaginal RLIS.

Aging effects on G-actin

In cultured hECE cells, estrogen modulation of the RLIS involves fragmentation of the cytoskeleton, and the effect is induced by shifting actin steady state from polymerized F-actin to monomeric G-actin (13, 23, 24, 25). The next experiment tested the hypothesis that the aging-dependent modulation of estrogen-induced decrease in RLIS is the result of inhibition of G-actin formation. The hypothesis was tested by measurements of total cellular actin and determinations of G-actin in hECE cells obtained from women aged 36–65 yr. Aging had relatively little effect on total cellular actin. In contrast, there was an age-related decrease in monomeric G-actin, which had decreased from about 29% in cells obtained from tissues of women aged 36–40 yr to 15% in cells obtained from tissues of women aged 61–65 yr (Fig. 7Go).



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  		FIG. 7. Age-related changes in total cellular actin ({circ}) and percent G-actin before (baseline, •) and after treatment with SNP ({diamondsuit}). Cultures of hECE cells were generated from women aged 35–65 yr. Cells were plated on filters, and after 2 d cells were shifted to steroid-free medium for an additional 3 d. At the completion of incubations, cells were treated for 20 min with 1 mM SNP. Total cellular actin and the percent G-actin were determined as described in Materials and Methods. Some filters with cells were preincubated for 15 min with 50 µM hemoglobin (not shown). Shown are means (± SD) of three to five specimens (i.e. women) at each age group with two to three filter repeats per subject. The age-related trends for baseline and post-SNP curves were significant (P < 0.01 for both). Total cellular actin is expressed as nanograms per 10–2 per total cellular protein (TP, in milligrams).

 
In hECE cells estrogen-induced increase in G-actin is mediated by estrogen receptor-{alpha}-dependent (26), nitric oxide (NO)-related activation of cGMP (27, 28, 29), and treatment with NO donors can bypass and mimic estrogen effect (27, 28). To determine whether aging affects the responsiveness to NO, cells were treated with sodium nitroprusside (SNP), a NO donor that can mimic cellular effects of NO (30). Treatment with SNP had no significant effect on total cellular actin (not shown), but it increased significantly G-actin regardless of the woman’s age (Fig. 7Go). However, the magnitude of SNP-induced increase in G-actin was age dependent: in cells obtained from tissues of women aged 36–40 yr SNP increased G-actin from 29 to about 42%; in contrast, in cells obtained from tissues of women aged 61–65 yr, SNP increased G-actin from 15 to only 21% (Fig. 7Go). Pretreatment of cells for 15 min with 50 µM of the NO scavenger hemoglobin (30) blocked the effect of SNP (not shown). These results suggest that aging blocks actin depolymerization in response to NO.

Figure 8Go summarizes the results as a schematic model, showing that the cervical-vaginal epithelial permeability is determined mainly by the contributions of the RTJ and RLIS to the paracellular resistance (RTE). Estrogen decreases the RTJ and the RLIS and would tend to decrease the RTE and increase the permeability. Aging affects the RTJ relatively little, but it blocks estrogen decrease in RLIS and increases the RLIS. The model predicts that in estrogen-deficient postmenopausal women, the decrease in cervical-vaginal epithelial is the result of lack of estrogen and the effect of aging on the RLIS.



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  		FIG. 8. Schematic representation of the results. The cervical-vaginal epithelial permeability is determined mainly by the contributions of the RTJ and RLIS to the paracellular resistance (RTE). Estrogen decreases both the RTJ and RLIS and therefore would tend to decrease the RTE and increase the permeability. Aging affects the RTJ relatively little, but it increases the RLIS by modulating estrogen effects on G-actin. The model predicts that in estrogen-deficient postmenopausal women, the cervical-vaginal epithelial permeability decreases as a result of lack of estrogen as well as the effect of aging, which would further attenuate estrogen modulation of the RLIS.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present results show that in cultured hECE cells, aging had little effect on the RTJ or the estrogen-induced decrease in RTJ. Estrogen decreased RTJ by about 12 {Omega}/cm2, possibly by modulation of occludin (19). Because aging per se did not significantly affect estrogen decrease in RTJ, it is suggested that estrogen can efficiently modulate occludin, irrespective of age. Aging increased maximal RLIS from eight to about 16 {Omega}/cm2 between ages 35 and 65 yr. In cells of women aged 35–55 yr, estrogen decreased the maximal RLIS to approximately 4 {Omega}/cm2, irrespective of age. However, in cells of women aged 61–65 yr, the estrogen decrease in RLIS was blocked. Because the main modulator of RLIS in vivo is the capillary blood pressure, which induces hydrostatic gradients in the subluminal to luminal direction (17, 18), it is possible that with advanced age the prevailing capillary pressure is less effective in decreasing the RLIS. Therefore, aging and estrogen deficiency act in concert to increase the epithelial resistance: hypoestrogenism would tend to increase the RTJ, whereas aging and hypoestrogenism would tend to increase the RLIS. The net result would be an increase in total transcervical-transvaginal paracellular resistance (hence, a decrease in paracellular permeability), leading to diminished transport of plasma from the blood into the lumen and to lumenal dryness.

