Departments of Reproductive Biology and Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106
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ABSTRACT |
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Estrogen increases baseline transepithelial permeability across CaSki cultures and augments the increase in permeability in response to hypertonic gradients. In estrogen-treated cells, lowering cytosolic calcium abrogated the hypertonicity-induced augmented increase in permeability and decreased baseline permeability to a greater degree than in estrogen-deprived cells. Steady-state levels of cytosolic calcium in estrogen-deprived cells were higher than in estrogen-treated cells. Increases in extracellular calcium increased cytosolic calcium more in estrogen-deprived cells than in estrogen-treated cells. However, in estrogen-treated cells, increasing cytosolic calcium was associated with greater increases in permeability in response to hypertonic gradients than in estrogen-deprived cells. Lowering cytosolic calcium blocked the estrogen-induced increase in nitric oxide (NO) release and in the in vitro conversion of L-[3H]arginine to L-[3H]citrulline. Treatment with estrogen upregulated mRNA of the NO synthase isoform endothelial nitric oxide synthase (eNOS). These results indicate that cytosolic calcium mediates the responses to estrogen and suggest that the estrogen increase in permeability and the augmented increase in permeability in response to hypertonicity involve an increase in NO synthesis by upregulation of the calcium-dependent eNOS.
paracellular permeability; transepithelial transport; cervical mucus; cytosolic calcium; nitric oxide; nitric oxide synthase; G-actin; cytoskeleton; endothelial nitric oxide synthase
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INTRODUCTION |
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THE MAIN FUNCTION OF THE UTERINE cervical epithelium is to control secretion of cervical mucus. The cervical mucus is important for reproduction and for woman's health. Abnormal secretion of cervical mucus may lead to infertility and to states of disease such as mucorrhea and dryness dyspareunia (14).
The cervical mucus is a mixture of mucins and cervical plasma. Mucins are secreted in the cervical canal by exocytosis from endocervical cells (14). The cervical plasma composes 80-99% of the total weight of the cervical mucus, and it originates by transudation of fluid from the blood (14). The driving force is the blood pressure: it generates a hydrostatic gradient between the capillaries and the cervical canal and compels movement of fluid through the intercellular (paracellular) space into the lumen (48). Cervical epithelial cells control the free movement of fluid through the cervix by forming two functional barriers: the tight junctional resistance (RTJ) and the resistance of the lateral intercellular space (RLIS; see Ref. 15). According to the equivalent electrical circuit model of ion transport the sum of RTJ and RLIS in series determines the overall permeability of the cervical epithelium (41, 47).
In human cervical epithelial cells, the RTJ and
RLIS can be independently regulated
(15), but the mechanisms of regulation are not entirely
understood. An example is the effect of estrogen on cervical
permeability. Estrogen increases cervical secretions in women
(14). Studies at the cellular level revealed that the effect of estrogen involves an increase in RLIS
(16) and is mediated by estrogen receptor -activation
of the nitric oxide (NO)/cGMP-dependent increase in G-actin (18,
24). The proposed molecular mechanism of increase in
permeability is modulation of the cytoskeleton. By shifting actin
steady state toward G-actin, estrogen stimulates transformation of the
cytoskeleton in a more flexible structure (16). This
renders cervical epithelial cells more deformable in response to the
prevailing blood pressure-induced hydrostatic pressure and results in a
greater decrease in RLIS and an increase in the
permeability (16, 17).
Calcium regulates paracellular permeability of cultured human cervical epithelia (23). Calcium increases the RTJ by maintaining the tight junctional elements in a closed state (27). Also, fluctuations in cytosolic calcium can modulate agonist-induced changes in RTJ and the RLIS (23, 25, 27). Until recently, little was known about the role of calcium in the responses to estrogen. Calcium levels in cervical secretions change during the cycle (14), suggesting that estrogen modulates transcervical transport of calcium. Changes in extracellular calcium can regulate paracellular permeability directly by changing the gating status of the tight junctions (27) or indirectly by modulating levels of cytosolic calcium (23). Consequently, if estrogen signaling depends on calcium signaling, changes in extracellular calcium can affect the estrogen increase in cervical secretions by modulation of intracellular calcium activity. The objective of the present study was to determine the degree to which changes in cytosolic calcium modulate the effect of estrogen on cervical permeability.
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METHODS |
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Cell cultures.
