NO increases permeability of cultured human cervical epithelia
by cGMP-mediated increase in G-actin
George I.
Gorodeski
Department of Reproductive Biology and Department of Physiology and
Biophysics, Case Western Reserve University School of Medicine,
Cleveland, Ohio 44106
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ABSTRACT |
Human cervical
epithelial cells express mRNA for the nitric oxide (NO) synthase (NOS)
isoforms ecNOS, bNOS, and iNOS and release NO into the extracellular
medium. NG-nitro-L-arginine methyl
ester (L-NAME), an NOS inhibitor, and Hb, an NO scavenger,
decreased paracellular permeability; in contrast, the NO donors sodium
nitroprusside (SNP) and
N-(ethoxycarbonyl)-3-(4-morpholinyl)sydnonimine increased
paracellular permeability across cultured human cervical epithelia on
filters, suggesting that NO increases cervical paracellular permeability. The objective of the study was to understand the mechanisms of NO action on cervical paracellular permeability. 8-Bromo-cGMP (8-BrcGMP) also increased permeability, and the effect was
blocked by KT-5823 (a blocker of cGMP-dependent protein kinase), but
not by LY-83583 (a blocker of guanylate cyclase). In contrast, LY-83583
and KT-5823 blocked the SNP-induced increase in permeability. Treatment
with SNP increased cellular cGMP, and the effect was blocked by Hb and
LY-83583, but not by KT-5823. Neither SNP nor 8-BrcGMP had modulated
cervical cation selectivity. In contrast, both agents increased
fluorescence from fura 2-loaded cells in the
Ca2+-insensitive wavelengths, indicating that SNP and
8-BrcGMP stimulate a decrease in cell size and in the resistance of the
lateral intercellular space. Neither SNP nor 8-BrcGMP had an effect on
total cellular actin, but both agents increased the fraction of
G-actin. Hb blocked the SNP-induced increase in G-actin, and KT-5823
blocked the 8-BrcGMP-induced increase in G-actin. On the basis of these
results, it is suggested that NO acts on guanylate cyclase and
stimulates an increase in cGMP; cGMP, acting via cGMP-dependent protein
kinase, shifts actin steady-state toward G-actin; this fragments the
cytoskeleton and renders cells more sensitive to decreases in cell size
and resistance of the lateral intercellular space and, hence, to
increases in permeability. These results may be important for
understanding NO regulation of transcervical paracellular permeability
and secretion of cervical mucus in the woman.
paracellular permeability; transepithelial transport; cervical
mucus; nitric oxide; nitric oxide synthase; cytoskeleton
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INTRODUCTION |
CERVICAL EPITHELIAL CELLS regulate secretion of
cervical mucus. The cervical mucus lubricates the lower genital canal
and prevents entry of microorganisms and cells into the uterus. During reproductive years, changes in cervical mucus in the preovulatory phase
allow for sperm penetration into the cervix and for sperm capacitation
and migration. Abnormal secretion of cervical mucus may lead to
infertility and to states of disease such as mucorrhea and dryness
dyspareunia (12).
The major component of cervical mucus, the cervical plasma, originates
by transudation of fluid and solutes from the blood into the cervical
canal (12). In vivo, the driving force for transudation through the
cervical epithelium is the transepithelial hydrostatic gradient, in the
blood-to-lumen direction (33, 37). Cultured human cervical cells form
leaky epithelia (17, 18), and recent studies suggest that movement of
fluid across cervical epithelia occurs mainly via the paracellular
pathway (13). Movement of molecules in the paracellular space is
restricted by resistances of tight junctions (RTJ)
and of the lateral intercellular space (RLIS) in
series. The regions of tight junctions are considered high-resistive
elements because of 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 proximity of the plasma membranes of neighboring cells and by length of the
intercellular space from tight junctions to the basal lamina (37).
Human cervical epithelial cells can actively regulate RTJ, as well as RLIS (13, 17),
and are therefore a useful model to study regulation of paracellular permeability.
Nitric oxide (NO) is an important regulator of cell functions, and it
can modulate permeability of endothelial cells (4, 24, 38, 39) and
epithelial cells (22, 23, 28, 31, 36). NO is synthesized from
L-arginine during the NO synthase (NOS)-catalyzed
conversion of L-arginine to L-citrulline (28, 31). Previous studies have identified NOS expression in human uterine
and vaginal tissues (20), but until recently little was known about the
role of NO in human cervical epithelium. The objective of the present
study was to determine the effects of NO on paracellular permeability
across cultured human cervical epithelia and to understand the
mechanisms of NO action.
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METHODS |
Cell cultures.
Three types of cell cultures were used. Human ectocervical epithelial
(hECE) cells, a model of the stratified ectocervical epithelium, were
obtained from minces of ectocervix and used in passage 3 (18).
ECE16/1 cells are a stable line of immortalized hECE cells with the
human papilloma virus 16 (1) (kindly provided by Dr. R. L. Eckert,
Dept. of Physiology and Biophysics, Case Western Reserve University
School of Medicine) and are a model of the squamous metaplastic
epithelium (18). CaSki cells are a stable line of transformed cervical
epithelial cells that express phenotypic markers of the endocervix
(18). Cells were grown and maintained in culture dishes at 37°C in
a 91% O2-9% CO2 humidified incubator and
plated on filters for experiments (17, 18). Cells were routinely tested
for mycoplasma. Before experiments, filters containing cells were
washed three times and preincubated for 15 min at 37°C in a
modified Ringer buffer (17, 18).
