Departments of 1 Reproductive Biology, 2 Pathology, and 3 Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106
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ABSTRACT |
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Estradiol had a
biphasic effect on permeability across cultures of human umbilical vein
endothelial cells (HUVEC): at nanomolar concentrations it decreased the
HUVEC culture permeability, but at micromolar concentrations it
increased the permeability. The objective of the present study was to
test the hypothesis that the changes in permeability were mediated by
nitric oxide (NO)-related mechanisms. The results revealed dual
modulation of endothelial paracellular permeability by estrogen.
1) An endothelial NO synthase (eNOS)-, NO-, and cGMP-related,
Ca2+-dependent decrease in
permeability was activated by nanomolar concentrations of estradiol,
resulting in enhanced Cl
influx, increased cell size, and increases in the resistance of the
lateral intercellular space
(RLIS) and in
the resistance of the tight junctions
(RTJ); these
effects appeared to be limited by the ability of cells to generate cGMP
in response to NO. 2) An inducible
NO synthase (iNOS)- and NO-related,
Ca2+-independent increase in
permeability was activated by micromolar concentrations of estradiol,
resulting in enhanced Cl
efflux, decreased cell size, and decreased
RLIS and
RTJ. We conclude that the net effect on transendothelial permeability across HUVEC depends on the relative contributions of each of these two systems to
the total paracellular resistance.
paracellular resistance; tight junctions; sex hormones; microcirculation; endothelial nitric oxide synthase; inducible nitric oxide synthase
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INTRODUCTION |
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ENDOTHELIAL CELLS FORM A barrier between the blood and tissue parenchyma and control the movement of blood cells, plasma fluid, and solutes between the vascular compartment and the extracellular space. The main route of transport across endothelia is via the intercellular (paracellular) space, and the driving forces are transendothelial hydrostatic and hypertonic gradients (41).
Endothelial cells are targets for the actions of the female hormone estrogen. Studies in vivo suggest that estrogens may regulate the permeability of endothelia in different vascular beds (44), but data are lacking about the effects of estrogen on endothelial paracellular permeability. We (15) and others (39) have recently developed experimental conditions to study transport phenomena across low-resistance epithelia in vitro (e.g., cultured endothelial cells), and we used cultured human umbilical vein endothelial cells (HUVEC) to study the effects of estrogen on paracellular permeability and the mechanisms involved.
In endothelia, some of the effects of estrogen are mediated by the
nitric oxide (NO) system. NO can modulate the permeability of
epithelial and endothelial tissues, but there is considerable controversy concerning the role of NO as a mediator of vascular permeability (e.g., Refs. 3, 24, 25). Some studies suggested the
involvement of downstream messengers from NO such as cGMP (23).
Estrogens can upregulate NO activity (e.g., Refs. 46, 49) in
endothelial cells (20) and in women (37), but there are no definitive
conclusions with regard to the mechanisms involved (2, 47) and whether
NO increases (42) or decreases (48) the per- meability. In
preliminary experiments with HUVEC, we found that
N-nitro-L-arginine methyl ester
(L-NAME), a NO
synthase (NOS) inhibitor, modulates the paracellular permeability,
suggesting that NO regulates the paracellular permeability across
HUVEC. In those studies, the magnitude of the
L-NAME effect was dependent on
previous treatment of the cells with estrogen, suggesting that estrogen
controls endothelial paracellular permeability by modulating the
activity of NO. One of our objectives was to test the hypothesis that
the effects of estrogen on HUVEC permeability are mediated by
NO-related mechanisms.
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METHODS |
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Isolation of HUVEC. The experiments were conducted on passage 3 HUVEC, which retain morphological and physiological characteristics of the native umbilical vein endothelium (51). We used cells obtained from females, to avoid gender-related differences in the response to estrogen. HUVEC were isolated from fresh human umbilical veins of newborn females using previously described methods (51). Briefly, segments of umbilical cords were washed with saline, and the distal end was clamped. An umbilical vein was filled with 20-30 ml of solution, composed of 1 mg/ml collagenase (type CLS, Worthington Biochemical, Freehold, NJ) in Hanks' balanced salt solution (HBSS) containing Ca2+ and Mg2+. The proximal portion of the vessel was ligated, and the vessel was incubated for 10-20 min at room temperature. The solution contained within the vessel, including cells detached from the umbilical vein, was collected and rinsed in culture medium composed of medium 199 (M.A. Bioproducts, Walkersville, MD) containing 20% heat-inactivated fetal bovine serum (FBS; HyClone, Logan, UT), 5 µg/ml insulin, 5 ng/ml transferrin, 5 µg/ml selenium, 75 µg/ml endothelial cell growth supplement (Upstate Biotechnology, Lake Placid, NY), 100 ng/ml heparin, and penicillin-streptomycin-fungizone solution. The mixture containing cells was centrifuged at 800 g for 5-10 min at room temperature. The cells in the pellet (mostly HUVEC) were resuspended in culture medium and plated onto human fibronectin (5 µg/cm2; Chemicon International, Tecumula, CA)-coated T-75 tissue culture flasks (Corning, Corning, NY). Cells were maintained at 37°C in 91% O2-9% CO2 in a humidified incubator and refed fresh culture medium every 3 days. Cells were grown to confluence (~2 wk; typical confluent density of 2-3 × 106 cells/flask). Identification of endothelial cells was confirmed by immunohistochemical staining for factor VIII antigen (factor VIII/von Willebrand factor) (51).
