Nitric oxide attenuates H2O2-induced endothelial barrier dysfunction: mechanisms of protection

Mahesh P. Gupta1, Michael D. Ober1, Carolyn Patterson1, Mohammed Al-Hassani1, Viswanathan Natarajan2, and C. Michael Hart1

1 Department of Medicine, Indiana University and Richard L. Roudebush Veterans Affairs Medical Centers, Indianapolis, Indiana 46202; and 2 Department of Medicine, Johns Hopkins University, Baltimore, Maryland 21224


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Nitric oxide (·NO) attenuates hydrogen peroxide (H2O2)-mediated injury in porcine pulmonary artery endothelial cells (PAECs) and modulates intracellular levels of cGMP and cAMP. We hypothesized that ·NO attenuates H2O2-induced PAEC monolayer barrier dysfunction through cyclic nucleotide-dependent signaling mechanisms. To examine this hypothesis, cultured PAEC monolayers were treated with H2O2, and barrier function was measured as transmonolayer albumin clearance. H2O2 caused significant PAEC barrier dysfunction that was attenuated by intracellular as well as extracellular ·NO generation. ·NO increased PAEC cGMP and cAMP levels, but treatment with inhibitors of soluble guanylate cyclase or protein kinase G did not abrogate ·NO-mediated barrier protection. In contrast, H2O2 decreased protein kinase A activity, and inhibiting protein kinase A abrogated the protective effect of ·NO. H2O2-induced barrier dysfunction was not associated with decreased levels of cGMP or cAMP. 3-Isobutyl-1-methylxanthine and the cGMP analog 8-bromo-cGMP had little effect on H2O2-mediated endothelial barrier dysfunction, whereas 8-bromo-cAMP plus 3-isobutyl-1-methylxanthine was protective. These results indicate that ·NO modulates vascular endothelial barrier function through cAMP-dependent signaling mechanisms.

vascular endothelium; hydrogen peroxide; adenosine 3',5'-cyclic monophosphate; guanosine 3',5'-cyclic monophosphate; nitric oxide synthase


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE VASCULAR ENDOTHELIUM serves as a barrier to regulate the exchange of fluids and macromolecules between the blood and extravascular tissue. Derangements in vascular endothelial barrier function contribute to the pathogenesis of a variety of clinical disorders. For example, impaired vascular endothelial cell (EC) barrier function in the lung leads to exudation of fluids and proteins into the alveoli and interstitial spaces, compromising pulmonary compliance and gas exchange during states of acute lung injury, e.g., acute respiratory distress syndrome. Partially reduced oxygen species released from activated neutrophils perturb EC barrier function and contribute to the pathogenesis of lung injury (9, 54). In vitro studies with cultured monolayers of endothelial cells have confirmed that oxidant stimuli including hydrogen peroxide (H2O2) (25, 56), hyperoxia (47), and oxidant-generating systems, e.g., activated granulocytes or xanthine-xanthine oxidase (53, 54), disrupt EC barrier function. Current evidence suggests that oxidant-mediated derangements of EC barrier function are partially reversible and can occur in the absence of cytotoxicity (25, 53). These findings suggest that oxidant-mediated alterations in vascular endothelial function represent a critical early event in the pathophysiology of vascular injury.

Oxidant-mediated derangements in EC barrier function likely result from the culmination of diverse oxidant-stimulated events. In addition to their potential to directly damage cell components, oxidants also activate a variety of signal transduction cascades to generate second messengers (59) that modulate the structure and organization of cytoskeletal proteins. These cytoskeletal alterations lead to changes in cell shape and the formation of paracellular gaps that impair endothelial barrier function (18). Numerous studies now suggest that nitric oxide (·NO) regulates the complex interactions among oxidant-stimulated signaling pathways and endothelial barrier function. For example, ·NO attenuated oxidant-induced endothelial barrier dysfunction in cultured macrovascular ECs (39), in isolated perfused lung preparations (23, 32, 49), in intact experimental animals (8, 17, 38), and in patients with acute respiratory distress syndrome (5). Additional studies (1, 29, 33, 34) have demonstrated that basal as well as stimulated alterations in vascular permeability are accentuated by inhibiting endothelial ·NO production. On the other hand, by reacting with superoxide to form peroxynitrite (50), ·NO may injure ECs (3, 4) to disrupt endothelial barrier dysfunction. However, the precise mechanisms by which ·NO modulates oxidant-induced endothelial barrier dysfunction are not clearly understood.

A major mechanism for ·NO-mediated effects involves the activation of soluble guanylate cyclase and increased cellular levels of cGMP. Therefore, we hypothesized that ·NO modulates H2O2-induced endothelial barrier dysfunction via cyclic nucleotide-dependent signaling mechanisms. To examine this hypothesis, the present study extends previous investigations in this area (5, 8, 17, 23, 32, 38, 39, 49) by examining the ability of both endogenous and exogenous sources of ·NO to modulate H2O2-induced endothelial barrier dysfunction and further clarifies the role of cyclic nucleotides in ·NO-mediated alterations of H2O2-induced endothelial barrier dysfunction.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. MEM-alpha , penicillin-streptomycin, and trypsin were obtained from GIBCO BRL (Life Technologies, Grand Island, NY). Collagenase was purchased from Worthington Biochemical (Freehold, NJ). Bovine serum albumin (BSA), fetal bovine serum (FBS), dimethyl sulfoxide (DMSO), phenylmethylsulfonyl fluoride, leupeptin, aprotinin, Tris, HEPES, pepstatin A, 3-isobutyl-1-methylxanthine (IBMX), cAMP, kemptide, ATP, digitonin, and trichloroacetic acid (TCA) were purchased from Sigma (St. Louis, MO). Gentamicin was purchased from SoloPak Laboratories (Elk Grove Village, IL). Amphotericin B was obtained from Formica Scientific (Columbus, OH). Hanks' balanced salt solution (HBSS) was purchased from Biofluids (Rockville, MD). RPMI 1640 medium was obtained from ICN Flow (Costa Mesa, CA). [gamma -32P]ATP triethylammonium salt, Biotrak cGMP assay kits, and [3H]cAMP-Biotrak radioimmunoassay kits were purchased from Amersham Life Science (Arlington Heights, IL). S-nitroso-N-acetylpenicillamine (SNAP), 8-bromo-cGMP, 8-bromo-cAMP, NG-nitro-L-arginine methyl ester (L-NAME), 2,4-diamino-6-hydroxypyrimidine (DAHP), KT-5823, and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) were procured from BIOMOL Research Laboratories (Plymouth Meeting, PA). Sepiapterin was obtained from Alexis Biochemicals (San Diego, CA). Plastic ring wells were obtained from Adaps (Dedham, MA).