Changes in paracellular permeability were determined in terms of
changes in permeability to pyranine (Ppyr) and in
terms of changes in transepithelial electrical conductance
(GTE). Methods, including conditions for optimal
determinations of Ppyr and
GTE across low-resistance epithelia, calibrations
and controls, potential pitfalls, and appropriate measures to prevent
artifacts, were described and discussed previously (16-18).
Changes in Ppyr were determined from unidirectional
(luminal-to-subluminal) fluxes across filters mounted vertically in
modified Ussing/diffusion chambers, as described elsewhere (16, 17), to
prevent hydrostatic gradients. Pyranine was added to the luminal compartment, and the amount of pyranine in the subluminal compartment was measured after 10 min. The transepithelial permeability coefficient (Ppyr) was calculated as described elsewhere (16,
17). Cytolysis of human cervical epithelial cells that were previously
incubated with 0.1 mM pyranine did not increase pyranine fluorescence
significantly above background (not shown).
Changes in GTE were determined continuously across
filters mounted vertically in a modified Ussing chamber from successive measurements of transepithelial electrical current (
I) and
transepithelial potential difference (
PD, lumen negative):
GTE =
I/
PD. All reagents used
for the Ussing chamber experiments were added from concentrated stocks
(300-1,000×) of 1% ethanol and/or DMSO and saline to the
luminal and subluminal solutions (17). Determinations of the dilution
potential and the interpretations of changes in the dilution potential
in terms of the ratio of mobilities of Cl
(uCl) and Na+ (uNa)
in the intercellular space
(uCl/uNa) were described
previously (16-18). Transepithelial hypertonic gradients of
325-285 mosmol/l in the subluminal-to-luminal direction were
established by adding 120 µl of 2 M sucrose solution to the
subluminal solution (13).
Molecular biology methods.
Total RNA from cultured cells was isolated with the Qiagen kit (Qiagen,
Chatsworth, CA) with use of lysis buffer plus
-mercaptoethanol at
350 µl/107 cells. The final total RNA
pellets were resuspended in 50 µl of diethyl pyrocarbonate-water and
quantitated by measuring optical density at 260 nm (15). The method for
RT-PCR was described previously (15). The following PCR conditions were
applied: for endothelial NOS (ecNOS), 35 cycles of 1-min denaturation
step at 94°C, 1-min of annealing step at 62°C, and 2-min
extension step at 72°C; for neuronal (brain) NOS (bNOS), 35 cycles
of 1 min at 94°C, 2 min at 56°C, and 2 min at 72°C; for
inducible NOS (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 ecNOS (25) 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 (30)
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 laser densitometer Sciscan 5000 (US Biochemical, Cleveland, OH)
and normalized relative to glyceraldehyde 3-phosphate dehydrogenase RNA.
Fluorescence of attached cells.
Cells on filters were incubated in culture medium with 7 µM fura 2-AM + 0.25% Pluronic F12 for 45 min at 37°C. After the incubation, cells were washed twice and reincubated with fresh culture medium for
10 min at 37°C to permit hydrolysis of the esters and to retain the
polar molecules intracellularly. Fluorescence was measured in a
custom-designed fluorescence chamber, as described previously (4, 19).
A filter with cells was placed in an enclosed dark chamber maintained
at a fixed temperature and under conditions that permit selective
perfusion of the luminal and subluminal compartments. Cells were
illuminated over the apical surface, and the intensity of the emitted
light from the apical surface was measured as described elsewhere (4,
19). Fura 2 fluorescence was measured at the isosbestic wavelengths
[360 nm excitation/510 nm emission (F360/510)]
(4, 19). Under these conditions, the leakage of fura 2, photobleaching,
and metabolism of fura 2 are minimal (16). The theoretical background
for the changes in F360/510 was discussed previously (4,
19).
Changes in cytosolic Ca2+ in cells attached on filters were
also determined in the fluorescence chamber by switching the excitation filters (4, 19) to record the maximal (340 nm excitation/510 nm
emission) and minimal (380 nm excitation/510 nm emission) fluorescence for cytosolic Ca2+ determinations and the isosbestic
fluorescence (360 nm excitation/510 nm emission) (4, 19). Changes in
cytosolic Ca2+ were calculated according to the following
formula: [Ca2+]i (nM) = [(R
Rmin)/(Rmax
R)] · Kd · (Sf2/Sb2),
where [Ca2+]i is the level of
cytosolic Ca2+, R is the ratio of fluorescence excitation
measurements at 340 nm to that at 380 nm, Rmin and
Rmax are the experimentally determined minimum and maximum
Ca2+ measurement ratios (i.e., 340 nm to 380 nm),
Kd is the dissociation constant for fura 2 (224 nM), and Sf2/Sb2 is the ratio
of fluorescence at 380 nm excitation determined at Rmin (0 Ca2+) to that determined at Rmax (maximal
Ca2+). Maximal Ca2+ fluorescence was obtained
by adding 10 µM ionomycin in the presence of 10 mM CaCl2,
and minimal Ca2+ fluorescence was obtained by competing
Ca2+ from fura 2 with 2.5 mM MnCl2. All agents
and solutions were added to the luminal and subluminal compartments.
