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Am J Physiol Cell Physiol 278: C942-C952, 2000;
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Vol. 278, Issue 5, C942-C952, May 2000

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (Delta I) and transepithelial potential difference (Delta PD, lumen negative): GTE = Delta I/Delta 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 beta -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).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Omega   ·  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.

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).

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.

                              
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  		Table 1.   Effects of L-NAME on NO release in hECE and CaSki cells

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).

                              
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  		Table 2.   Effects of SNP on Ppyr across cultures of hECE and CaSki cells on filters

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.

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.

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.

                              
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  		Table 3.   Effects of SNP on cellular cGMP in CaSki cells

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.

                              
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  		Table 4.   Effects of SNP and 8-BrcGMP on uCl/uNa and on GTE across CaSki cultures

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 (open circle )], 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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DISCUSSION
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