For some experiments with estrogens, cells were shifted to a steroid-free medium. This medium was composed of phenol-red-deficient medium 199 (M. A. Bioproducts) containing 20% heat-inactivated FBS that was previously treated with charcoal to remove steroids, and supplemented with all the other reagents as described above for the regular culture medium. Preparation of charcoal-treated serum was described (7). Briefly, dextran-coated charcoal was dissolved at 8% in 0.15 M NaCl, autoclaved, mixed by stirring, and spun, and the pellet was resuspended as 1 g/1.25 ml in H2O. FBS (HyClone) was mixed with the activated charcoal-dextran at 20:1 (vol/vol) and incubated for 45 min at 55°C. At the completion of incubation, the mixture was spun twice at 800 g for 20 min and the supernatant (serum) was decanted and collected.Preparation of cultures on filters. Anocell filters (ceramic base; Anocell-10, Anotec, Oxon, UK; surface area of 0.6 cm2, pore size of 0.02 µm, and width of 50 µm) were coated on their upper (luminal) surface with 5 µg/cm2 collagen type I and incubated at 37°C overnight. The excess collagen solution was aspirated, the filter was dried at 37°C, and both sides of the filter were rinsed three times with HBSS. 3T3 murine fibroblasts were irradiated as described (16) and were used as feeders to improve plating efficiency of HUVEC on filters. 3T3 fibroblasts were plated on the luminal surface of the filter at 4 × 104 cells/cm2 24 h before plating HUVEC. The plating efficiency of 3T3 fibroblasts was relatively low, ~10%. At this low density, the feeder cells were scattered and did not form a confluent cell layer and therefore had no significant effect on the permeability.
Before being plated on filters, HUVEC were washed twice with HBSS containing 1 mM EDTA and harvested in the same medium supplemented with 0.025% trypsin. Detached cells were collected in a conical tube and mixed with an equal volume of culture medium to inactivate the trypsin. Tubes were spun at 800 g for 10 min at room temperature, and HUVEC in the pellet were resuspended in culture medium and plated on the luminal surface of the filter at a high density (3 × 105 cells/cm2) in 0.5 ml of culture medium. Under these conditions, the plating efficiency of HUVEC was 50-70% (in contrast to ~15% on filters without fibroblasts). This resulted in a ratio of ~50:1 HUVEC to 3T3 fibroblasts after plating, but this ratio tripled after 2-3 days due to the proliferation of HUVEC and lack of proliferation of the irradiated 3T3 fibroblasts. After the plating of the HUVEC, 1 ml of medium was added to the bottom (subluminal) compartment. HUVEC cultures became confluent within 12-24 h after plating. In all experiments we also included control filters seeded only with 3T3 fibroblasts.Changes in paracellular permeability. Changes in paracellular permeability were determined in terms of changes in the permeability to pyranine (Ppyr) and in terms of changes in the transendothelial electrical conductance (GTE). Before experiments, filters containing cells were washed three times and preincubated for 15 min at 37°C in a modified Ringer buffer composed of (in mM) 120 NaCl, 5 KCl, 10 NaHCO3 (before saturation with 95% O2-5% CO2), 1.2 CaCl2, 1 MgSO4, 5 glucose, and 10 HEPES (pH 7.4), with 0.1% BSA in volumes of 4.7-5.2 ml in the luminal and subluminal compartments.
Determinations of Ppyr. The Ppyr was determined from unidirectional (luminal to subluminal) fluxes across filters mounted vertically in the modified Ussing-diffusion chamber as described (16), to prevent hydrostatic gradients. Pyranine is a trisulfonic acid with a molecular mass of 0.51 kDa; it was chosen as a probe to assess paracellular permeability because it traverses epithelia via the paracellular pathway, and its concentration can be measured down to nanomolar levels by fluorescence techniques (16). Cytolysis of HUVEC that were previously incubated with 0.1 mM pyranine did not increase pyranine fluorescence significantly above background (not shown). Ppyr was determined from unidirectional (luminal to subluminal) fluxes: pyranine was added to the luminal compartment, and the amount of pyranine in the subluminal compartment was measured after 10 min. The transendothelial permeability coefficient (Ppyr) was calculated as described (16).
Determinations of GTE.
GTE was chosen as
an end point to assess paracellular permeability because this method is
sensitive to small changes in transendothelial permeability, and it
allows for real-time measurements of changes in
GTE. The
permeability of the paracellular pathway is
104 to
105 times higher than
that of the plasma membranes (34), and the former determines the
overall conductive properties of the cultured endothelium. Changes in
GTE therefore
reflect changes in paracellular permeability. The electrophysiological
methods, including appropriate measures to prevent artifacts, were
previously discussed by us (15) and by others (34). Changes in
GTE were
determined continuously across filters mounted vertically in a modified
Ussing chamber as described (15). This chamber, which also served as a
diffusion chamber, was originally designed by Dr. Grass (Precision
Instrument Design), was obtained through Costar (no. 3430, Cambridge,
MA), and was custom modified by Analytical Bioinstrumentation (Case Western Reserve University School of Medicine, Cleveland, OH). The
potential electrodes consist of
Ag-AgCl2 in 2 M KCl, are enclosed in Teflon tubing ending with a porcelain porous plug, and are situated
at close proximity (<1 mm) to the center of the luminal and
subluminal surfaces of the filter. This enabled us to measure precisely
the transendothelial potential difference (PD). The current electrodes
were made of Pt-Pt black and installed via existing inlets. Electrical
measurements were made with a conventional four-electrode voltage clamp
(DVC 1000, World Precision Instruments, Sarasota, FL), with a fluid
resistance compensation range of 0-1,000 , which was determined
each time with a blank filter.