Release of NO was determined as the accumulation of nitrite
(NO
2) and nitrate
(NO
3) in the extracellular medium
by a modified Greiss method, as described elsewhere (4). Detection
limit of the assay was 2 µM, and results were expressed as picomoles
per minute per milligram protein. For determinations of cGMP, cells on
filters were homogenized in TCA, and cGMP content within the cell
homogenate was assayed using a commercially available RIA kit
(Amersham, Arlington Heights, IL) (4). Results were expressed as
picomoles per minute per milligram DNA. For DNase-I inhibition assay,
cells on filters were lysed in situ, and DNase-I activity in the lysate
was assayed by measuring DNase-I-dependent degradation of DNA. Total
actin was measured by the guanidine-HCl method after depolymerization of F-actin to monomeric G-actin, as recently described (14). Cellular
DNA and total protein were measured as described previously (18).
Cell viability was determined by mitochondrial respiration assay by use
of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
staining (35). 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 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 the protonophore
uncoupler carbonyl cyanide m-chlorophenylhydrazone (50 µM;
Aldrich, Milwaukee, WI). Viability was defined as <5% positive
staining compared with control (carbonyl cyanide
m-chlorophenylhydrazone-treated) cells.
Statistical analysis of the data.
Values are means ± SD, and significance of differences among means
was estimated by ANOVA. Trends were calculated using GB-STAT (version
5.3, Dynamic Microsystems, Silver Spring, MD) and 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 analyzed using ANOVA.
Chemicals and supplies.
Anocell (Anocell 10) filters were obtained from Anotec (Oxon, UK).
Fluorescent microspheres (FluoresBrite beads, calibration grade) were
obtained from Polysciences (Warrington, PA). All other chemicals were
obtained from Sigma Chemical (St. Louis, MO).
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RESULTS |
NO increases permeability of cervical cultures.
Baseline levels of GTE across cultures of hECE,
ECE16/1, and CaSki cells ranged from 45 to 85 mS · cm
2 (Figs.
1 and
2; ~12-22
· cm2). These results confirm our
previous studies (17, 18) and indicate that human cervical epithelial
cells form a relatively permeable epithelium on filters (33).

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Fig. 1.
Modulation of transepithelial electrical conductance
(GTE) across cultured CaSki cells on filters.
Similar trends were obtained with hECE and ECE16/1 cells, and data are
summarized in Fig. 2. A: effects of 1 mM
NG-nitro-L-arginine methyl ester
(L-NAME, arrow) in absence or presence of 1 mM
L-arginine (added to medium 10 min before
L-NAME). B: effects of 1 mM sodium nitroprusside
(SNP), 50 µM Hb, or Hb followed by SNP (arrows). C: effects
of 50 µM 8-bromo-cGMP (8-BrcGMP, arrow) in absence or presence of 25 µM KT-5823 (added to medium 30 min before 8-BrcGMP).
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Fig. 2.
GTE across cultured hECE, ECE16/1, and CaSki cells
before (Baseline) and 30 min after addition of L-NAME,
L-arginine (L-Arg), SNP, or Hb. Experiments
were done as described in Fig. 1, with 3-6 filters of each cell
type in each treatment group. In A, when indicated,
L-Arg was added to medium 10 min before L-NAME.
In B, when indicated, Hb was added to medium 5 min before SNP.
Values are means ± SD. * P < 0.01.
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Treatment with the NOS inhibitor
NG-nitro-L-arginine methyl ester
(L-NAME, 1 mM) (9, 10) decreased GTE in
all three types of cervical cells (Figs. 1A and 2A).
NG-nitro-L-arginine, a
more-selective NOS inhibitor for the Ca2+-dependent ecNOS
(8), had a similar effect on GTE; in contrast, NG-monomethyl-L-arginine, which
inhibits mainly the Ca2+-independent iNOS (10, 26), had no
effect (not shown). Preincubation of cells for 1 h with 1 mM
L-arginine, a naturally occurring substrate for NOS that
can competitively block the effect of L-NAME (28, 31), had
no significant effect on GTE, but it blocked the
L-NAME-induced decrease in permeability (Figs. 1A
and 2A). These results indicate that L-NAME
decreases GTE by a mechanism that involves blocking NOS and suggest that NO is involved in regulation of
GTE across cervical cultures.
To better understand how NO regulates permeability of cultured human
cervical epithelia, five additional experiments were done. The first
experiment identified expression of NOS isoforms in human cervical
cells. Experiments were done on extracts of human endocervical and
ectocervical tissues, as well as on lysates of cultured hECE, ECE16/1,
and CaSki cells, by use of the RT-PCR technique. With use of
oligonucleotide primers complementary to cloned human ecNOS (25), bNOS
(30), and iNOS (11), single cDNA fragments of 465, 415, and 485 bp,
respectively, were amplified by RT-PCR from endocervical and
ectocervical tissues (Fig. 3A) and
from lysates of hECE, ECE16/1, and CaSki cells (Fig. 3B). The
cDNA fragments were isolated, amplified, and purified, and the products
were sequenced by the dideoxy chain termination method. Sequence
analysis of the cloned segments revealed homologies of 98-99%
(sense and antisense) with the human ecNOS, bNOS, and iNOS (the
differences were sequence errors). These results indicate that human
cervical epithelial cells express mRNA for ecNOS, bNOS, and iNOS.

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Fig. 3.