Pulmonary artery EC isolation and culture. ECs were isolated from the main pulmonary artery of pigs as previously reported (24, 26). In brief, pulmonary arteries were isolated immediately after death and transported on ice from the slaughterhouse to the laboratory. The lumen of each vessel was treated with 0.3% collagenase for 15 min at 37°C. The ECs were then gently scraped from the vessel lumen with a sterile scalpel and dispersed in growth medium consisting of MEM-alpha containing 10% FBS, 100 U/ml of penicillin-streptomycin, 20 µg/ml of gentamicin, and 2 µg/ml of amphotericin B. The pulmonary artery ECs (PAECs) were then transferred to 60-mm plastic culture dishes precoated with 0.2% gelatin and maintained in a humidified 95% air-5% CO2 atmosphere at 37°C until primary confluence was reached. Once confluent, the PAECs were passaged by treatment with 0.05% trypsin for 1-2 min and transferred to 35-mm dishes for cyclic nucleotide assays or microporous polycarbonate filters for EC monolayer barrier function studies. After confluence was reached, the concentration of FBS was decreased to 4% (maintenance medium). In all experiments, the PAECs were studied within 1-3 days after confluence. Control and experimental dishes were matched according to cell line, number of passages, and number of days postconfluence. Monolayers were identified as ECs by phase-contrast microscopy and periodically by immunofluorescence staining for factor VIII antigen (7).

Measurement of PAEC monolayer barrier function. The ability of PAEC monolayers to prevent the transendothelial passage of albumin was measured as an index of PAEC barrier function as previously reported (25, 26). In brief, cells were grown to confluence on microporous polycarbonate filters (0.8-µm pore size) mounted on plastic ring wells. Each filter was filled with luminal medium composed of Evans blue dye bound to BSA [4% (wt/vol) in RPMI 1640 culture medium]. The filters were floated in 45 ml of RPMI 1640 medium (abluminal medium) that was stirred continuously at 37°C. Aliquots of abluminal medium were taken every 10 min for 1-3 h. Reagents were added to the luminal chamber of designated filters as indicated in Examining the role of ·NO and cyclic nucleotides in H2O2-mediated PAEC barrier dysfunction. The impact of interventions on EC barrier function was determined by comparison to matched filters treated under control conditions or by comparison with baseline function measured for 1 h immediately before the introduction of the experimental interventions. The passage of Evans blue-albumin across the PAEC monolayers was determined by measuring absorbance of the abluminal medium at 620 nm with a V-max spectrophotometric plate reader (Molecular Devices, Melnon, CA). The raw absorbance data were transferred to a spreadsheet program, and the net abluminal sample absorbance was used to calculate the equivalent theoretical volume of luminal medium cleared to the abluminal space (46). Albumin clearance values, expressed as microliters per minute, were calculated by linear regression analysis of changes in absorbance over 1-h intervals.

Examining the role of ·NO and cyclic nucleotides in H2O2-mediated PAEC barrier dysfunction. To examine the role of ·NO in H2O2-induced PAEC monolayer barrier dysfunction, reagents were employed to either stimulate or inhibit endogenous PAEC ·NO production or to deliver ·NO exogenously to the cells. To stimulate endogenous ·NO production, PAECs were treated with vehicle (HBSS) or 250 µM sepiapterin for 16 h at 37°C. To inhibit endogenous PAEC ·NO production, monolayers were treated for 16 h with vehicle (HBSS) or 1 mM DAHP, a tetrahydrobiopterin synthesis inhibitor (52), or for 6 h with vehicle (HBSS) or the nitric oxide synthase inhibitor L-NAME (1 mM). Although L-NAME lowered the pH of the incubation solution from 7.46 to 7.23, DAHP had no significant effect. After being washed, each monolayer was treated under control (HBSS) or oxidant (100 µM H2O2 in HBSS) conditions for 30 min at 37°C. After the wash, albumin clearance was measured for 1 h. To examine the ability of exogenous ·NO to modulate H2O2-mediated PAEC barrier dysfunction, albumin clearance of the PAEC monolayers in control medium (RPMI 1640 medium plus 4% BSA) was measured for 1 h (baseline). H2O2 (100 µM) and SNAP (0-250 µM) were then added to the luminal medium, and transmonolayer albumin clearance was measured for an additional 2 h.

The ability of cyclic nucleotides to regulate H2O2-mediated PAEC barrier dysfunction was examined by treating PAECs with either vehicle (0.1% DMSO), 1 mM IBMX, 1 mM 8-bromo-cGMP plus 1 mM IBMX, or 8-bromo-cAMP plus 1 mM IBMX in RPMI 1640 medium containing 4% BSA. Albumin clearance was determined for 1 h (baseline) before H2O2 (100 µM) was added to all filters and was then recorded for an additional 2 h. In addition, to explore the role of guanylate cyclase activation in ·NO-mediated barrier regulation, PAECs were pretreated with the soluble guanylate cyclase inhibitor ODQ (5 µM) or with 0.1% DMSO vehicle for 30 min at 37°C (11). Baseline PAEC monolayer barrier function was measured for 1 h before the filters were treated with 100 µM H2O2 with and without 100 µM SNAP. Endothelial barrier function was then measured for an additional 2 h. The role of protein kinase (PK) G in ·NO-mediated attenuation of H2O2-induced endothelial barrier dysfunction was examined by pretreating PAEC monolayers with the PKG inhibitor KT-5823 (16). PAEC monolayers grown on microporous filters were treated with 10 µM KT-5823 or with an equivalent volume of vehicle (0.1% DMSO) in RPMI 1640 medium containing 4% BSA. Baseline barrier function was recorded for 1 h, and then each monolayer was treated with 100 µM H2O2 with and without 100 µM SNAP, and transmonolayer albumin clearance was monitored for an additional 2 h.