GTE was determined continuously from successive measurements of the
transendothelial electrical current
(I; obtained by measuring the current
necessary to clamp the offset potential to zero and normalized to the
0.6-cm2 surface area of the
filter) and the transendothelial PD (lumen negative), switching between
I (pulses of 200-1,400
µA · cm
2)
and PD at a rate of 20 Hz:
GTE =
I/
PD. The volumes and content of
the buffer solutions used in the Ussing chamber experiments were kept
constant. The temperature in the chamber was maintained at 37°C,
and the medium in each compartment was continuously bubbled with an air
lift of 95% O2-5%
CO2, which flowed parallel to the surface of the culture. The output of the voltage clamp was recorded in
parallel on a strip-chart recorder (Linseis L6514, Cleveland, OH) and
on an IBM PC 286-30 equipped with a DI-220 analog-to-digital converter
board, a 1995 version of AT/MCA CODAS hardware and software (DATA Q,
Akron, OH), and a Bernoulli 90-megabyte hard disk. The CODAS board
allows acquisition of up to 50,000 samples/s with gap-free display.
AT-playback provides for data analysis.
Determinations of dilution potential.
Determinations of the transendothelial dilution potential
(Vdil) were
performed in the Ussing chamber as described (15). Vdil were
determined by measuring the effect of lowering NaCl in the luminal
solution on changes in voltage generated across the endothelial
culture. This was done by replacing the Ringer buffer in the luminal
compartment (130 mM NaCl) with low-NaCl (10 mM) solution. The latter
buffer was similar to the Ringer solution except that it lacked the 120 mM NaCl and was supplemented with 240 mM sucrose to compensate for
osmolarity. The methods of electrophysiological data evaluation were
previously described and discussed (15).
Vdil was the
measured PD (voltage in subluminal solution voltage in luminal
solution) after lowering of NaCl in the luminal solution, corrected for
the asymmetry of the potential electrodes. The Henderson diffusion
equation (15) was used to interpret the
Vdil in terms of
ionic permeabilities. With the assumption that
Na+ and
Cl
are the major permeant
ionic species and all other components can be neglected, the
Vdil can be
expressed in terms of the ratio of the mobilities of the monoions as
uCl/uNa,
where uCl and
uNa are the
mobilities of Na+ and
Cl
in the intercellular space.
Assay of NO.
Release of NO was determined as the accumulation of nitrite
(NO2) and nitrate
(NO
3) in the extracellular medium by a
modified Greiss method (30, 10). Before experiments, cells were washed
three times with PBS (37°C) and incubated in Ringer solution for 2 h at 37°C. At the end of incubation, the buffer was collected and
centrifuged to remove dead cells. Aliquots (20 µl) were mixed 1:1
vol/vol with 2× Greiss reagent (composed of 0.5% sulfanilamide
and 0.05% naphthalene diamine dihydrochloride in 2.5%
H3PO4)
and incubated for 10 min at room temperature. The fluorescence was read
at 365-nm excitation and 450-nm emission against a
NaNO2 standard curve, using the buffer as blank fluorescence. The detection limit of the assay was 2 µM. Results were expressed as picomoles per minute per milligram protein. Total cellular protein was determined as described (16).
Determinations of cGMP.
Cells were washed with ice-cold PBS, the buffer was aspirated, and 1.0 ml of 6% TCA was added and vigorously pipetted. The cell homogenate
was collected in a microcentrifuge tube; this step was repeated, and
the two homogenates were combined. The tube was vortexed for 1 min and
then centrifuged at 9,000 g for 15 min
at room temperature, and the aqueous phase was stored at 20°C until assayed. The cGMP content within the aqueous
phase was assayed using a commercially available RIA kit (Amersham, Arlington Heights, IL), after addition of a 25:1 ratio of
acetylation reagent (1 vol of acetic anhydride with 2 vol of
triethylamine) to each 500-µl sample. Results were expressed as
picomoles per minute per milligram protein.
Fluorescence of attached cells. The fluorescence experiments were conducted in a newly designed fluorescence chamber (18). In this apparatus, 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. The cells were illuminated over the apical surface, and the intensity of the emitted light from the apical surface was measured as described (18). Cells on filters were incubated in culture medium with 7 µM fura 2-AM plus 0.25% Pluronic F-12 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. Measurements of fura 2 fluorescence were made at the isosbestic wavelengths (360-nm excitation and 510-nm emission; F360/510) (40). Under these conditions, the leakage, photobleaching, and metabolization of fura 2 are minimal (18).