Human cervical cells express mRNA for endothelial, neuronal, and
inducible nitric oxide synthase (ecNOS, bNOS, and iNOS, respectively).
Experiments utilized RT-PCR technique and were done on extracts of
endocervical (EndoCx) and ectocervical (EctoCx) minces (A) or
on lysates of cultured CaSki, ECE16/1, and hECE cells (B).
Oligonucleotide primers complementary to cloned ecNOS, bNOS, and iNOS
were used to amplify single cDNA fragments of 465, 415, and 485 bp,
respectively. Experiment was repeated twice. In mock reactions
[lacking oligo(dt) and avian myeloblastosis virus, see
METHODS], no detectable bands were found (not
shown).
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The second experiment studied release of NO by cultured human cervical
epithelial cells. All three types of cervical cells release NO
constitutively into the medium (Table 1) at
levels that are similar to other cell types (3). L-NAME
attenuated release of NO (Table 1); preincubation of cells for 1 h with 1 mM L-arginine had no effect on baseline release of NO,
but it blocked the L-NAME-induced decrease in NO release
(Table 1). These results suggest that cultured human cervical
epithelial cells release NOS-derived NO constitutively into the
extracellular medium.
The third experiment tested the effect of sodium nitroprusside (SNP) on
GTE. SNP is an NO donor, and it can mimic cellular effects of NO (28, 31). Treatment with 1 mM SNP resulted in an acute
and transient increase in GTE, followed by a slower
and more sustained increase in permeability (Fig. 1B). The
acute increase in GTE began within 1 min of
addition of SNP and was followed by fast return of
GTE to near-baseline values (Fig. 1B). The
late increase in GTE began 5 min after addition of
SNP, and it reached a plateau after 25-30 min (Fig. 1B). A
similar effect was obtained with the NO donor molisidomine
[N-(ethoxycarbonyl)-3-(4-morpholinyl)sydnoneimine (SIN-1)] in the presence of superoxide dismutase (to degrade
superoxide anions; not shown). Viability tests using MTT staining
revealed that treatment with SNP resulted in <3% positive staining.
These results indicate that the increase in GTE in
response to SNP is not the result of toxic effects on the cells but is
mediated by NO or a related metabolite.
The fourth experiment tested the effect of SNP on
Ppyr. The objective was to determine effects on
paracellular permeability by use of an additional end point to
GTE measurements. Pyranine is a 510-Da trisulfonic
acid; it traverses epithelia via the paracellular pathway, and its
concentration can be measured down to nanomolar levels by fluorescence
techniques (18). Similar to the effect on GTE, SNP
also increased the Ppyr (Table
2).
The fifth experiment studied the effect of the NO scavenger Hb (4) on
GTE. Treatment with 50 µM Hb decreased
GTE acutely (Figs. 1B and 2B), and
the permeability remained low for the duration of presence of Hb in the
bathing medium (Fig. 1B). In cells pretreated with Hb, SNP did
not evoke the acute transient increase in GTE (Fig.
1B), and the amplitude of the late increase in permeability was
markedly attenuated (Figs. 1B and 2B). Hb did not have
a significant effect on Ppyr, but it blocked the
SNP-induced increase in Ppyr (Table 2).
Collectively, the results shown in Figs. 1-3 and Tables 1 and 2
suggest that cultured human cervical epithelial cells produce NO; NO
constitutively increases the paracellular permeability, and Hb
antagonizes the NO-induced increase in permeability.
NO-induced increase in permeability is mediated by cGMP.
In some biological systems, effects of NO are mediated by cGMP. This
signaling cascade involves activation of soluble guanylate cyclase,
upregulation of cellular cGMP, and activation of cGMP-dependent protein
kinase (9, 21, 28, 31). The hypothesis that was tested in this section
was that the NO-induced increase in cervical permeability is mediated
by cGMP. To test the hypothesis, the effect on GTE
of 8-bromoguanosine-cGMP (8-BrcGMP) was determined. 8-BrcGMP is a
stable cell-permeable analog of cGMP previously used in similar studies
to mimic cellular effects of cGMP (4, 28, 31). Treatment of cultured
human cervical epithelial cells on filters with 8-BrcGMP caused a slow
increase in GTE (Fig. 1C). The increase in
GTE began 5 min after addition of 8-BrcGMP, and it
reached a plateau after 25-30 min, similar to the late effect of
SNP (cf. Fig. 1, B and C). Hb had no significant effect
on the response to 8-BrcGMP (not shown).
A possible explanation for the similar time course of the increases in
GTE in response to SNP (the late response) and to
8-BrcGMP is that the two effects are interrelated. To clarify the
mechanism of action of NO and cGMP on GTE, four
additional experiments were done. First, cells were pretreated with 25 µM LY-83583, a blocker of guanylate cyclase (21, 29), or with 25 µM
KT-5823, a blocker of cGMP-dependent protein kinase (9, 10, 31). When
administered alone, neither LY-83583 nor KT-5823 had a significant
effect on GTE (Fig. 4).
LY-83583 blocked the SNP-induced increase in GTE, but it had no significant effect on the 8-BrcGMP increase in
permeability (Fig. 4). KT-5823 attenuated the SNP-induced increase in
GTE and the 8-BrcGMP-induced increase in
permeability (Fig. 4). These results indicate that LY-83583 blocks a
necessary signaling step for SNP, while KT-5823 blocks a necessary
signaling step for 8-BrcGMP.