The experiments utilized CaSki cells, which are a stable line of
transformed cervical epithelial cells that express phenotypic markers
of the endocervix (26). Cells were grown and maintained in
a culture dish at 37°C in a 91% O2-9% CO2
humidified incubator and were routinely tested for mycoplasma. For
experiments, cells were plated on filters for 8 h and then shifted
to steroid-free medium (16). After 3 days, cells were
treated with 10 nM 17-estradiol (estrogen-treated cells) or the
vehicle (estrogen-deprived cells) for two additional days. Before
assays, filters containing cells were washed three times and
preincubated for 15 min at 37°C in a modified Ringer buffer
(16, 24).
Changes in paracellular permeability.
Changes in paracellular permeability were determined in terms of
changes in transepithelial electrical conductance
(GTE). Changes in GTE
were determined continuously across filters mounted vertically in a
modified Ussing chamber from successive measurements of transepithelial
electrical current (I) and of the transepithelial potential difference (
PD, lumen negative):
GTE =
I/
PD.
Transepithelial hypertonic gradients in the subluminal to luminal
direction were established by adding aliquots of 2 M sucrose solution
to the subluminal solution (15, 20).
Determinations of cytosolic calcium.
Determinations of cytosolic calcium in cells attached on filters were
published previously (28). Briefly, cells on filters were
loaded with 7 µM fura 2, and measurements of fluorescence were
conducted in a custom-designed fluorescence chamber as described (4, 28). Cells were illuminated over the apical surface, and the intensity of the emitted light from the apical surface was
measured. Changes in cytosolic calcium were determined by switching the
excitation filters to record the maximal (340-nm excitation/510-nm
emission) and minimal (380-nm excitation/510-nm emission) fluorescence
for cytosolic calcium determinations (4, 28) and were
calculated according to the formula [Ca2+]i
(nM) = [(R Rmin)/(Rmax
R)] · Kd · (Sf2/Sb2)
(30), where [Ca2+]i is the level
of cytosolic calcium, R is the ratio of fluorescence excitation
measurements at 340 to 380 nm, Rmin and Rmax
are the experimentally determined minimum and maximum calcium
measurement ratios at 340 and 380 nm, respectively,
Kd is the dissociation constant for fura 2 (224 nM), and Sf2/Sb2 is the ratio of fluorescence value at 380-nm excitation determined at Rmin (0 calcium)
and Rmax (maximal calcium). Maximal calcium fluorescence
was obtained by adding 10 µM ionomycin in the presence of 10 mM
CaCl2, and minimal calcium fluorescence was obtained by
competing calcium from fura 2 with 2.5 mM MnCl2.
Determinations of free calcium. Levels of calcium in the extracellular buffer were manipulated using the calcium chelator EGTA. Concentrations of free calcium in the extracellular buffer were calculated as described (23, 25).
DNase I inhibition assay. DNase I inhibition assay was described (16). G-actin content in lysates of cells grown on filters was determined in terms of DNase I inhibition of DNA degradation, based on the fact that under the conditions of the experiment the main inhibitor of DNase I is G-actin (2). Total actin was measured by incubating lysates with guanidine hydrochloride to depolymerize F-actin to monomeric G-actin. Data of the G-actin and total cellular actin were expressed per milligram total protein.
Release of NO. Release of NO was determined as the accumulation of nitrite and nitrate in the extracellular medium by a modified Greiss method as described (4). The detection limit of the assay was 2 µM, and results were expressed as picomoles per minute per milligram of protein.