The theoretical background for the changes in F360/510 was recently discussed (18). Similar to the principle of F360/510 fura 2 microfluorescence imaging of attached cells (7), changes in F360/510 are not influenced by cytosolic Ca2+ but rather reflect changes in the intracellular concentration of the fura 2 and subsequently reflect changes in cell volume. The explanation is that, in attached and confluent cells, changes in volume are the result of changes both in the cross-sectional plane (the x-y plane, parallel to basal lamina) and in height (the z-axis, from basal lamina to the apical surface). It was suggested that in the new fluorescence chamber the emitted light stems from a single (or from a few) section(s) at the x-y plane. Changes in cell volume, and in particular in cell height, will draw more fura 2 molecules into, or away from, the monitored x-y plane(s), depending on whether the cell volume decreases or increases, respectively. Therefore, if the cell volume decreases, the signal will be stronger; if the cell volume increases, the signal will be weaker.Viability of cells attached on filters. Viability of cells attached on filters was determined by incubation of cells with 10 µg/ml propidium iodide. Positive staining with propidium iodide indicates late apoptosis or necrosis. A stock solution of propidium iodide (100 µg/ml) was prepared in PBS and kept in the dark at room temperature. For experiments, HUVEC on filters were washed with Ringer buffer and reincubated for 3 min at room temperature with propidium iodide; cultures were washed with PBS, and the fluorescence (540-nm excitation, 625-nm emission) was determined immediately in the fluorescence chamber at room temperature. Propidium iodide fluorescence was normalized to maximal fluorescence obtained in permeabilized cells that were preincubated with 5 µg/ml digitonin.
Preparation of total RNA.
Total RNA from HUVEC on filters was isolated in situ on the filter with
the Qiagen kit (Qiagen, Chatsworth, CA), using 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
(OD260). The purity of the RNA
sample was judged by determination of
OD260/OD280.
RT-PCR.
A Perkin-Elmer DNA thermal cycler (Cetus, Norwalk, CT) was utilized for
the assays using a RT-PCR kit (Boehringer Mannheim, Indianapolis, IN).
Total RNA (1.5 µg) denatured at 65°C for 5 min was reverse
transcribed in a final volume of 20 µl of reaction mixture containing
10 mM Tris · HCl (pH 8.3), 50 mM KCl, 10 mM MgCl2, 1 mM dNTPs, 5 µM
oligo(dT)15 (Promega, Madison,
WI), 40 units RNase inhibitor, and 25 units avian myeloblastosis virus (AMV) RT (Boehringer Mannheim, Mannheim, Germany). For mock reaction, a
similar tube was made up without the oligo(dT) and without the AMV RT.
The reaction was allowed to proceed at 42°C for 60 min and was
terminated by heating to 99°C for 2 min. The sample was diluted 1:5
with deionized distilled water. PCR was then performed in
a 50-µl volume using 5 µl of the diluted sample, 5× PCR
buffer (Amersham), 1 µM each primer, 0.01 mM dNTPs, 5 units
Taq polymerase (Amersham), and 1.4 µM Taq antibody (Clontech) in a
Perkin-Elmer 460 DNA thermal cycler (Cetus). Samples were heated for 5 min at 94°C, and then the following conditions were applied: for
endothelial NOS (eNOS), 35 cycles of 1 min of denaturation step at
94°C, 1 min of annealing step at 62°C, and 2 min of extension
step 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; and for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 30 cycles of 1 min at
94°C, 1 min at 60°C, and 1 min at 72°C. Samples were then
kept 7 min at 72°C, cooled at 4°C (soak file), and frozen at
80°C to facilitate removal of the mineral oil. The following
oligonucleotide primers were used: for eNOS (26), forward (sense)
5'-CAG TGT CCA ACA TGC TGC TGG AAA TTG-3' and reverse
(antisense) 5'-TAA AGG TCT TCT TGG TGA TGC C-3'; for iNOS
(11), forward (sense) 5'-GCC TCG CTC TGG AAA GA-3' and
reverse (antisense) 5'-TCC ATG CAG ACA ACC TT-3'; and for
GAPDH, forward (sense) 5'-TGA AGG TCG GAC TCA ACG GAT TTG
GT-3' and reverse (antisense) 5'-GTG GTG GAC CTC ATG GCC CAC ATG-3'. The sequences used to amplify the primers were
synthesized by the Molecular Biology Core Laboratory (Case Western
Reserve University School of Medicine) and were prepared as 10 µM
stocks. Amplified samples (20 µl) were analyzed on 1.5% agarose gel,
stained with ethidium bromide, and photographed. Parallel experiments were routinely done using DNase-I before RT, to exclude amplification of genomic cDNA contaminants. The DNA molecular mass markers were from
Hinc II digest of OX174 DNA (United
States Biochemical, Cleveland, OH) or 1-kb ladder (GIBCO, Grand Island, NY).
Densitometry. The X-ray films were analyzed with a laser densitometer (Sciscan 5000; United States Biochemical) and normalized relative to GAPDH RNA.
Statistical analysis of data. Data are presented as means ± SD. Significance of differences among means within groups was estimated by ANOVA. Significance of differences among means of the ratio uCl/uNa was estimated by a randomization test for two independent nonparametric samples (36). Changes in trends were calculated using GB-STAT version 5.3 (1995, Dynamic Microsystems, Silver Spring, MD) and analyzed with ANOVA. Best fit of regression equations (least squares criterion) was achieved with SlideWrite Plus 1995 (Advanced Graphics Software, Carlsbad, CA), which uses the Levenberg-Marquardt algorithm, and analyzed using ANOVA. Variance among experiments (interassay variability) ranged 15%, but variance within experiments (intra-assay variability) ranged <5%, and all trends were consistent among experiments.
Chemicals and supplies. Anocell-10 filters were obtained from Anotec (Oxon, UK). All chemicals were obtained from Sigma Chemical (St. Louis, MO), unless stated otherwise.
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RESULTS |
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Estrogen effects on GTE
and on Ppyr.