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Fig. 4.
GTE across cultured hECE, ECE16/1, and CaSki cells
before (Baseline) and 30 min after addition of 1 mM SNP, 50 µM
8-BrcGMP, 25 µM LY-83583, or 25 µM KT-5823, alone or in
combination. Experiments were done as described in Fig. 1, with
3-4 filters of each cell type for each treatment group. For
combined treatments, LY-83583 or KT-5823 was added 30 min before
addition of SNP or 8-BrcGMP. Values are means ± SD. * P < 0.01.
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The second experiment tested the degree to which treatment with SNP + 8-BrcGMP exerts an additive effect on GTE compared
with the effects of each drug alone. As shown in Fig.
5, SNP and 8-BrcGMP produced dose-related
increases in GTE. In the range of
concentrations that were used, 0.5 mM SNP and 20 µM 8-BrcGMP
produced a half-maximal increase in GTE (Fig. 5,
A and B, respectively). These concentrations were used
to determine the degree of additivity of the increases in
GTE in response to SNP + 8-BrcGMP. In cells that
were pretreated with 20 µM 8-BrcGMP, SNP increased
GTE in a dose-related manner (Fig. 5A), but
the effect of 8-BrcGMP + SNP was smaller than that expected from the
calculated combined effects of both agents (Fig. 5B). In cells
that were pretreated with 0.5 mM SNP, 8-BrcGMP increased GTE in a dose-related manner (Fig. 5B), but
the effect of SNP + 8-BrcGMP was also smaller than that expected from
the calculated combined effects of both agents (Fig. 5A). These
results indicate that the effects of SNP and 8-BrcGMP on
GTE do not summate.

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Fig. 5.
Dose effects of SNP and 8-BrcGMP on GTE across
CaSki cells on filters. A: 30 min before experiments, cells
were treated with SNP in absence ( ) or presence of 20 µM 8-BrcGMP
(+8-BrcGMP, ). B: 30 min before experiments, cells were
treated with 8-BrcGMP in absence ( ) or presence of 0.5 mM SNP (+SNP,
). In some experiments, cells were treated with 1 mM SNP (A)
or 100 µM 8-BrcGMP (B) and then exposed to hypertonic
gradient (+HTG) in subluminal-to-luminal direction by addition of 120 µl of 2 M sucrose solution to subluminal compartment ( ).
* P < 0.01.
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A possible explanation for the lack of additivity of SNP and 8-BrcGMP
effects on GTE is that 1 mM SNP or 100 µM
8-BrcGMP has already produced maximal increases in
GTE; subsequently, treatments with
8- BrcGMP or SNP, respectively, could not evoke an additional increase in permeability. This possibility was tested in the third experiment, in which a hypertonic challenge was used to increase GTE. Hypertonic gradients in the
subluminal-to-luminal direction increase GTE by
decreasing the RLIS (13). In the present study, cells treated with 1 mM SNP or 100 µM 8-BrcGMP were also exposed to
hypertonic gradients of 325 to 285 mosmol/l in the
subluminal-to-luminal direction. Hypertonic gradients increased
GTE significantly in cultures treated with SNP
(Fig. 5A) or 8-BrcGMP (Fig. 5B). Thus, in contrast to
lack of an additive effect of SNP and 8-BrcGMP on
GTE, hypertonic gradients produced additive
increases in permeability with SNP and 8-BrcGMP. Therefore, the lack of
additivity in response to SNP + 8-BrcGMP cannot be explained by the
fact that each of these agents alone simulates a maximal increase in
GTE. Another explanation is that the effects of NO
and cGMP on GTE are interrelated and that cGMP
mediates the effect of NO.
To better understand the possible role of cGMP as mediator of the
SNP-induced increase in GTE, the fourth experiment
studied effects of SNP on cGMP accumulation in human cervical cells.
Treatment with SNP increased cGMP, and cotreatment with Hb blocked the
effect of SNP (Table 3). LY-83583 alone had
no significant effect on cGMP, but it also blocked the effect of SNP
(Table 3). KT-5823 had no significant effect on cellular levels of cGMP
or on the response to SNP (Table 3). Collectively, the results shown in Figs. 3-5 and Table 3 support the hypothesis that the effect of NO
on GTE is mediated by cGMP.
NO and cGMP decrease the RLIS.
Increases in paracellular permeability, such as those produced by SNP
and 8-BrcGMP (Fig. 1, B and C), can be the result of decreases in RTJ or RLIS (37).
To determine the degree to which NO and cGMP modulate
RTJ or RLIS, two experiments
were done. In the first experiment, the effects of SNP and 8-BrcGMP on
uCl/uNa across the cultured
epithelium were determined. This parameter was chosen because tight
junctions influence the mobilities of monoions in the intercellular
space, and the cation selectivity reflects the degree of occlusion of
the paracellular space by the tight junctions (17, 33, 37). Neither SNP
nor 8-BrcGMP affected uCl/uNa
(Table 4), suggesting lack of a significant effect on RTJ. The positive control in this
experiment was incubation of cells in low (0.6 mM) extracellular
Ca2+; in low extracellular Ca2+ the
permeability increases as a result of decreases in
RTJ, and uCl/uNa increases (Table 4)
(17). The results shown in Table 4 suggest that RTJ
is not involved in the responses to SNP or 8-BrcGMP.