Chemiluminescence analysis of PAEC ·NO release. PAEC ·NO release was determined by measuring ·NO and its oxidation products NO2- and NO3- (collectively referred to as NOx) in the culture medium above confluent PAEC monolayers as previously described (25). After experimental manipulations, aliquots of PAEC culture medium were aspirated and transferred to glass tubes prewashed with ultrapure H2O. All samples were centrifuged at 1,500 g for 15 min before 40-100 µl of the supernatant were transferred to a purge vessel containing 0.8% vanadium chloride in 1 N HCl at 95°C. These conditions convert NOx to ·NO that was carried with N2 into a Sievers chemiluminescence nitric oxide analyzer (model 280, Sievers, Boulder, CO). In the detector, ·NO in the gas phase reacts with ozone to generate an excited state of nitrogen dioxide (NO2*) plus O2. The NO2* decays to give a signal at 600 nm that is proportional to ·NO concentration. Standard curves were generated daily with 0.1-10 µM NaNO3. The detector response was linearly related to NaNO3 concentrations between 0.5 and 3.0 µM, with all sample concentrations falling within this range. All experimental NOx measurements were corrected for NOx attributable to culture medium components. Stimulation of PAEC ·NO production by sepiapterin was confirmed by measurement of NOx during a 1-h incubation in DMEM immediately after treatment with 250 µM sepiapterin for 16 h. Inhibition of nitric oxide synthase (NOS) activity with L-NAME was confirmed by measuring PAEC ·NO release into DMEM during a 1-h incubation after treatment with HBSS or 1 mM L-NAME for 6 h.

Measurement of ·NO with an amperometric electrode. ·NO production by SNAP was measured at 37°C for 2 h after the addition of 100 or 250 µM SNAP to RPMI 1640 medium containing 4% BSA in the absence of PAECs with an amperometric electrode (ISONO P200 sensor, World Precision Instruments, Sarasota, FL). The sensor was calibrated daily according to the protocol provided by the manufacturer. In brief, ·NO was generated by adding 50-2,500 nM SNAP to an air-equilibrated 0.1 M CuSO4 solution (pH 4.0) at 37°C. A standard curve was prepared by plotting the electrode response (in pA) versus the SNAP concentration, assuming that 53.9% of SNAP under these conditions generates ·NO capable of detection by the electrode (data provided by the manufacturer). The electrode response was linearly related to ·NO concentrations between 0.01 and 1.0 µM. The concentration of ·NO generated by 100 or 250 µM SNAP at 37°C was determined by comparison to standard curves after baseline current generated by culture medium alone was subtracted.

Measurement of PAEC cyclic nucleotide levels. The effect of H2O2 on PAEC cGMP and cAMP levels was examined in monolayers treated with and without SNAP under conditions identical to those employed in PAEC barrier function studies. Confluent PAECs were treated under control (RPMI 1640 medium plus 4% BSA) or oxidant (100 µM H2O2 in RPMI 1640 medium plus 4% BSA) conditions in the presence and absence of 100 µM SNAP for 0.5 or 2 h at 37°C. Next, the monolayers were thoroughly washed with chilled HBSS and collected in ice-cold 0.8 M perchloric acid. The samples were vortexed and centrifuged at 1,000 g for 15 min, and the supernatants were immediately stored at -70°C as previously described (60). The samples were neutralized with 2 M potassium hydroxide in 0.8 M Tris and subjected to an enzyme immunoassay for cGMP and a radioimmunoassay for cAMP, both performed according to protocols supplied by the manufacturer (Amersham). In selected experiments, to prevent phosphodiesterase (PDE)-dependent cyclic nucleotide degradation, PAEC monolayers were treated with 1 mM IBMX for 30 min before the effect of H2O2 and SNAP on PAEC cyclic nucleotide levels was investigated.

Measurement of PKA activity. Confluent PAEC monolayers grown in 96-well plates were treated with control medium (RPMI 1640 medium containing 4% BSA) or control medium containing either 100 µM H2O2, 100 µM SNAP or 100 µM SNAP plus 100 µM H2O2 for 2 h at 37°C. The PAECs were then rinsed, and PKA activity was measured as Garcia et al. (19) have previously described, with minor modifications. In brief, after treatment with H2O2 or ·NO, PAECs were incubated for 15 min with 200 µl of PKA assay buffer (0.3 mM Na2HPO4, 0.4 mM K2HPO4, 5.4 mM NaCl, 135 mM KCl, 10 mM MgCl2, 20 mM HEPES, 100 µM ATP, 500-600 counts · min-1 [32P]ATP · pmol unlabeled ATP-1, 50 µg/ml of digitonin, 1 mM IBMX, and 64 µM kemptide, pH 7.0). Assay buffer without kemptide was added to selected wells in each experiment to measure nonspecific endogenous phosphorylation of PAEC proteins. The reaction was terminated by the addition of 10% chilled TCA. The lysates were then collected and applied to phosphocellulose filters under continuous suction. The filters were washed sequentially with 1% (wt/vol) o-phosphoric acid (H3PO4), 95% ethanol, and diethyl ether. 32P incorporation was determined by liquid scintillation counting, and PKA activity, expressed as picomoles of kemptide phosphorylated per minute per milligram of protein, was calculated by subtracting the nonspecific protein phosphorylation detected in the absence of kemptide.

Measurement of endothelial electrical resistance. PAECs were grown to confluence on gold electrodes in an eight-well electrical cell substrate impedance-sensing system (Applied Biophysics, Troy, NY). The total electrical resistance was measured dynamically across the monolayer as the combination of resistance between the cells and the electrode and the resistance between the cells as previously reported (19). Changes in electrical resistance therefore represent alterations in cell-cell adhesion and/or cell-matrix adhesion. Experiments were conducted only on wells that achieved >5,000-Omega steady-state resistance. Electrical resistance was dynamically recorded for 45 min under the same conditions used to measure PAEC barrier function by albumin clearance. PAECs were incubated in RPMI 1640 culture medium with 4% BSA at 37°C. After the baseline electrical resistance was recorded for 45 min, the PKA inhibitor Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS; 1.7 mM) was added to selected wells. After 15 min, the monolayers were treated with H2O2 and/or SNAP as described in the studies of albumin clearance in Examining the role of ·NO and cyclic nucleotides in H2O2-mediated PAEC barrier dysfunction. Changes in electrical resistance were then monitored continuously for an additional 2 h. The slope of the changes in electrical resistance over time during the second hour of treatment with H2O2 and/or SNAP was calculated for each well.