Nitric oxide synthase activity. Nitric oxide synthase (NOS) activity was assayed by following the conversion of L-[2,3,4,5-3H]arginine to L-[2,3,4,5-3H]citrulline as described (43) with modifications. Before experiments, cells on filters were washed three times with PBS (37°C) and lysed on ice for 20 s with buffer (1 µl/104 cells at 20°C, pH 7.4) containing 320 mM sucrose, 0.1 mM EDTA, 1 mM dithiothreitol, 10 mM HEPES, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml leupeptin, and 2 µg/ml aprotinin. Aliquots were removed for DNA. The lysates were centrifuged (104 g for 20 min at 4°C), and the supernatants were assayed in triplicate. The supernatant (100 µl, about 200 µg of cell protein) was incubated with 100 µl of buffer (40 mM potassium phosphate, pH 7.4, 8 mM L-valine, 100 µM NADPH, 1 mM MgCl2, 1 mM CaCl2, 100 µM L-arginine, 25 µM FAD, 25 µM FMN, 5 µM tetrahydrobiopterin, 100 µM phenylmethylsulfonyl fluoride, and 0.2 µM calmodulin) plus L-[2,3,4,5-3H]arginine monohydrochloride. Assays were also carried out in the presence of 1.2 mM EGTA, 1 mM NG-nitro-L-arginine methyl ester (L-NAME), 1 mM NG-nitro-L-arginine (L-NA), or 1 mM NG-monomethyl-L-arginine (L-NMMA). Samples were incubated for 30 min at 37°C before the reaction was terminated by adding 500 µl of H2O-Dowex-50W (1:1 vol/vol; Na+ form). The resin-incubated mixture was dispersed and diluted by adding 860 µl of 2dH2O. The resin was allowed to settle for 15 min, and 975 µl of supernatant were pipetted into scintillation vials. Scintillation counting fluid (10 ml) was added to each vial, and the radioactivity corresponding to L-[2,3,4,5-3H]citrulline was measured. NOS activity was expressed as picomoles per minute per milligram of DNA. The calcium-dependent NOS activity was determined from the difference between activities obtained in control and in EGTA (or L-NAME, L-NA, or L-NMMA) buffers; total activity of the NOS was determined by subtracting the activities obtained in EGTA plus L-NAME buffers.
Molecular biology methods. Molecular biology methods were described (19). Total RNA from cultured cells was isolated with the Qiagen kit (Qiagen, Chatsworth, CA; see Ref. 19). The method for RT-PCR was described (19). The following PCR conditions were applied: for endothelial nitric oxide synthase (eNOS), 35 cycles of 1-min denaturation step at 94°C, 1 min of annealing step at 62°C, and 2 min of extension step at 72°C; for neuronal (brain) nitric oxide synthase (bNOS), 35 cycles of 1 min at 94°C, 2 min at 56°C, and 2 min at 72°C; for the inducible nitric oxide synthase (iNOS), 35 cycles of 1 min at 94°C, 2 min at 56°C, and 2 min at 72°C. The following oligonucleotide primers were used: human eNOS (34) 5'-forward (sense) 5'-CAG TGT CCA ACA TGC TGC TGG AAA TTG-3', 3'-reverse (antisense) 5'-TAA AGG TCT TCT TGG TGA TGC C-3'; human bNOS (38) 5'-forward (sense) 5'-TTT CCG AAG CTT CTG GCA ACA GCG GCA ATT-3', 3'-reverse (antisense) 5'-GGA CTC AGA TCT AAG GCG GTT GGT CAC TTC-3'; iNOS (11) 5'-forward (sense) 5'-GCC TCG CTC TGG AAA GA-3', 3'-reverse (antisense) 5'-TCC ATG CAG ACA ACC TT-3'. X-ray films were analyzed with a laser densitometer Sciscan 5000 (United States Biochemical, Cleveland, OH) and normalized relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (19).
Total protein and total DNA. Total protein and total DNA were measured as described (21, 22).
Cell viability. Cell viability was determined by mitochondrial respiration assay using dimethylthiazol diphenyl tetrazolium (MTT) staining (46). Cells on filters were incubated for 60 min at 37°C in Ringer buffer containing 1 mg/ml MTT. Cultures were washed with PBS and solubilized in isopropanol containing 0.1 M HCl and 1% Triton X-100. Lysates were mixed by pipetting to dissolve the reduced MTT crystals and were spun at 10,000 g for 5 min. The solubilized formazan was measured by determining absorption at 575 nm minus background absorbance at 690 nm for each sample. In control experiments, cells were treated for 30 min with 50 µM of the protonophore uncoupler carbonyl cyanide m-chlorophenylhydrazone (mClCCP; Aldrich, Milwaukee, WI). Viability was defined as <5% positive staining compared with control (mClCCP-treated) cells.
Drugs treatments. For experiments with cells on filters, all reagents, except sucrose, were added from concentrated (1,000-300×) stocks (pH 7.2) of saline, PBS, ethanol, or DMSO to both the luminal and subluminal solutions. Aliquots of sucrose solution were added only to the subluminal solution.
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, Silver Spring, MD) and were analyzed with ANOVA. Best fit of regression equations (least squares criterion) was achieved with SlideWrite Plus (Advanced Graphics Software, Carlsbad, CA), which uses the Levenberg-Marquardt Algorithm, and was analyzed using ANOVA.