The second experiment tested the degree to which SNP and 8-BrcGMP
modulate the RLIS. In an intact epithelium the
RLIS usually depends on the geometry of the
intercellular space and, in turn, on the size of the cells surrounding
this space (37). The objective was therefore to determine the degree to
which SNP and 8-BrcGMP modulate the size of cervical epithelial cells
attached on filters. CaSki cells on filters were loaded with the
fluorescent dye fura 2, and changes in cell size were determined by
measuring changes in fluorescence in the Ca2+-insensitive
wavelengths (F360/510) (4, 19). Changes in cytosolic Ca2+ were also determined in the same cells by switching
the excitation filters between the Ca2+-sensitive (340 and
380 nm) and Ca2+-insensitive (360 nm) wavelengths. Such an
experiment is shown in Fig. 6, in which
effects of SNP (Fig. 6A) and 8-BrcGMP (Fig. 6B) on
F360/510 and cytosolic Ca2+ are shown.

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Fig. 6.
Effects of 1 mM SNP (A) and 50 µM 8-BrcGMP (B) on
changes in ratio of fluorescence at 350 nm to fluorescence at 510 nm
(F360/510), in cytosolic Ca2+ concentration
([Ca2+]i), and in
GTE in CaSki cells attached on filters. Experiments
were conducted in parallel in fluorescent chamber (for measurements of
F360/510 and [Ca2+]i)
and in Ussing chamber (for measurements of GTE).
Experiments were repeated 3 times.
|
|
SNP had a bimodal effect on F360/510: an acute and
transient increase with return of F360/510 levels to
baseline, followed by a slow and steady increase in
F360/510 (Fig. 6A). An increase in
F360/510 is the result of a decrease in cell size (4, 19), and the data shown in Fig. 6A indicate that SNP stimulated a
bimodal decrease in cell size: an acute transient decrease with fast
return to baseline, followed by a sustained prolonged decrease in cell size. Also shown in Fig. 6A are changes in
GTE that were determined in parallel filters in the
Ussing chamber. The changes in F360/510 had a time course
similar to the changes in GTE. Because a decrease in cell size causes a decrease in RLIS (37), the
results shown in Fig. 6A suggest that the SNP-induced increase
in GTE is mediated by decreases in
RLIS. Interestingly, SNP induced a transient
increase in cytosolic Ca2+ that had a time course similar
to the transient increases in F360/510 and
GTE but not to the late sustained increase in
F360/510 or GTE (Fig. 6A).
These results suggest that changes in cytosolic Ca2+ do not
mediate the late increases in F360/510 and
GTE.
8-BrcGMP also stimulated an increase in F360/510, but in
contrast to SNP, the effect was monotonous and resembled only the late
increase in fluorescence induced by SNP (Fig. 6B). The increase in F360/510 induced by 8-BrcGMP had a time course similar
to the effect of 8-BrcGMP on GTE (Fig. 6B),
suggesting that 8-BrcGMP increases the permeability by decreasing the
RLIS. Furthermore, the effects of 8-BrcGMP on
F360/510 and GTE (Fig. 6B) had
a time course similar to the effects of SNP on F360/510 and
GTE (Fig. 6A). However, 8-BrcGMP did not
change significantly levels of cytosolic Ca2+ (Fig.
6B). A possible explanation for the results shown in Fig. 6 and
Table 4 is that the SNP-induced late increase in
GTE is mediated by a cGMP-dependent decrease in
RLIS.
SNP and 8-BrcGMP increase G-actin.
Changes in cell size, such as those described in Fig. 6, depend on the
ability of cells to change their shape in response to stimuli. Two
types of cellular mechanisms are usually involved in cell size
decrease: changes in size secondary to loss of cellular water (7) and
changes in size secondary to alterations of the cytoskeleton (7, 34).
In secretory epithelial cells, the former mechanism is usually acute
and transient; it is mediated by active Cl
secretion, followed by water efflux to compensate for osmolar changes
(7). Such a mechanism can explain the acute transient decrease in
cervical cell size in response to SNP but is an unlikely mechanism for
the prolonged decrease in cervical cell size in response to SNP and
8-BrcGMP. The hypothesis that was tested in the present section was
that the NO-induced prolonged decrease in cell size is mediated by
cGMP-dependent changes of the cytoskeleton.
The major component of the cytoskeleton in eukaryotic cells is actin
filaments. In most cells, density of actin filaments depends on
equilibrium between polymerization of monomeric G-actin and
depolymerization of filamentous F-actin (5). Enhanced polymerization of
G-actin to form F-actin is usually associated with a dense and rigid
cytoskeleton, whereas depolymerization of F-actin is associated with a
more dynamic (fragmented) cytoskeleton (5, 6). A G-actin-associated
cytoskeleton is likely to render cells more deformable. On the basis of
these considerations, the objective was to determine the effect of NO
and cGMP on total cellular actin and on the fraction of G-actin.
Neither SNP nor 8-BrcGMP had a significant effect on total cellular
actin, but both agents significantly increased G-actin (Table
5). Neither Hb nor KT-5823 had a
significant effect on baseline total cellular actin levels or on
G-actin (Table 5). In contrast, Hb blocked the SNP-induced increase in
G-actin, and KT-5823 blocked the 8-BrcGMP-induced increase in G-actin
(Table 5).