Statistical analysis. All analyses were performed in duplicate or triplicate in three to nine different cell lines. Comparisons between multiple groups were made with analysis of variance followed by Bonferroni analysis. The level of significance was taken as P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of ·NO on H2O2-induced PAEC barrier dysfunction. Treatment with H2O2 resulted in PAEC barrier dysfunction as indicated by significant increases in transmonolayer albumin clearance (Fig. 1). Using identical treatment protocols in this experimental model, Gupta et al. (25) and Hart et al. (26) have previously shown that these H2O2-mediated alterations in PAEC barrier function are reversible and not associated with cytotoxicity. Treatment with sepiapterin significantly enhanced PAEC ·NO production (Fig. 1, inset) and had little effect on barrier function in control PAECs but significantly attenuated H2O2-induced PAEC barrier dysfunction (Fig. 1). In separate experiments, endothelial NOS activity was inhibited with DAHP (·NO production, 623 ± 77 and 211 ± 20 pmol · min-1 · mg protein-1 for control and DAHP-treated cells, respectively; P < 0.05) or L-NAME (Fig. 2, inset). Inhibiting endogenous ·NO production failed to significantly alter PAEC barrier function in either control or H2O2-treated cells (Fig. 2). These findings demonstrate that H2O2-induced PAEC barrier dysfunction was altered by stimulation but not by inhibition of endogenous NOS activity.


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Fig. 1.   Effect of sepiapterin on hydrogen peroxide (H2O2)-induced pulmonary artery endothelial cell (PAEC) monolayer barrier dysfunction. Confluent PAEC monolayers were treated with Hanks' balanced salt solution (HBSS; vehicle) or 250 µM sepiapterin for 16 h at 37°C. After being washed, monolayers were treated under control (HBSS alone) or oxidant (100 µM H2O2 in HBSS) conditions for 30 min. PAECs were washed again, and endothelial barrier function was monitored for 1 h by measuring transmonolayer albumin clearance. Values are means ± SE from 3 experiments, each performed in triplicate. * P < 0.05 vs. vehicle control. ** P < 0.05 vs. H2O2. In separate experiments (inset), the effect of sepiapterin on endothelial nitric oxide (·NO) production was determined by treating confluent PAEC monolayers with HBSS or 250 µM sepiapterin for 16 h. Monolayers were then washed and incubated for 1 h in serum-free DMEM, and medium from each monolayer was subjected to chemiluminescence measurement of NOx production. Values are means ± SE from 5 experiments. * P < 0.05 vs. vehicle control.



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Fig. 2.   Effect of nitric oxide synthase (NOS) inhibition on H2O2-induced PAEC monolayer barrier dysfunction. Confluent PAEC monolayers were treated for 16 h with HBSS or the tetrahydrobiopterin synthesis inhibitor 2,4-diamino-6-hydroxypyrimidine (DAHP; 1 mM) at 37°C or for 6 h with vehicle (HBSS) or 1 mM NG-nitro-L-arginine methyl ester (L-NAME). Monolayers were then washed and treated under control (HBSS) or oxidant (100 µM H2O2) conditions for 30 min at 37°C. Cells were washed again, and PAEC monolayer barrier function was measured for 1 h. Values are means ± SE from 9-11 experiments. * P < 0.05 vs. vehicle control. Effect of L-NAME on endothelial ·NO production was determined after PAECs were treated with HBSS or 1 mM L-NAME for 6 h at 37°C (inset). PAEC monolayers were then washed with HBSS and incubated for 60 min in serum-free DMEM. ·NO in aliquots of culture medium was measured by chemiluminescence analysis (see METHODS). Values are means ± SE from 3 experiments. * P < 0.05 vs. vehicle.

The effect of exogenous ·NO on H2O2-induced endothelial barrier dysfunction was investigated with use of the ·NO donor compound SNAP (Fig. 3). Compared with barrier function under control conditions, H2O2 caused time-dependent increases in albumin clearance as Gupta et al. (25) and Hart et al. (26) have previously reported. SNAP (100 µM) significantly attenuated H2O2-induced PAEC barrier dysfunction. Further attenuation of H2O2-induced barrier dysfunction was not accomplished by increasing the concentration of SNAP to 250 µM, likely due to the comparable levels of ·NO generated by 100 and 250 µM SNAP during the first hour of H2O2 treatment (~0.7 µM; Fig. 3, inset). Gupta et al. (24) have previously shown that the ability of SNAP to prevent H2O2-mediated PAEC injury was specifically attributable to ·NO. The protective effects of SNAP were lost when SNAP was incubated for prolonged periods, thereby exhausting its ·NO-generating ability. Furthermore, the protective effects of SNAP were replicated with an unrelated ·NO-generating compound, diethylamine nitric oxide. The baseline albumin clearance rates reported in the present study (see Figs. 1-5 and 7) are well within the range of basal albumin clearance rates (0.05-0.1 µl/min) previously reported for PAEC monolayers (19, 26). The interexperimental fluctuations in H2O2-stimulated albumin clearance rates in PAEC monolayers in the current investigation (see Figs. 1-4) are likely attributable to physiological variation in the populations of PAECs comprising each series of experiments. To control for this variability, each experiment included paired monolayers treated under control conditions or included measurements of baseline barrier function of each monolayer before the introduction of experimental interventions.


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Fig. 3.   Effect of S-nitroso-N-acetylpenicillamine (SNAP) on H2O2-induced PAEC monolayer barrier dysfunction. PAEC albumin clearance was measured for 1 h in RPMI 1640 medium plus 4% BSA (baseline). H2O2 (+H2O2; 100 µM) was then added to the luminal medium of all filters along with SNAP, and transmonolayer albumin clearance was measured for an additional 2 h. Values are means ± SE from 3 experiments, with each observation performed in triplicate. * P < 0.05 vs. vehicle baseline. ** P < 0.05 vs. vehicle 3 h. Inset: ·NO production by SNAP was measured in RPMI 1640 medium plus 4% BSA at 37°C in the absence of PAEC with an amperometric electrode (see METHODS). After a baseline signal was established, levels of ·NO generated by 100 or 250 µM SNAP were monitored for 2 h. Curves are representative of 3 observations.