Chemicals and supplies. L-[2,3,4,5-3H]arginine monohydrochloride was obtained from American Radiolabled Chemicals (St. Louis, MO). Anocell (Anocell-10) filters were obtained from Anotec (Oxon, UK). Fluorescent microspheres (FluoresBrite beads, calibration grade) were obtained from Polysciences (Warrington, PA). All other chemicals were obtained from Sigma Chemicals.
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RESULTS |
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Experimental design to test effects of cytosolic calcium on paracellular permeability. The main objective of the study was to determine the degree to which changes in cytosolic calcium affect the estrogen-induced increases in paracellular permeability. To manipulate cytosolic calcium, CaSki cells attached on filters were exposed to the following conditions.
To increase cytosolic calcium, extracellular calcium was increased above the physiological level of 1.2 mM to augment calcium influx. In cells grown in regular medium, raising extracellular calcium from 1.2 to 4.0 mM increased cytosolic calcium from 89 to 97 nM (P < 0.01, paired t-test), but it had no effect on GTE (Fig. 1A).
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Estrogen increases baseline permeability and augments effects of
hypertonicity and ionomycin.
To determine the degree to which changes in cytosolic calcium affect
the estrogen-induced increase in paracellular permeability, CaSki cells
on filters were treated with 17-estradiol at the physiological
concentration of 10 nM. The following two control conditions were used:
1) cells grown in estrogen-deficient medium and considered
to be estrogen depleted and 2) cells exposed to a hypertonic
gradient in the subluminal to luminal direction. Both treatments with
estrogen and hypertonic gradients in the subluminal to luminal
direction increase permeability across cultured human cervical
epithelia by decreasing the resistance of the lateral intercellular
space RLIS (16, 20). However, the
effects have different time courses and involve different
cellular/molecular mechanisms: treatment with estrogen requires hours
(16), whereas hypertonic gradients increase
GTE instantaneously (Fig. 1B and Refs. 15 and 20). Also, estrogen increases GTE
by fragmenting the cytoskeleton (16), whereas hypertonic
gradients in the subluminal to luminal direction dilate the lateral
intercellular space by stimulating water efflux (15, 20).
Calcium depletion decreases GTE. The ionomycin-induced depletion of cytosolic calcium decreased GTE. The effect was small in estrogen-deficient cells (Fig. 3), but in estrogen-treated cells GTE decreased significantly by ~20 mS/cm2 30-40 min after adding EGTA-Mn2+-ionomycin (Fig. 3, 285 mosmol/l). These results indicate that depletion of intracellular calcium decreases permeability to a greater degree in estrogen-treated cells than in estrogen-deficient cells.
Restitution of extracellular calcium to 1.2 mM resulted in a slow but complete reversal of baseline cytosolic calcium and GTE to levels that were observed before the treatments with EGTA-Mn2+-ionomycin (data not shown). Increasing extracellular calcium above 1.2 mM had no effect on baseline GTE (data not shown).Lowering cytosolic calcium alters the responses to hypertonicity. In estrogen-deficient cells, treatment with EGTA-Mn2+-ionomycin slightly attenuated the responses to hypertonic gradients (Fig. 3). In estrogen-treated cells, treatment with EGTA-Mn2+-ionomycin abrogated the responses to hypertonic gradients; for instance, in estrogen-treated, calcium-depleted cells, a hypertonic gradient of 45 mosmol/l increased GTE only by 25 mS/cm2 compared with 85 mS/cm2 in cells bathed in normal calcium (Fig. 3, P < 0.01). This result indicates that in estrogen-treated cells most of the increase in permeability in response to hypertonic gradient depends on calcium.
Increasing extracellular calcium above 1.2 mM had no significant effect on the responses to hypertonic gradients, neither in estrogen-deficient nor in estrogen-treated cells (data not shown).Estrogen modulates cytosolic calcium.
To better understand the effect of cytosolic calcium on the responses
to hypertonicity, levels of cytosolic calcium were determined in the
range of extracellular calcium of 0.6-4.0 mM, and levels of
cytosolic calcium were correlated with levels of
GTE in response to a hypertonic gradient of 45 mosmol/l in the subluminal to luminal direction. In estrogen-deprived
cells, steady-state levels of cytosolic calcium were higher than in
estrogen-treated cells (Figs. 4 and
5A). Changing extracellular
calcium in the range of 0.6-4.0 mM increased cytosolic calcium
both in estrogen-deprived cells and in estrogen-treated cells (Figs. 4
and 5A), but cytosolic calcium increased more in
estrogen-deprived cells than in estrogen-treated cells (Fig.
5A).