The present results also show that levels of cellular G-actin declined with age. The actin-cytoskeletal network is involved in events regulating cell proliferation, differentiation, apoptosis, and malignant transformation (31, 32, 33) as well as in aging (34, 35). The present study is the first to show a correlation between age-related changes in actin function (decreased RLIS) and mechanisms (decreased G-actin). The actin-cytoskeleton determines cell rigidity and the degree to which cells can deform in response to stimuli (10, 11, 12). In hECE cells the estrogen-induced decrease in RLIS involves cGMP-dependent ADP-ribosylation of monomeric G-actin and shift in actin steady state toward depolymerization (13, 26, 27, 28, 29). Cells become more deformable and tend to decrease their size more readily in response to transepithelial hydrostatic gradients. Decreases in cell size would cause reciprocal increase in the size of the intercellular space, i.e. a decrease in RLIS, and an increase in the paracellular permeability (3, 13). A decrease in G-actin, such as that induced by aging, would tend to shift actin steady state toward polymerization and formation of rigid cytoskeleton, leading to an increase in RLIS and a decrease in permeability. The concept of aging-related formation of rigid cytoskeleton has been previously suggested in other tissues such as the skin (36) and blood vessels (37, 38). However, the present data of aging-related shift in actin stead state toward polymerization are novel.

The magnitude of estrogen decrease in RLIS was smaller in cells obtained from women aged 56 yr and older. The estrogen-induced increase in G-actin is mediated by the estrogen receptor-{alpha}, NO, and cGMP (26, 27, 28, 29), but it is unlikely that the aging-dependent attenuation of the estrogen decrease in RLIS involved the proximal arm of the estrogen signaling cascade because activation of estrogen receptor-{alpha} in hECE cells of older women is similar to that in cells of younger women (25). Furthermore, the estrogen occludin effect also involves estrogen receptor-{alpha} (19), and the present study showed a similar pattern of estrogen modulation of occludin in cells of older and younger women. A more likely explanation is that the aging process involves modulation of steps downstream to the point of NO interaction. This speculation is supported by the finding of age-related decrease in actin depolymerization in response to NO.

The present results have three take-home messages. First, the RTJ was traditionally considered the main determinant of epithelial resistance and hence epithelial permeability. This statement is generally true for occlusive epithelia such as the frog’s skin (5), but in leaky epithelia such as the cervical-vaginal epithelium, the RLIS plays an equally important role in the occlusion of the paracellular route (3). Second, estrogen regulation of the RLIS was evident earlier than the RTJ (Fig. 4Go) (25), and it could be activated by signaling elements downstream the estrogen receptor such as NO and cGMP (27, 28). Therefore, whereas RTJ is the basic gatekeeper of the paracellular route, the RLIS is the more readily modulatable resistance. During premenopausal years plasma estrogen activity would suffice to decrease RTJ and RLIS to the range of minimal resistances, and additional increases in estrogen would fine-tune the permeability by decreasing the RLIS further. Third, the present findings correlate with clinical observations in which short-term treatment with estrogen is more effective in alleviation of vaginal dryness in younger than older postmenopausal women (39). Short-term treatment with estrogen would target initially the RLIS, but changes in RLIS are age dependent (present data) and are predicted to be less effective in older women. Longer treatment with estrogen could increase the permeability by decreasing the RTJ as well (present data). Based on the present results, a possible strategy could be to target signaling cascade checkpoints downstream to the estrogen receptor such as NO or cGMP.