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Fig. 4.   Effect of cyclic nucleotide analogs on H2O2-induced PAEC monolayer barrier dysfunction. PAEC monolayers were pretreated for 1 h with vehicle (0.1% DMSO), 1 mM 3-isobutyl-1-methylxanthine (IBMX), IBMX plus 1 mM 8-bromo-cGMP, or IBMX plus 1 mM 8-bromo-cAMP. After baseline albumin clearance was measured for 1 h, each monolayer was treated with 100 µM H2O2, and albumin clearance was measured for an additional 2 h. Values are means ± SE from 4 experiments, each performed in triplicate. * P < 0.05 vs. baseline vehicle, 1 h. ** P < 0.05 vs. vehicle H2O2, 3 h.



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Fig. 5.   Effect of inhibiting soluble guanylate cyclase on ·NO-mediated attenuation of H2O2-induced PAEC monolayer barrier dysfunction. Confluent PAEC monolayers were treated with 10 µM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or vehicle (0.1% DMSO) in RPMI 1640 medium plus 4% BSA for 1 h during measurement of baseline albumin clearance. Next, 100 µM H2O2 was added to the luminal medium of each monolayer with (+) and without (-) 100 µM SNAP, and PAEC monolayer barrier function was monitored for an additional 2 h. Values are means ± SE from 4 experiments, each performed in triplicate. * P < 0.05 vs. baseline -ODQ, -SNAP. ** P < 0.05 vs. 3 h -ODQ, -SNAP.

Effect of cyclic nucleotide analogs on H2O2-induced PAEC barrier dysfunction. To determine the ability of cyclic nucleotides to regulate oxidant-induced endothelial barrier dysfunction, PAECs were treated with cell-permeable analogs of cGMP and cAMP (8-bromo-cGMP and 8-bromo-cAMP, respectively) for 1 h before exposure to control or oxidant conditions (Fig. 4). Treatment with IBMX alone or in combination with either 8-bromo-cGMP or 8-bromo-cAMP had no effect on basal barrier function. Similarly, neither IBMX alone nor IBMX plus 8-bromo-cGMP significantly altered H2O2-induced PAEC barrier dysfunction. In contrast, IBMX plus 8-bromo-cAMP significantly attenuated H2O2-mediated barrier dysfunction.

Effect of ·NO and H2O2 on PAEC cyclic nucleotide content. Numerous studies in a variety of experimental models have demonstrated the ability of cyclic nucleotides to modulate EC barrier function responses (14, 28, 31, 33, 35, 55, 57, 58, 65, 67). Because stimulation of guanylate cyclase comprises a major downstream effector mechanism for ·NO signaling, the effect of H2O2 and SNAP on PAEC cyclic nucleotide content was determined either 0.5 or 2 h after the addition of H2O2 (Table 1). As Gupta et al. (25) have previously reported, H2O2 treatment conditions sufficient to induce PAEC barrier dysfunction were not associated with significant alterations in PAEC cGMP content (Table 1). This lack of a H2O2 effect on PAEC cGMP content was observed in either the presence or absence of treatment with the PDE inhibitor IBMX. H2O2 also failed to alter PAEC cAMP content. As expected, pretreatment with IBMX caused substantial increases in both cGMP and cAMP levels in PAECs regardless of treatment condition. In the presence of IBMX, PAEC cGMP content was higher 0.5 h compared with 2 h after the addition of 100 µM SNAP, consistent with decreasing levels of extracellular ·NO (Fig. 3, inset). Treatment with SNAP in the absence of IBMX increased PAEC cGMP levels by 69 and 126% in control and oxidant-treated cells, respectively. SNAP increased PAEC cAMP content in the absence of IBMX pretreatment at 2 h and in the presence of IBMX at 0.5 h, an effect that was not significantly altered by simultaneous treatment with H2O2.

                              
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Table 1.   Effect of SNAP and H2O2 on PAEC cyclic nucleotide content

Role of cGMP-dependent pathways in ·NO-mediated attenuation of H2O2-induced PAEC barrier dysfunction. To further examine the role of cyclic nucleotides in ·NO-mediated regulation of PAEC barrier dysfunction, monolayers were treated with the soluble guanylate cyclase inhibitor ODQ (10 µM) or DMSO (vehicle) for 1 h followed by the addition of H2O2 (100 µM) and/or SNAP (100 µM) for 2 h (Fig. 5). In these studies, H2O2 caused significant barrier dysfunction after 2 h (P < 0.05 vs. baseline without ODQ, without SNAP) but not after 1 h of H2O2 treatment. Comparable to studies in Fig. 3, the addition of SNAP substantially reduced H2O2-mediated barrier dysfunction in the absence of ODQ (P < 0.05 for without ODQ and without SNAP at 3 h vs. without ODQ and with SNAP at 3 h). The presence of ODQ slightly diminished the protective effects of SNAP against H2O2-mediated barrier dysfunction and appeared to slightly enhance H2O2-mediated barrier dysfunction, but these trends did not achieve significance. These findings indicate that guanylate cyclase inhibition does not abrogate the barrier protective effects of SNAP against H2O2-mediated barrier dysfunction.