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Cytosolic calcium does not modulate the effects of sodium
nitroprusside and bromo-cGMP on GTE.
A possible explanation for the results shown in Figs. 4 and 5 is that
calcium regulates critical steps along the signaling cascade that
mediates the estrogen increase in permeability. Estrogen increases
transcervical paracellular permeability by a mechanism that involves
estrogen receptor -activation of the NO/cGMP-dependent increase in
G-actin (18, 24). The objectives of the following two
experiments were to test the degree to which the effects of NO and cGMP
on GTE are dependent on calcium.
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Calcium modulates the estrogen increase in NO synthesis. A possible explanation for the lack of effect of lowering calcium on the SNP- or 8-Br-cGMP-induced increase in G-actin and in the hypertonicity-induced increase in GTE is that calcium regulates signaling steps more proximal to the action of NO. The three experiments in this section tested the hypothesis that the calcium-sensitive step is estrogen upregulation of NO.
NO is synthesized from L-arginine during the NOS-catalyzed conversion of L-arginine to L-citrulline (1, 36, 39). The first experiment tested the effects of estrogen and calcium on NO release. CaSki cells release NO constitutively in the extracellular medium (Fig. 7A and Ref. 17). In estrogen-deficient cells, L-NAME, an NOS inhibitor (10), decreased NO release slightly; in estrogen-treated cells, L-NAME decreased NO release markedly to levels that were observed in estrogen-deficient cells. This result indicates that most of the increase in NO release in estrogen-treated cells originates from NOS(s).
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DISCUSSION |
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The results of the present study provide novel data about the
regulation of transcervical permeability by estrogen and by cytosolic
calcium. Treatment with estrogen augmented increases in permeability
that were induced by ionomycin and hypertonicity. Although all three
agents/conditions increase permeability by decreasing the resistance of
the lateral intercellular space, RLIS (15,
20, 28), the cellular mechanisms of action are different:
estrogen increases permeability by fragmenting the cytoskeleton
(present results and Ref. 16); ionomycin and hypertonicity generate a
subluminal to luminal hydrostatic gradients. Ionomycin stimulates
Cl secretion, followed by water efflux (15,
20); hypertonicity stimulates water efflux directly
(15). In both cases, water moves from the cells into the
intercellular space; water that accumulates in the basal compartment of
the intercellular space dilates the space and generates a subluminal to
luminal hydrostatic gradient (32, 45). The effects of
estrogen on G-actin and on the cytoskeleton can explain how estrogen
augments the responses to ionomycin and hypertonicity; changes in cell
size depend on the flexibility of the cytoskeleton, and cells with a
fragmented cytoskeleton can adapt more readily to changes in cell
volume (6, 7, 9, 44).
Estrogen, ionomycin, and hypertonicity activate a common paracellular mechanism, namely, a decrease in RLIS (15, 20, 28). This finding has physiological relevance because in vivo the main driving force for fluid transudation across the cervical epithelium is the pressure generated by the blood in the subepithelial space (14), which is equivalent to a hydrostatic gradient in the subluminal to luminal direction.
The experiments of the present study also showed that baseline permeability and the responses to hypertonicity depend on cytosolic calcium. The results showed that 1) increases in cytosolic calcium had little effect on baseline permeability; in contrast, lowering cytosolic calcium decreased baseline permeability, suggesting that physiological resting steady-state cytosolic calcium levels induce a maximal degree of permeability. 2) Lowering cytosolic calcium attenuated the increase in permeability in response to hypertonicity, whereas increasing cytosolic calcium augmented the response. The critical factor relative to the status of cytosolic calcium was resting levels of cytosolic calcium, as determined by cellular calcium content, rather than the cell's ability to increase cytosolic calcium by mobilization from intracellular stores, since treatment with the endoplasmic reticulum calcium pump inhibitor thapsigargin had no significant effect on permeability (results not shown). 3) Lowering cytosolic calcium blocked the estrogen-induced increase in G-actin, and it attenuated the augmented increase in permeability in response to hypertonicity, indicating that the estrogen-related, hypertonicity-induced increase in permeability is calcium dependent.