   Acknowledgments
 
The technical support of Kim Frieden, Brian De Santis, and Dipika Pal is acknowledged. Cervical tissue samples were provided by the Cooperative Human Tissue Network, which is funded by the National Cancer Institute.


   Footnotes
 
This work was supported in part by National Institutes of Health Grants HD29924 and AG15955 (to G.I.G.).

First Published Online October 13, 2004

Abbreviations: diC8, sn-1,2-Dioctanoyl diglyceride; hECE, human ectocervical-vaginal (cells); NO, nitric oxide; Ppyr, permeability to the cell-impermeant pyranine; RLIS, resistance of the lateral intercellular space; RTE, transepithelial electrical resistance; RTJ, resistance of the intercellular tight junctions; SNP, sodium nitroprusside; UCl/UNa, relative mobilities of Na+ and Cl– in the intercellular space.

Received June 27, 2004.

Accepted October 7, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Bachman GA, Ebert GA, Burd ID 1999 Vulvovaginal complaints. 2nd ed. In: Lobo RA, ed. Treatment of the postmenopausal woman. Philadelphia: Lippincott, Williams, Wilkins; 195–202
  2. Gorodeski GI, Romero MF, Hopfer U, Rorke E, Utian WH, Eckert RL 1994 Human uterine cervical epithelial cells grown on permeable support—a new model for the study of differentiation and transepithelial transport. Differentiation 56:107–118[Medline]
  3. Gorodeski GI 1996 The cultured human cervical epithelium: a new model for studying transepithelial paracellular transport. J Soc Gynecol Invest 3:267–280[CrossRef][Medline]
  4. Ussing HH, Zerahn K 1951 Active transport of sodium as the source of electric current in the short-circuited isolated frog skin. Acta Physiol Scand 23:110–127[Medline]
  5. Reuss L 1991 Tight junction permeability to ions and water. In: Cereijido M, ed. Tight-junctions. Boca Raton, FL; Ann Arbor, MI; and London: CRC Press; 49–66
  6. Gorodeski GI, De Santis BJ, Goldfarb J, Utian WH, Hopfer H 1995 Osmolar changes regulate the paracellular permeability of cultured human cervical epithelium. Am J Physiol 269:C870–C877
  7. Gorodeski GI, Peterson D, De Santis BJ, Hopfer U 1996 Nucleotide-receptor mediated decrease of tight-junctional permeability in cultured human cervical epithelium. Am J Physiol 270:C1715–C1725
  8. Gorodeski GI, Hopfer U, Wenwu J 1998 Purinergic receptor induced changes in paracellular resistance across cultures of human cervical cells are mediated by two distinct cytosolic calcium related mechanisms. Cell Biochem Biophys 29:281–306[CrossRef][Medline]
  9. Gorodeski GI 2002 Regulation of transcervical permeability by two distinct P2-purinergic receptor mechanisms. Am J Physiol 282:C75–C83
  10. Korn ED 1982 Actin polymerization and its regulation by proteins from nonmuscle cells. Physiol Rev 62:672–737[Free Full Text]
  11. Pollard TD, Cooper JA 1986 Actin and actin-binding proteins. A critical evaluation of mechanisms and functions. Annu Rev Biochem 55:987–1035[CrossRef][Medline]
  12. Cooper JA 1991 The role of actin polymerization in cell motility. Annu Rev Physiol 53:585–605[CrossRef][Medline]
  13. Gorodeski GI 1998 Estrogen increases the permeability of the cultured human cervical epithelium by modulating cell deformability. Am J Physiol 275:C888–C899
  14. Gorodeski GI, Eckert RL, Utian WH, Rorke EA 1990 Maintenance of in vivo-like keratin expression, sex steroid responsiveness and estrogen receptor expression in cultured human ectocervical epithelial cells. Endocrinology 126:399–406[Abstract/Free Full Text]
  15. Gorodeski GI, Eckert RL, Utian WH, Sheean L, Rorke EA 1990 Cultured human ectocervical epithelial cell differentiation is regulated by the combined direct actions of sex steroids, glucocorticoids and retinoids. J Clin Endocrinol Metab 70:1624–1630[Abstract/Free Full Text]