HUVEC grown on filters formed epithelia that significantly
decreased GTE and
Ppyr compared
with levels across blank filters without cells (Table
1). Preplating of 3T3 fibroblasts had no significant effect on permeability (Table 1). In EGTA-treated cells,
the levels of GTE
and of Ppyr were
similar to those across blank filters (Table 1), indicating that
lowering extracellular Ca2+ to
<0.1 mM abolished the paracellular resistance, probably by disrupting
the tight junctions (13, 17, 21). The levels of
GTE and
Ppyr that were
conferred by HUVEC were ~140
mS · cm2
and 11 cm · s
1 · 10
6
(for fluxes of 0.51 kDa), respectively (Table 1, Fig.
1). These values compare well
with previously published results in endothelial cells (3, 5, 27, 29,
33, 41) and confirm that HUVEC form confluent endothelia on filters and
restrict the free movement of solutes through the intercellular
space.
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Effects of NOS inhibitors on
GTE.
In HUVEC grown in steroid-free medium,
L-NAME increased
GTE acutely by 31 mS · cm2
(Fig. 2); preincubation of cells with
L-Arg blocked the
L-NAME effect (Table
2). In cells treated with 1 nM estradiol L-NAME increased
GTE by only 8 mS · cm
2,
and in cells treated with 1 µM estradiol
L-NAME decreased
GTE by 8 mS · cm
2
(Fig. 2, Table 2). L-NAME is a
relatively nonspecific NOS inhibitor (10); to better understand the
effect of NOS inhibition on
GTE, cells were
treated also with
NG-nitro-L-arginine
(L-NNA), a more selective NOS
inhibitor for the Ca2+-dependent
eNOS (9), or with
NG-monomethyl-L-arginine
(L-NMMA), which inhibits mainly
the Ca2+-independent iNOS (28).
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Estrogen effects on NO release and on cellular levels of
cGMP.
Levels of NO in the extracellular fluid bathing filters seeded with 3T3
fibroblasts were undetectable by our method, regardless of treatment
with estradiol (not shown). In contrast, HUVEC plated on filters
released measurable amounts of NO into the extracellular fluid (2.5 ± 0.2 pmol · min1 · mg
protein
1 in control cells;
Fig.
3A). At
concentrations of <1 nM, estradiol had no significant effect on NO
release into the extracellular fluid by HUVEC; however, at estradiol
concentrations of more than 10 nM, there was a dose-dependent increase
in NO release (Fig. 3A). Lowering
extracellular Ca2+ from 1.2 to
<0.1 mM by adding aliquots of 0.3 M EGTA increased GTE across HUVEC
cultures but had no effect on cell viability for up to 3 h after
addition of the Ca2+ chelator;
furthermore, replenishment of extracellular
Ca2+ to 1.2 mM reversed the
increase in permeability (not shown). Lowering extracellular
Ca2+ tended to increase NO
release, but it did not influence the estrogen effect. Incubation of
cells with 1 mM L-NMMA inhibited
the estradiol-induced increase in NO release;
L-NNA had no significant effect
(Fig. 3A).
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Effects of 8-bromoguanosine 3'5'-cyclic monophosphate and of SNP on GTE. To clarify the roles of cGMP and of NO on GTE across HUVEC, two additional experiments were conducted. First we determined the effects of 8-bromoguanosine 3'5'-cyclic monophosphate (8-BrcGMP) and of SNP on permeability. 8-BrcGMP is a stable, cell-permeant analog of cGMP, and it can mimic cellular effects of cGMP; SNP is a NO donor, and it can mimic cellular effects of NO (30, 31).
8-BrcGMP decreased GTE acutely across cells grown in steroid-free medium, as well as across cells treated with low (1 nM) or with high (1 µM) concentrations of estradiol. In all cases, GTE decreased by ~29 mS · cm
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Estrogen effects on eNOS and iNOS RNA. Likely sources for the NO involved in the estrogen effects shown in Figs. 2 and 3 are eNOS and iNOS (30, 31). To test this hypothesis, we determined the effects of estrogen treatment on the expression of eNOS and iNOS RNA. Total RNA was isolated from cells treated with 1 nM or 1 µM estradiol and analyzed by RT-PCR, using specific primers for the respective NOS isoforms.
RT-PCR analysis of RNA from the murine 3T3 fibroblasts using the primers of human eNOS and iNOS did not reveal expression of NOS. In contrast, HUVEC expressed RNA of both eNOS and iNOS isoforms (Fig. 5). Treatment with estradiol had no significant effect on the expression of RNA for the ubiquitous enzyme GAPDH, but it modulated the expression of RNA of eNOS and iNOS relative to GAPDH (Fig. 5). Analysis of the results using densitometry revealed that treatment with 1 nM estradiol increased the expression of eNOS/GAPDH RNA by 6-fold (P < 0.05), but treatment with 1 µM estradiol increased it only 1.5-fold (P > 0.1). These results indicate that estradiol had a biphasic effect on the expression of eNOS RNA; at nanomolar concentrations the hormone increased eNOS RNA, but at micromolar concentrations estradiol had no significant effect on eNOS RNA. Treatment with 1 nM estradiol increased the expression of iNOS/GAPDH RNA by 1.5-fold, and treatment with 1 µM estradiol increased it by 5-fold, significantly more than the effect in cells treated with 1 nM estradiol (P < 0.05, Fig. 5). Thus, in contrast to eNOS, the estrogen-induced increase in iNOS RNA occurred mainly at micromolar concentrations of the hormone.
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Effects on Vdil and on
uCl/uNa.