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Table 5.
Effects of SNP and 8-BrcGMP on total cellular actin and on fraction of
G-actin in hECE and CaSki cells attached on filters
|
|
To better understand the effect of 8-BrcGMP on actin steady state,
CaSki cells were pretreated with L-NAME [to
downregulate NO (Table 1) and possibly cGMP (Table 3)] and then
treated with 8-BrcGMP. At different time intervals after addition of
8-BrcGMP, changes in G-actin were determined. 8-BrcGMP increased the
ratio of G-actin to total cellular actin in a time-related manner: the increase began 10 min after addition of 8-BrcGMP and plateaued after
~30 min (Fig. 7). This time course
resembles the effect of 8-BrcGMP on GTE (Fig.
1C). KT-5823 blocked the 8-BrcGMP-induced increase in G-actin
(Fig. 7), whereas LY-83583 had no effect (not shown). These results
suggest that cGMP, via its action on a cGMP-dependent protein kinase,
upregulates G-actin in human epithelial cervical cells.

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Fig. 7.
Effects of 8-BrcGMP on ratio of G-actin to total cellular actin. CaSki
cells on filters were treated for 30 min with 1 mM L-NAME,
then with 50 µM 8-BrcGMP [in absence ( ) or presence of 25 µM KT-5823 ( )], in continued presence of L-NAME.
At indicated time intervals, filters containing cells were removed for
determinations of G-actin and total cellular actin (see
METHODS). Values are means ± SD of 3 filters at each
point. * P < 0.03-0.01.
|
|
 |
DISCUSSION |
The results of the present study indicate that NO increases cervical
epithelial paracellular permeability by two mechanisms: an acute
transient increase, followed by a prolonged sustained increase in
permeability. The transient increase is mediated possibly by an
increase in cytosolic Ca2+. The paracellular mechanism of
the acute transient increase in GTE is unknown but,
on the basis of studies in other cell types, it may involve
Cl
secretion and water efflux (7). Human cervical
epithelial cells restore acutely water loss by volume regulatory
increase (13), and this can explain the prompt termination of the
response and fast return of GTE to baseline levels.
The main effort in the present study was to understand the mechanisms
involved in the NO-induced late sustained increase in GTE. The results suggest that the effect is
mediated by cGMP and that it involves alterations of the cytoskeleton.
The statement that NO directly stimulates an increase in paracellular
permeability is supported by the following experimental findings:
1) The NOS inhibitors L-NAME and
NG-nitro-L-arginine decreased
GTE. 2) The effect of L-NAME
was blocked by pretreatment with L-arginine. 3) The
NO donors SNP and SIN-1 increased GTE. 4)
The SNP-induced increase in GTE was dose dependent and could be blocked by the NO scavenger Hb.
Hb blocked the SNP-induced transient and sustained increases in
GTE. This effect of Hb can be explained by
deactivation of SNP-donated NO. However, Hb alone also decreased the
permeability. A possible explanation is that Hb
deactivated NO produced constitutively by the cells. This speculation
is supported by the findings that human cervical epithelial cells
release constitutively NO into the extracellular medium,
L-NAME decreases the NO release, and L-arginine
blocks the effect of L-NAME. These findings suggest that
cervical cells autoregulate permeability and maintain increased paracellular permeability by continuously secreting NO. According to
this hypothesis, the effect of NO involves autocrine/paracrine regulation of permeability: cervical epithelial cells secrete NO, and
NO can act on the same cell (autocrine regulation) or diffuse into
neighboring cells (paracrine regulation).
In addition to blocking the SNP-induced increase in permeability, Hb
also blocked the SNP-induced increase in cGMP. Both effects can be
explained by deactivation of SNP-derived NO. However, as was mentioned
earlier, Hb also decreased the permeability when administered alone.
If, as speculated below, NO increases permeability by activating
cGMP-dependent signaling, then the effect involves activation of
intracellular guanylate cyclase (28, 31). Because Hb does not permeate
cells, the Hb-induced decrease in GTE cannot be
explained by deactivation of intracellular NO. An alternative explanation is that Hb decreased intracellular NO by deactivating NO in
the extracellular medium. NO is a volatile gas that can permeate cell
membranes (28, 31), and in biological systems, such as cultured cells
on filters, it is in equilibrium between the intracellular and
extracellular fluids. Consequently, by deactivating NO in the
extracellular medium, intracellular activity of NO is secondarily
decreased, and lowered intracellular NO activity results in lower
cellular cGMP.
NO is synthesized from L-arginine during the NOS-catalyzed
conversion of L-arginine to L-citrulline (28,
31). Three broad categories of NOS isozymes have been characterized,
ecNOS, bNOS, and iNOS, and all three are products of different genes
(11, 25, 28, 30, 31). The ecNOS and bNOS isoforms are constitutively expressed, and their expression can be regulated by physiological stimuli (3, 23, 28, 31); in contrast, iNOS is usually expressed at low
levels, but it can be upregulated during exposure to pathological
conditions (11, 28, 31). Until recently, little was known about the
expression of NOS in the human cervix. The present study shows that
cultured human cervical epithelial cells, as well as human endocervical
and ectocervical tissues, express mRNA for all three NOS isoforms. It
is unknown which of the three NOS isoforms contributes to the NO pool
that is released constitutively into the extracellular medium.