The estrogen increase in G-actin and in the permeability involves four
key signaling steps: the -estrogen receptor, eNOS, NO, and cGMP
(16, 17, 18, 24). Agents that mimic effects of NO (e.g.,
SNP) and cGMP (e.g., 8-Br-cGMP) can increase G-actin and the
permeability (present results and Refs. 17, 18, 28). Lowering cytosolic
calcium had no effect on the responses to SNP or to 8-Br-cGMP; in
contrast, lowering cytosolic calcium abrogated the estrogen-induced
increase in NO release from intact cells, and it attenuated the
estrogen-induced increase in the conversion of L-citrulline
to L-arginine. In addition, estrogen increased expression
of mRNA for the calcium-dependent eNOS. Collectively, these results
suggest that the calcium-dependent rate-limiting step is NO synthesis
by the calcium-dependent eNOS.
In estrogen-deficient cells bathed in low calcium, the responses to hypertonic gradients were mildly attenuated compared with responses in cells bathed in normal calcium, suggesting some involvement of calcium also in the response to hypertonicity. A possible explanation is that lowering calcium blocked the activity of calcium-dependent NOS(s) that produce(s) NO constitutively, even in cells grown in steroid-free conditions. This statement is supported by the findings that CaSki cells (present study) and other types of cultured human cervical epithelial cells express mRNA for all three NOS isoforms and that the cells constitutively secrete NO (present results and Ref. 17). Another source of NO is from calcium-independent NOS; lowering cytosolic calcium abrogated the estrogen-induced increase in the conversion of L-citrulline to L-arginine, and the remaining NO activity could be blocked by L-NAME, indicating that some of the NO activity is contributed by calcium-independent mechanisms, possibly by the iNOS (33). These results support our hypothesis (17) that cervical cells autoregulate permeability and maintain increased paracellular permeability by continuously secreting NO. Accordingly, the effect of NO involves autocrine/paracrine regulation of permeability: cells secrete NO, and NO can either act on the same cell (autocrine regulation) or diffuse into neighboring cells (paracrine regulation). This hypothesis explains the slow decrease in permeability in cultures treated with EGTA-Mn2+-ionomycin; depletion of calcium may have decreased NO synthesis and abrogated its effect on permeability (4). It also explains the greater decrease in permeability in estrogen-treated cells than in estrogen-deficient cells because estrogen increases NO (present results and Refs. 8, 12, 42), and blocking NO synthesis would have a greater impact on permeability.
Previous studies in smooth muscle cells reported that estrogen could directly block calcium channels and attenuate calcium influx, thus lowering cytosolic calcium (5, 35, 37, 48). The present experiments show similar trends in human cervical epithelial cells in that baseline levels of cytosolic calcium were lower in estrogen-treated cervical cells than in estrogen-deficient cells. The mechanism by which estrogen lowers cytosolic calcium in cervical epithelial cells is unclear. Changing extracellular calcium in the range of 0.6-4.0 mM increased cytosolic calcium in a concentration-related manner, both in estrogen-treated cells and in estrogen-deficient cells. Extracellular calcium levels >1.2 mM increased cytosolic calcium more in estrogen-deficient cells than in estrogen-treated cells, raising the possibility that estrogen blocks calcium channels also in cervical cells.
Relatively little is known about the biological role of the lowered cytosolic calcium in estrogen-treated cells. Because both estrogen and calcium upregulate NO (present results), it is possible that the lowered cytosolic calcium controls NO activity and prevents apoptosis (3, 29). However, more studies are needed to clarify this issue.
The results of the present study may be important for understanding regulation of cervical secretions in vivo because agonists that elevate cytosolic calcium can activate the eNOS (36, 39). Secretagogues, neurotransmitters, and agonists that participate in the inflammatory response can stimulate an increase in cytosolic calcium in human cervical cells (23). These agents may stimulate NO production, increase G-actin, and lead to increased paracellular permeability. Increased permeability will allow greater fluxes of fluids and solutes from the blood into the cervical canal and augment secretion of cervical mucus. In cervical cells, estrogen increases NO by upregulation of the calcium-dependent eNOS (present study). Subsequently, changes in cytosolic calcium are likely to affect permeability more in estrogen-treated cells than in estrogen-deprived cells. This conclusion may have pharmacological significance in the sense of modulating cervical secretions directly by NO-related agents, but more studies are needed to clarify the effects in women.
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ACKNOWLEDGEMENTS |
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The study was supported by National Institutes of Health Grants HD-00977, HD-29924, and AG-15955.
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FOOTNOTES |
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Address for reprint requests and other correspondence: G. I. Gorodeski, Univ. MacDonald Women's Hospital, Univ. Hospitals of Cleveland, 11100 Euclid Ave., Cleveland, OH 44106 (E-mail: gig{at}po.cwru.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 24 January 2000; accepted in final form 5 June 2000.
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