The results shown in Fig. 1 indicate that estradiol modulates the
paracellular permeability across HUVEC. The paracellular permeability
is usually determined by two main mechanisms: the tight junctional
resistance
(RTJ) and
resistance of the lateral intercellular space
(RLIS) (34). To
determine whether estrogen modulates the
RTJ, we studied
the effect of treatment with estrogen on
uCl/uNa
in the paracellular space (34). Treatment with estradiol had a biphasic
effect on
uCl/uNa:
levels of
uCl/uNa
were lower in cells treated with 1 nM estradiol (1.28 ± 0.01) than
in untreated cells (1.35 ± 0.01; Fig.
6A,
inset; Table
3; P < 0.05). In cells treated with 1 µM estradiol, levels of
uCl/uNa
were similar to those across untreated cells (Fig.
6A,
inset). These results indicate a
biphasic effect on
uCl/uNa,
similar to the biphasic effects of estradiol on
GTE and on
Ppyr (Fig. 1).
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Modulation of cell size. To determine whether estrogen also modulates the RLIS, we determined the effects of 8-BrcGMP and of SNP on cell size. The rationale was as follows. 1) 8-BrcGMP and SNP appear to mediate, respectively, the estrogen-induced decrease and increase in permeability (Figs. 1 and 4A). 2) In an intact epithelium, such as HUVEC, the RLIS usually depends on the geometry of the intercellular space and in turn on the volume of the surrounding cells that form this space (34). HUVEC 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). To quantify the changes in F360/510, measurements of fluorescence were expressed relative to the fluorescence obtained when cells were perfused with Ringer buffer.
Experiments done on filters seeded only with 3T3 fibroblasts revealed that the cells were able to load fura 2, but the fluorescence signal was ~100-fold weaker than that obtained from filters plated with HUVEC on top of 3T3 fibroblasts (not shown), indicating that the changes in fluorescence are contributed mainly by the HUVEC. We have previously shown that changes in F360/510 induced by changing the buffer osmolarity in attached human cervical cells were linear in the range from 0.5 to 1.5 F360/510 (14). A similar relationship was also found in HUVEC, using the same method (not shown). 8-BrcGMP decreased F360/510 across HUVEC in a time-related manner [Fig. 6B; half time (t1/2) of ~1.5 min]. It was previously shown by us (18) and by others (7) that a decrease in F360/510 correlates with an increase in cell size, and the response to 8-BrcGMP shown in Fig. 6B is interpreted as an increase in HUVEC cell size. SNP had an opposite effect, and it increased F360/510, i.e., it decreased cell size (Fig. 6B; t1/2 of ~1 min); in those experiments, the background autofluorescence of SNP was minimal and did not affect the interpretation of the results (not shown). The fluorescence method was not sensitive enough to determine actual changes in cell size. Collectively, the results shown in Fig. 6B indicate that 8-BrcGMP-induces an acute increase in HUVEC size, whereas SNP induces an acute decrease in cell size, and they therefore suggest that 8-BrcGMP increases RLIS and SNP decreases RLIS.Role of extracellular Cl.
One of the mechanisms by which cells can change their size acutely is
by modulating transcellular osmotic gradients secondary to changes in
transcellular ion transport. To determine whether the effects of
8-BrcGMP and of SNP involve acute changes in ion transport, we
determined the effects of lowering luminal NaCl on the
GTE responses to
8-BrcGMP or to SNP. Changes in
GTE under conditions of asymmetrical NaCl concentrations were determined by
correcting the measured levels of
GTE for fluid
conductivity (15). Lowering luminal NaCl attenuated the
8-BrcGMP-induced decrease in
GTE and augmented
the SNP-induced increase in conductance (Fig.
7, Table 4).
Lowering NaCl in the luminal solution of filters seeded only with 3T3
fibroblasts had no effect on
GTE (not shown). These results suggest that Na+
and/or Cl
may be
involved in the changes in cell size induced by 8-BrcGMP or by SNP.
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DISCUSSION |
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Estradiol had a biphasic effect on paracellular permeability across HUVEC cultures: at nanomolar concentrations it decreased the permeability, whereas at micromolar concentrations it increased the permeability. Our results suggest that the decrease in permeability was mediated by a Ca2+-dependent eNOS-NO-cGMP mechanism for five reasons. 1) 8-BrcGMP decreased the permeability, whereas KT-5823, a cGMP-dependent protein kinase inhibitor, blocked the effect. 2) The effect of estradiol on cGMP correlated with the decrease in permeability. 3) Incubation of cells in low extracellular Ca2+ decreased the efficacy and potency of the estrogen effect on cellular cGMP. 4) L-NNA, a Ca2+-dependent NOS inhibitor, but not L-NMMA, increased the permeability and attenuated the estrogen-induced increase in cGMP. L-NNA also increased acutely the permeability, and treatment with 1 nM estradiol augmented the response to L-NNA. A possible explanation is that estrogen upregulated and L-NNA inhibited a NO-related mechanism proximal to cGMP. 5) Nanomolar but not micromolar concentrations of estradiol increased the expression of eNOS RNA, which is a Ca2+-dependent NOS (30, 31).