The present study suggests that the NO-induced sustained increase in
permeability in cultured human cervical epithelia is mediated by cGMP.
This statement is supported by the following experimental findings:
1) SNP increased cGMP, and the effect was blocked by Hb.
2) 8-BrcGMP increased GTE, and the effect
had a time course similar to that of SNP. 3) Neither
L-NAME nor Hb modulated the 8-BrcGMP-induced increase in
GTE. 4) LY-83583, a blocker of guanylate
cyclase (21, 29), blocked the SNP-induced increase in permeability, but
it did not modulate the effect of 8-BrcGMP on GTE.
5) KT-5823, a blocker of the cGMP-dependent protein kinase (9,
31), blocked the SNP- and 8-BrcGMP-induced increases in permeability.
6) Treatment with SNP + 8-BrcGMP produced a smaller increase in
permeability than was expected from the calculated combined effects of
both agents. Collectively these results suggest that cGMP mediates the
NO-induced increase in permeability.
SNP and 8-BrcGMP increased cervical epithelial permeability by
decreasing RLIS. Decreases in
RLIS are the result of decreases in cell size (32,
37). Prolonged decreases in paracellular permeability usually involve
rearrangement of cytoskeletal proteins (34). In cervical cells, SNP and
8-BrcGMP increased G-actin; the effect of 8-BrcGMP correlated in time
with the 8-BrcGMP-induced increase in GTE, and it
could be blocked by KT-5823, suggesting that the increase in cellular
G-actin is mediated by activation of cGMP-dependent protein kinase.
The molecular mechanism by which cGMP increases G-actin is unknown. NO
can ADP-ribosylate actin in broken cell preparations (2, 27).
ADP-ribosylation of monomeric actin sequesters G-actin and decreases
its availability for polymerization, thus inhibiting formation of
F-actin and increasing G-actin (2, 27). The present results suggest
that cGMP has a similar effect in human cervical epithelial cells, but
more studies are needed to test this hypothesis.
On the basis of the present results, a new model of NO regulation of
cervical permeability is proposed. The model suggests that NO acts on
soluble guanylate cyclase and stimulates an increase in cGMP; cGMP,
acting via cGMP-dependent protein kinase, shifts actin steady state
toward G-actin; this fragments the cytoskeleton (5, 6) and renders
cells more sensitive to decreases in cell size in response to intrinsic
and external stimuli.
NO/cGMP-dependent modulation of the cytoskeleton may be important for
our understanding of regulation of paracellular permeability. Human
cervical epithelia, like other types of epithelia, form a confluent
layer of cells in which neighboring cells are bound into a single
functional sheet by intercellular junctions (33). The intercellular
connections are usually located near the apical border of the
epithelium (33). This asymmetric location of intercellular connections
results in an uneven distribution of plasma membrane between the larger
"basolateral" and the smaller "apical" cell surface (33).
In vivo, cervical epithelial cells are exposed to a blood pressure
gradient in the subluminal-to-luminal direction. Because the
basolateral cell surface is greater than the apical cell surface, the
hydrostatic gradient will exert a net vectorial positive pressure on
the cell. Epithelial cells respond to such vectorial pressure by
decreasing their size, mainly in the region that is determined by the
basolateral plasma membrane (32). A decrease in cell size involves a
parallel increase in the volume of the intercellular space, i.e., a
decrease in RLIS and an increase in the permeability.
The present results suggest that NO stimulates cGMP-mediated
rearrangement of the cytoskeleton. This can render cervical cells more
deformable in response to the hydrostatic pressure and result in an
increase in permeability. Consequently, NO modulation of permeability
may be an important mechanism for regulation of cervical mucus
secretion in vivo. NO is a naturally occurring signaling transmitter in
the uterus in vivo (20). The present results indicate that human
cervical epithelial cells produce NO constitutively. In other types of
cells, NO activity can be increased by upregulation of NOS(s)
transcription in response to normal (e.g., estrogen) or abnormal (e.g.,
bacterial toxin) stimuli (3, 28, 31), but little is known about NOS(s)
regulation in the human cervix. Another mode of NO regulation is by
agents that elevate cytosolic Ca2+. Elevated levels of
intracellular Ca2+ can activate release of NO by
Ca2+-dependent NOS (28, 31). In human cervical epithelial
cells, secretagogues, such as ATP and histamine, can increase cytosolic Ca2+ (16); they can lead to higher NO activity and to
increased permeability. This conclusion may have pharmacological
significance in the sense of modulating cervical secretions by
NO-related agents, but more studies are needed to clarify the effects
in women.
The new model of NO regulation of cervical permeability may also
contribute to our understanding of abnormal secretions in the cervix.
Excessive cervical secretion is a common complaint among women of all
ages. In many cases, an infective organism can be found, but the
mechanism of excessive secretion is unclear. A possible explanation,
based on the present study, is upregulation of an NOS mechanism
(possibly iNOS), followed by increased NO activity and increased
epithelial permeability.
 |
ACKNOWLEDGEMENTS |
This study was supported by National Institutes of Health Grants
HD-00977, HD-29924, and AG-15955.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: G. I. Gorodeski,
University MacDonald Women's Hospital, University Hospitals of
Cleveland, 11100 Euclid Ave., Cleveland, OH 44106 (E-mail:
gig{at}po.cwru.edu).
Received 13 October 1999; accepted in final form 23 November 1999.
 |
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