Role of cyclic nucleotide-dependent PKs in ·NO-mediated attenuation of H2O2-induced PAEC barrier dysfunction. To further determine whether ·NO modulates H2O2-induced PAEC barrier dysfunction by activating PKG, PAECs were treated with the PKG inhibitor KT-5823 before treatment with H2O2 in the presence and absence of SNAP (Fig. 6). H2O2 caused time-dependent increases in PAEC barrier dysfunction that were significantly attenuated by SNAP 2 h after treatment with H2O2. The PKG inhibitor KT-5823 failed to abrogate the protective effect of SNAP, indicating that PKG activation is not required for ·NO-mediated barrier protection. Treatment with KT-5823 alone also attenuated H2O2-induced PAEC barrier dysfunction. We speculate that this barrier protective effect of KT-5823 may be attributable to its PKC-inhibitory activity (IC50 = 4 µM), a signaling system previously demonstrated to contribute to H2O2-mediated endothelial barrier dysfunction (56). In contrast, Fig. 7 illustrates that inhibiting PKA had significant effects on the ability of ·NO to attenuate H2O2-mediated barrier dysfunction. In these studies, transmonolayer electrical resistance was measured as an alternative index of endothelial barrier function. H2O2 caused barrier dysfunction as indicated by decreased monolayer electrical resistance. SNAP attenuated H2O2-mediated barrier dysfunction, and this protective effect was abrogated by the presence of the PKA inhibitor Rp-cAMPS (Fig. 7). In separate studies, PAEC PKA activity was measured (Table 2). Compared with control monolayers, treatment with H2O2 significantly decreased PKA activity. Treatment with SNAP had no effect on PKA activity in control PAECs and slightly attenuated H2O2-mediated inhibition of PKA activity, although this trend did not achieve significance. In contrast, treatment with cholera toxin (positive control) caused large increases in PKA activity.


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Fig. 6.   Effect of PKG inhibition on ·NO-mediated attenuation of H2O2-induced PAEC monolayer barrier dysfunction. Confluent PAEC monolayers were treated with 10 µM KT-5823 or vehicle (0.1% DMSO) in RPMI 1640 medium plus 4% BSA, and transmonolayer albumin clearance was measured for 1 h (baseline). Next, 100 µM H2O2 with and without 100 µM SNAP was added to the luminal medium, and PAEC monolayer barrier function was monitored for an additional 2 h. Values are means ± SE from 2 experiments, each performed in triplicate. * P < 0.05 vs. vehicle baseline. ** P < 0.05 vs. vehicle 3 h.



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Fig. 7.   Effect of a protein kinase (PK) A inhibitor on ·NO-mediated barrier protection. PAECs were grown on gold electrodes for measurements of electrical resistance as described in METHODS. After baseline electrical resistance was measured for 45 min, selected monolayers were treated the PKA inhibitor Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS; 1.7 mM) followed by treatment with H2O2 (100 µM), SNAP (100 µM), or both reagents. Resistance was monitored for an additional 120 min. Decreases in resistance were then calculated over the 2nd hour of treatment with SNAP and/or H2O2. Values are averages ± SE from 7 experiments.


                              
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Table 2.   Effect of SNAP and H2O2 on PAEC PKA activity


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

·NO has been implicated in the regulation of oxidant-induced vascular endothelial barrier dysfunction (8, 17, 23, 32, 38, 39, 49). To better define the mechanisms of ·NO-mediated regulation of endothelial barrier function, the present study employed a well-characterized model to examine the role of cyclic nucleotide-dependent pathways in ·NO signaling. Our results confirm the ability of exogenous sources of ·NO to regulate H2O2-mediated EC barrier dysfunction and provide new evidence that stimulation of endogenous ·NO production attenuates barrier dysfunction. Sepiapterin, an analog of the NOS cofactor tetrahydrobiopterin, stimulated PAEC ·NO production as previously reported (52) and attenuated H2O2-induced EC barrier dysfunction (Fig. 1).

H2O2-induced barrier dysfunction was also attenuated by the ·NO donor SNAP, which produced submicromolar concentrations of exogenous ·NO, concentrations comparable to those generated by stimulated endothelial cells (6, 37) (Fig. 3). Although it is difficult to estimate the intracellular levels of ·NO generated by treatment with either sepiapterin or SNAP, sepiapterin clearly stimulated the production and release of ·NO by PAECs. SNAP-induced increases in exogenous ·NO concentration during the initial 30-60 min after the onset of oxidant stress appeared critical because 100 µM SNAP was protective despite rapidly declining levels of ·NO after 45 min. Furthermore, additional barrier protection was not provided by 250 µM SNAP even though this higher SNAP concentration produced a sustained elevation in ·NO during the latter half of the 120-min interval studied. Compared with Fig. 1, the results in Fig. 3 further illustrate that in the presence of albumin, H2O2-induced barrier dysfunction develops more slowly, and significant protection is not observed until the second hour of H2O2 exposure. These findings indicate that both endogenous as well as exogenous sources of ·NO can regulate oxidant-mediated endothelial barrier dysfunction. The constitutive production of ·NO, however, played little role in the regulation of barrier function. Inhibiting constitutive ·NO production with either DAHP or L-NAME had no effect on basal PAEC barrier function and exacerbated H2O2-induced barrier dysfunction to a limited and insignificant degree.

Several potential mechanisms could account for the barrier protective effect of ·NO. At concentrations produced by vascular endothelial cells under physiological conditions, a major effector arm of ·NO signaling involves activation of soluble guanylate cyclase and cGMP formation (10, 30). The role of cGMP in the regulation of EC barrier function remains controversial because cGMP may promote (14, 35, 45, 65), disrupt (40, 67, 68), or have little effect (2) on endothelial barrier function. Although these discrepancies may be attributable, in part, to differences between the species or the vascular origin of the ECs studied, the barrier-promoting or -disrupting effects of cGMP also depend on intracellular Ca2+ concentration ([Ca2+]i) (31). For instance, concurrent elevations in cGMP and [Ca2+]i enhanced barrier dysfunction in porcine aortic ECs, whereas elevated cGMP levels promoted barrier function under conditions associated with normal, basal [Ca2+]i (31). The current findings are consistent with this hypothesis. cGMP analogs not only failed to attenuate barrier dysfunction during the first hour of H2O2 treatment but tended to exacerbate barrier dysfunction slightly during that interval (Fig. 4), consistent with transient H2O2-induced elevations in PAEC [Ca2+]i levels as Hart et al. (27) have previously reported. This effect was less pronounced during the second hour of H2O2 treatment, consistent with time-dependent recovery of Ca2+ homeostasis and a tendency for development of less barrier dysfunction relative to monolayers treated with H2O2 alone.