Our results also suggest that the estrogen-dependent increase in permeability was mediated by a Ca2+-independent iNOS-NO mechanism for the following reasons. 1) SNP and SIN-1, NO donors, produced an acute increase in permeability that was blocked by hemoglobin but not by KT-5823. 2) The effect of estradiol on NO release correlated with the increase in permeability. 3) The increase in NO release was not affected by lowering extracellular Ca2+. 4) L-NMMA (which inhibits mainly the Ca2+-independent NOS), but not L-NNA, blocked the NO release that was induced with high concentrations of estradiol. L-NMMA also decreased the permeability in cells treated with 1 µM estradiol, but not in cells grown in steroid-free media or in cells treated with nanomolar estradiol. 5) Estradiol increased iNOS RNA, but, in contrast to the biphasic effect on eNOS RNA, micromolar concentrations of estradiol produced a significant increase in iNOS RNA.
The results of the experiments using L-NNA and L-NMMA should be interpreted with caution, because these agents may have effects other than those previously suggested (9, 10, 28) or have nonspecific effects. In cells grown in steroid-free medium, L-NNA did increase GTE but had no significant effect on cGMP. This may suggest that L-NNA acts on permeability via an additional pathway unrelated to cGMP. On the other hand, the data in other experiments support our hypothesis, and our results agree with previous publications in the field (9, 10, 28).
Our results showed that the eNOS-NO-cGMP and iNOS-NO systems differed
in their sensitivity to estradiol with regard to the expression of eNOS
and iNOS RNA, NO activity, and the cellular accumulation of cGMP. The
expression of eNOS RNA and the accumulation of cGMP were maximal at
nanomolar estradiol concentrations; the latter conclusion is supported
by the finding that, in cells pretreated with SNP at a high
concentration (1 mM), already 0.1 nM estradiol had no additional effect
on cGMP. In contrast, iNOS RNA expression and NO release did not reach
saturation even at micromolar estradiol. SNP increased cGMP
significantly in cells grown in steroid-free medium, but 0.1 nM
estradiol did increase cGMP further. Although the additional effect of
estradiol was mild compared with that of SNP and with that of 10 nM
estradiol, the results overall suggest that SNP per se does not
increase cGMP maximally. Regardless, however, our data showed that
levels of cGMP in cells treated with 10 nM estradiol were similar to
those in cells treated with SNP plus 0.1 nM estradiol, suggesting that
saturation of the soluble guanylate cyclase had occurred.
Although changes in the activities of either cGMP or NO can explain the biphasic effect of estrogen on permeability, the limiting factor for the estrogen-dependent decrease in permeability appears to be cGMP: 8-BrcGMP decreased the permeability even in cells treated with 1 µM estradiol; in these cells, levels of cGMP were presumably maximal, because (as stated above) cGMP accumulation was saturated already at 10 nM estradiol. Our results therefore suggest that the limiting factor for the estrogen-dependent decrease in permeability is the NO-induced turnover of guanylate cyclase.
One of our objectives was to determine the mechanism by which estrogen modulates the paracellular resistance across HUVEC. The results suggest that estrogen and cGMP (the presumed mediator of the estrogen-induced decrease in permeability) and NO (the presumed mediator of the estrogen-dependent increase in permeability) modulate both RTJ and RLIS. A possible explanation, based on the Ussing-Zerahn model of transepithelial transport (43), is that the changes in cell size (i.e., in RLIS) lead to changes in RTJ. According to the Ussing-Zerahn model, movement of molecules in the intercellular space (paracellular pathway) is restricted by the RTJ and RLIS. The regions of the tight junction are considered high-resistance elements, due to the occlusion of the intercellular space by the tight junctional complexes. In contrast, RLIS is considered a low-resistance element, and it is determined by the proximity of the plasma membranes of neighboring cells and by the length of the intercellular space from the tight junctions to the basal lamina (34). The intercellular space, both within the regions of the tight junctions and outside these regions, can be modeled as a series of narrow tubes, whose diameters depend on the proximity of plasma membranes of neighboring cells. Because the resistance to flow in a tube depends on the fourth power of the radius of the tube, even minor changes in the cross-sectional area of the intercellular space can greatly affect the resistance. In attached cells, changes in the geometry of the intercellular space are usually secondary to changes in cell size in the opposite direction. The present results showed that both 8-BrcGMP and SNP induced acute changes in the size of HUVEC, and it is therefore suggested that the changes in cell size affect both the RLIS, i.e., the resistance of the intercellular space outside the regions of the tight junctions, and the RTJ, i.e., the resistance of the intercellular space within the tight junctions.
Acute changes in cell size can be the result of two main mechanisms:
rearrangement of cytoskeletal proteins (35) and acute changes in
osmotic gradients across the plasma membrane that shift water out of or
into the cell (8). The changes in
GTE induced by
8-BrcGMP and by SNP were influenced by the concentrations of extracellular Na+ and
Cl.
Na+ transport blockers, including
furosemide (an inhibitor of the Na+-Cl
and
Na+-K+-Cl
cotransporters), had no effect on the responses, but
Cl
channel blockers
modulated the changes in conductance: DPC and NPPB, as well as low
luminal NaCl, blocked the decrease in
GTE in response
to 8-BrcGMP, but they augmented the increase in conductance in response
to SNP. Furthermore, most of the effects of DPC and NPPB were observed
when the agents were administered in the luminal solution. These
results suggest that Cl
transport via the apical plasma membrane determines the full magnitude
of changes in GTE
in response to 8-BrcGMP and to SNP. Consequently, the effect of
8-BrcGMP can be the result of augmented Cl
influx via apically
located Cl
channels,
followed by water influx to compensate for hyperosmolarity. The end
result would be an increase in cell size. The finding that low luminal
NaCl attenuated the response to 8-BrcGMP can be explained by a decrease
in the driving force for Cl
influx (lower extracellular-intracellular
Cl
gradient). The effect of
SNP can be explained by a similar mechanism, but in the opposite
direction: SNP may have induced
Cl
secretion via apically
located Cl
channels, with
subsequent loss of cellular water and a decrease in cell size. Low
luminal NaCl may have augmented the response by increasing the driving
force for Cl
efflux.