  16. Schultz SG 1980 Diffusion potentials. In: Basic principles of membrane transport. Cambridge, UK: Cambridge University Press; 42–56
  17. Lentner C 1990 Heart and circulation Geigy scientific tables. Vol 5. West Caldwell, NJ: Ciba Pharmaceutical Co.; 220–229
  18. Spring KR, Hope A 1978 Size and shape of the lateral intercellular spaces in a living epithelium. Science 200:54–58[Abstract/Free Full Text]
  19. Zeng R, Li X, Gorodeski GI2004 Estrogen abrogates transcervical tight junctional resistance by acceleration of occludin degradation. J Clin Endocrinol Metab 89:5145–5155
  20. Cereijido M, Gonzales-Mariscal L, Contreras RG, Gallardo JM, Garcia-Villergas R, Valdes J 1993 The making of a tight junction. J Cell Sci 17:127–132
  21. Jovov B, Lewis SA, Crowe WE, Berg JR, Wills NK 1994 Role of Ca2+ in modulation of tight junction resistance in A6 cells. Am J Physiol 266:F775–F784
  22. Gorodeski GI, Wenwu J, Hopfer U 1997 Extracellular Ca2+ directly regulates tight junctional permeability in the human cervical cell line CaSki. Am J Physiol 272:C511–C524
  23. Gorodeski GI 2000 Effects of menopause and estrogen on cervical epithelial permeability. J Clin Endocrinol Metab 85:2584–2595[Abstract/Free Full Text]
  24. Gorodeski GI 2001 Vaginal-cervical epithelial permeability decreases after menopause. Fertil Steril 76:753–761[CrossRef][Medline]
  25. Gorodeski GI 2001 Estrogen biphasic regulation of paracellular permeability of cultured human vaginal-cervical epithelia. J Clin Endocrinol Metab 86:4233–4243[Abstract/Free Full Text]
  26. Gorodeski GI, Pal D 2000 Involvement of estrogen receptors {alpha} and ß in the regulation of cervical permeability. Am J Physiol 278:C689–C696
  27. Gorodeski GI 2000 Role of nitric oxide and cGMP in the estrogen regulation of cervical epithelial permeability. Endocrinology 141:1658–1666[Abstract/Free Full Text]
  28. Gorodeski GI 2000 NO increases permeability of cultured human cervical epithelia by cGMP-mediated increase in G-actin. Am J Physiol 278:C942–C952
  29. Gorodeski GI 2000 cGMP-dependent ADP-depolymerization of actin mediates estrogen increase in human cervical epithelial permeability. Am J Physiol 279:C2028–C2036
  30. Nathan C 1992 Nitric oxide as a secretory product of mammalian cells. FASEB J 6:3051–3064[Abstract]
  31. Kirkpatrick FH 1979 New models of cellular control: membrane cytoskeletons, membrane curvature potential, and possible interactions. Biosystems 11:93–109[CrossRef][Medline]
  32. Hollan S 1996 Membrane fluidity of blood cells. Haematologia 27:109–127[Medline]
  33. Gourlay CW, Carpp LN, Timpson P, Winder SJ, Ayscough KR 2004 A role for the actin cytoskeleton in cell death and aging in yeast. J Cell Biol 164:803–809[Abstract/Free Full Text]
  34. Rao KM, Cohen HJ 1991 Actin cytoskeletal network in aging and cancer. Mutat Res 256:139–148[CrossRef][Medline]
  35. Moxham BJ, Webb PP, Benjamin M, Ralphs JR 1998 Changes in the cytoskeleton of cells within the periodontal ligament and dental pulp of the rat first molar tooth during ageing. Eur J Oral Sci 106(Suppl l):376–383
  36. Kennedy DF, Cliff WJ 1979 A systematic study of wound contraction in mammalian skin. Pathology 11:207–222[Medline]
  37. Plante GE 2003 Impact of aging on the body’s vascular system. Metabolism 52(Suppl 2):31–35
  38. Safar ME, Benetos A 2003 Factors influencing arterial stiffness in systolic hypertension in the elderly: role of sodium and the renin-angiotensin system. Am J Hypertens 16:249–258[CrossRef][Medline]
  39. Burger HG 2001 Physiological principles of endocrine replacement: estrogen. Horm Res 56(Suppl 1):82



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X. Li, L. Zhou, and G. I. Gorodeski
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