Despite the inability of cGMP to promote PAEC barrier function, cyclic nucleotide analysis clearly showed that exogenous, SNAP-derived ·NO increased PAEC cGMP levels (Table 1). cGMP regulates cell responsiveness, in part, through stimulation of cGMP-dependent PKG, which is expressed in bovine aortic ECs and comprises a major effector mechanism for cGMP action (36). cGMP also modulates cell function via PKG-independent regulation of PDE- and cyclic nucleotide-gated ion channel activities (30). Therefore, to further examine the role of cGMP in ·NO-mediated barrier regulation, the proximal upstream end of ·NO-mediated, cyclic nucleotide-dependent signaling mechanisms was blocked by inhibiting soluble guanylate cyclase with ODQ. ODQ is a highly specific inhibitor of soluble guanylate cyclase that does not affect the activities of particulate guanylate cyclase, NOS, or adenylate cyclase (20). ODQ (10 µM) was employed in the present study because it was previously shown (11) to completely abolish the rise in cGMP caused by S-nitrosoglutathione in cultured PAECs. However, ODQ failed to abrogate ·NO-mediated attenuation of oxidant-induced PAEC barrier dysfunction (Fig. 5). Thus despite the tendency for soluble guanylate cyclase to mediate the effects of low, physiological concentrations of ·NO (66), these findings indicate that ·NO attenuated oxidant-mediated endothelial barrier dysfunction by cGMP-independent pathways. This conclusion was further supported by studies employing PKG inhibitors. PAECs were treated with a high concentration of the PKG inhibitor KT-5823 (10 µM) to increase the likelihood of fully inhibiting PKG activity in intact PAECs. This concentration of KT-5823 inhibited PKG in cultured smooth muscle cells (42) and substantially exceeds the inhibition constant of 0.234 µM (64). The inability of KT-5823 treatment to abrogate the barrier protective effects of ·NO indicates that ·NO-induced PKG stimulation constitutes an unlikely mechanism for the attenuation of barrier dysfunction by ·NO in H2O2-treated PAECs (Fig. 6). These findings are consistent with other reports demonstrating PKG-independent pathways for ·NO effects in the vasculature (16).

These results suggest that the observed barrier protection is independent of ·NO-mediated guanylate cyclase or PKG activation. As shown in Table 1, ·NO not only stimulated PAEC cGMP levels but also significantly increased PAEC cAMP levels at 2 h in the absence and at 0.5 h in the presence of the PDE inhibitor IBMX. These findings indicate that SNAP-induced increases in PAEC cAMP content are mediated by two distinct mechanisms. At the early (0.5-h) time point after SNAP treatment, increases in cAMP content are PDE independent because they occur despite inhibition of PDE activity with IBMX. The direct activation of adenylate cyclase by submicromolar concentrations of ·NO was recently described in cardiac myocytes (63). The importance of this early, PDE-independent increase in cAMP is further suggested by the data in Fig. 5 that illustrate that guanylate cyclase-induced cGMP elevations were not required for the barrier protective effects of ·NO.

In contrast, at a later (2-h) time point, SNAP-induced increases in cAMP occur only in the absence of IBMX. This abrogation of ·NO-mediated cAMP accumulation by IBMX suggests the involvement of cGMP-induced inhibition of PDE isotypes that degrade cAMP. cGMP regulates the activities of several PDE isoforms (43) and could enhance cAMP accumulation by inhibiting PDE III or PDE IV (15). Such cross talk between cGMP- and cAMP-dependent intracellular signaling has been previously described in ECs (62). Therefore, our data indicate that ·NO increased PAEC cAMP content through both cGMP-dependent and -independent pathways. cAMP almost uniformly prevents endothelial barrier dysfunction caused by oxidants (55, 58) or agonists (14, 22). Coupled with the observation that cAMP, but not cGMP, analogs attenuated H2O2-induced PAEC barrier dysfunction (Fig. 4), these results suggest that ·NO-induced barrier protection is mediated by cAMP rather than by cGMP. The feasibility of this postulate is supported by the recent demonstration (48) that ·NO-mediated protection against TNF-alpha -induced PAEC cytotoxicity was mediated by cAMP.

Although previous studies (28, 58) demonstrated that oxidant-mediated endothelial barrier dysfunction was associated with decreases in cAMP content, our results provide new evidence that H2O2-induced PAEC barrier dysfunction can occur without associated decrements in PAEC cAMP levels (Table 1). These discrepancies are not surprising because the ability of H2O2 to decrease endothelial cAMP levels as well as the rapidity and extent of associated barrier dysfunction undoubtedly depends on the oxidant concentration as well as on the treatment conditions employed. In the present study, 100 µM H2O2 in RPMI 1640 culture medium containing 4% BSA caused no significant alteration in PAEC cAMP levels, and barrier dysfunction developed within 30-90 min after treatment with H2O2 was initiated. In contrast, higher concentrations of H2O2 (1 mM in HBSS with 0.25% BSA) caused significant decrements in PAEC cAMP content and barrier dysfunction within 5 min (58). In another report (28), H2O2 (100 µM in HBSS without BSA) decreased cAMP levels after 10 min and caused intercellular gap formation within 15-20 min in human umbilical vein endothelial cells. These studies illustrate that more severe oxidative stress can reduce endothelial cAMP content and cause rapid alterations in endothelial barrier function. Our results extend these reports by demonstrating that less severe, and perhaps more physiologically relevant, oxidative stimuli can cause endothelial barrier dysfunction that develops more slowly in the absence of reduced cAMP levels. Furthermore, our results suggest that even though ·NO and cGMP attenuate PAEC barrier dysfunction caused by 1 mM H2O2 through PDE-dependent pathways (57), these mechanisms cannot be extrapolated to barrier dysfunction caused by lower concentrations of H2O2.