On the basis of these results, we suggest that estradiol activates two
NO-related mechanisms in HUVEC. At nanomolar concentrations, estradiol
upregulates mainly the eNOS. The generated NO activates guanylate
cyclase and produces cGMP. cGMP stimulates enhanced Cl and water influx, an
increase in cell size, an increase in
RLIS and in
RTJ, and
subsequently a decrease in permeability. At nanomolar concentrations,
estrogen also upregulates NO production by inducing iNOS; however, the
total amount of NO produced at these low concentrations of estradiol is
low and could not be detected by the Greiss method. At micromolar
concentrations, estradiol stimulates a substantial increase in NO from
iNOS: one of the targets of NO, the guanylate cyclase, becomes
saturated already at nanomolar estradiol concentrations, and this
allows NO to act on other cellular targets, including Cl
channels. Our results
suggest that, in contrast to cGMP, which activates
Cl
influx, NO itself or
acting via another mediator stimulates
Cl
efflux. This leads to
water efflux, to decreases in cell size, in
RLIS, and in
RTJ, and
subsequently to an increase in permeability. The mechanisms by which
cGMP and NO activate influx and efflux of
Cl
, respectively, in HUVEC
are unknown.
The relevance of the present results to understanding how estrogens modulate transendothelial transport in vivo should be discussed with regard to the experimental model (cultured endothelial cells) and with regard to the concentrations of estradiol that were used in the present study. The measured permeability of cultured HUVEC was similar to that of other cultured endothelia but was higher than that determined for endothelia in vivo. This topic has been recently discussed (3, 5, 27, 29, 33, 41), and one explanation for the higher permeability in vitro is the lack of matrix and stromal cells that are present in vivo and can contribute to the paracellular resistance. Despite these considerations, experiments with cultured HUVEC can yield important mechanistic information, as in the present study.
The estrogen-induced decrease in permeability occurred at physiological (nanomolar) concentrations of estradiol in women. Because estrogen response elements were previously described in the eNOS gene (4), the modulation of the eNOS can be explained by the classical nuclear estrogen receptor mechanism (1). In contrast, the increase in permeability required micromolar concentrations of estradiol, which are supraphysiological for women. Free estrogens usually do not reach micromolar levels in the plasma; however, in certain conditions tissues may be exposed to high levels of estrogens. For instance, women treated with the birth control pill consume high doses of synthetic estrogens. It has been shown that one of the effects of the pill is tissue edema, as a result of increased transudation of fluid from the plasma through the capillary endothelium into the extracellular space (38). Based on the present study, it is possible that this effect is the result of high-dose, estrogen-induced increase in endothelial permeability. Another example is pregnancy, which is also a condition of high-estrogen milieu. During pregnancy, the permeability of the utero-placental tissues is high; a possible explanation is that the high levels of estrogens increase the permeability of both fetal (e.g., HUVEC) and maternal endothelial cells.
An argument raised by a number of previous studies was that micromolar estrogen concentrations can modulate cell function via nongenomic mechanisms; examples are changes in intracellular pH and in cytosolic Na+, K+, or Ca2+ levels (6, 19, 21, 45, 50). Because changes in cytosolic levels of these ions can modulate gene transcription, it is possible that the upregulation of iNOS RNA in HUVEC (present study) was a result of similar nongenomic effects of estrogen. However, more studies are needed to elaborate these findings.
To summarize, the present results in HUVEC revealed dual modulation of
endothelial paracellular permeability by estrogen. 1) An eNOS-, NO-, and cGMP-dependent
decrease in permeability is activated by nanomolar concentrations of
estradiol, resulting in enhanced
Cl influx, increased cell
size, and increased
RLIS and
RTJ; these effects appear to be limited by the ability of cells to generate cGMP
in response to NO. 2) An iNOS- and
NO-dependent increase in permeability was activated by micromolar
concentrations of estradiol, resulting in enhanced
Cl
efflux, decreased cell
size, and decreased
RLIS and
RTJ. The net
effect on transendothelial permeability will depend on the relative
contributions of each of these two systems. Understanding the
mechanisms by which estrogens modulate endothelial permeability may be
important for development of drugs that can target the specific
mechanisms involved in the actions of estrogen and may provide the
pharmacological means to selectively regulate the permeability.
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ACKNOWLEDGEMENTS |
---|
This study was supported in part by grants from Bristol Myers Squibb (US Pharmaceuticals) to G. I. Gorodeski and by National Heart, Lung, and Blood Institute Grant HL-48771 to N. P. Ziats.
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FOOTNOTES |
---|
Present address of M. M. Cho: Dept. of OB-GYN, Div. of Reproductive Endocrinology and Infertility, USC/LAC, Women's and Children's Hospital of Los Angeles, Rm. IM2, 1240 N. Mission Rd., Los Angeles, CA 90033.
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: G. I. Gorodeski, University MacDonald Women's Hospital, University Hospitals of Cleveland, 11100 Euclid Ave., Cleveland, OH 44106.
Received 28 May 1998; accepted in final form 20 October 1998.
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