The present study provides several lines of evidence supporting the critical role of cAMP in the barrier protective effect of ·NO. First, ·NO-mediated barrier protection was associated with increased PAEC cAMP content (Table 1). Second, treatment with cAMP analogs provided protection against H2O2-mediated barrier dysfunction similar to that provided by ·NO (Fig. 4). Third, inhibition of guanylate cyclase or PKG did not abrogate the barrier protective effects of ·NO (Figs. 5 and 6). Finally, because PKA activation constitutes the sole effector pathway for cAMP signaling, we examined the ability of the PKA inhibitor Rp-cAMPS to modulate the protective effect of ·NO. As shown in Fig. 7, inhibition of PKA substantially reduced ·NO-mediated barrier protection. However, increases in cAMP content in SNAP-treated PAECs did not translate into concomitant increases in PKA activity (Table 2). Furthermore, H2O2-induced reductions in PKA activity were not associated with reduced levels of cAMP. We speculate that this dissociation between cAMP levels and PKA activity stems from compartmentalization of cyclic nucleotides within distinct, subcellular pools (41). As a result, localized alterations in PKA activity may not be mirrored by analyses of whole cell cAMP levels. Taken together, our findings indicate that ·NO provides protection against oxidant-mediated barrier dysfunction by cAMP-dependent mechanisms.

In addition to increased cAMP levels, other mechanisms could contribute to the ability of ·NO to attenuate endothelial barrier dysfunction. For instance, ·NO may protect the endothelial barrier against H2O2 through its antioxidant effects by directly reacting with and scavenging lipid peroxides (44, 51) and through modulation of Fe2+-dependent formation of reactive oxygen species (12). Gupta et al. (24) have previously reported that ·NO donors dramatically attenuate PAEC lipid peroxidation induced by concentrations of H2O2 comparable to those employed in the present study. Alternatively, ·NO could regulate oxidant-induced alterations in [Ca2+]i that cause cytoskeletal derangements, changes in cell shape, and, ultimately, barrier dysfunction through direct actions on Ca2+ channel activity (13). Finally, ·NO could also inhibit or interrupt oxidant-stimulated signaling pathways. For example, H2O2 stimulates endothelial protein tyrosine phosphorylation through alterations in the activity of tyrosine kinases or phosphatases (61). The precise targets that undergo tyrosine phosphorylation to effect alterations in barrier function have not been defined. Similarly, the targets of cAMP-induced PKA activation and phosphorylation that promote barrier function remain to be established. Because of their central role in the maintenance of EC shape and barrier function, cytoskeletal proteins constitute plausible targets of kinase activity. H2O2 causes cytoskeletal remodeling (26), and ·NO inhibits tyrosine phosphorylation of components of focal adhesion complexes and stress fiber formation (21). These reports indicate that further studies examining the relationship between ·NO, intracellular signaling events, and endothelial barrier function represent a promising area for future study.

In summary, the present study demonstrates that both exogenous as well as endogenous sources of ·NO can attenuate H2O2-mediated endothelial barrier dysfunction. Although ·NO increases PAEC cGMP levels, the mechanisms for barrier protection depend on cAMP signaling pathways. The data presented herein suggest that ·NO-induced increases in cAMP are mediated by both PDE-independent and -dependent pathways. In addition, these results clarify that H2O2-mediated decreases in whole cell cAMP content are required for neither barrier dysfunction nor ·NO-induced barrier promotion. These findings emphasize the complexity of ·NO-mediated signaling pathways in vascular endothelium during oxidant stress and suggest that additional study will be required to fully understand and optimize the application of ·NO as a therapeutic agent in inflammatory vascular disorders involving increased levels of oxidative stress.


    ACKNOWLEDGEMENTS

We gratefully acknowledge the expert technical assistance of Dean Kleinhenz.


    FOOTNOTES

This work was supported by grants from the Research Service of the Roudebush Veterans Affairs Medical Center (C. M. Hart and C. Patterson); National Heart, Lung, and Blood Institute Grants HL-57260 (to V. Natarajan) and HL-58064 (to V. Natarajan and C. M. Hart); and the American Diabetes Association (C. M. Hart).

Present address of and address for reprint requests and other correspondence: C. M. Hart, Pulmonary and Critical Care Medicine Section, Atlanta VA Medical Center (151-P), 1670 Clairmont Rd., Decatur, GA 30033 (E-mail: Michael.Hart3{at}med.va.gov).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 28 October 1999; accepted in final form 9 August 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Baldwin, AL, Thurston G, and Naemi HA. Inhibition of nitric oxide synthesis increases venular permeability and alters endothelial actin cytoskeleton. Am J Physiol Heart Circ Physiol 274: H1776-H1784, 1998[Abstract/Free Full Text].

2.   Baron, DA, Lofton CE, Newman WH, and Currie MG. Atriopeptin inhibition of thrombin-mediated changes in the morphology and permeability of endothelial monolayers. Proc Natl Acad Sci USA 86: 3394-3398, 1989[Abstract].

3.   Beckman, JS, Beckman TW, Chen J, Marshall PM, and Freeman BA. Apparent hydroxyl radical production from peroxynitrite: implications for endothelial injury by nitric oxide and superoxide. Proc Natl Acad Sci USA 87: 1620-1624, 1990[Abstract].

4.   Beckman, JS, and Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and the ugly. Am J Physiol Cell Physiol 271: C1424-C1437, 1996[Abstract/Free Full Text].

5.   Benzig, A, Brautigam P, Geiger K, Loop T, Beyer U, and Moser E. Inhaled nitric oxide reduces pulmonary transvascular albumin flux in patients with acute lung injury. Anesthesiology 83: 1153-1161, 1995[ISI][Medline].

6.   Blatter, LA, Taha Z, Mesaros S, Shacklock PS, Wier WG, and Malinski T. Simultaneous measurements of Ca2+ and nitric oxide in bradykinin-stimulated vascular endothelial cells. Circ Res 76: 922-924, 1995[Abstract/Free Full Text].

7.   Block, ER, Patel J, and Sheridan NP. Effect of oxygen and endotoxin on lactate dehydrogenase release, 5-hydroxytryptamine uptake, and antioxidant enzyme activities in endothelial cells. J Cell Physiol 122: 240-248, 1985[ISI][Medline].

8.   Bloomfield, GL, Holloway S, Ridings PC, Fisher BJ, Blocher CR, Sholley M, Bunch T, Sugerman HJ, and Fowler AA. Pretreatment with inhaled nitric oxide inhibits neutrophil migration and oxidative activity resulting in attenuated sepsis-induced acute lung injury. Crit Care Med 25: 584-593, 1997[ISI][Medline].

9.   Brigham, KL. Oxidant stress and the adult respiratory distress syndrome. Eur Respir J 3: 482s-484s, 1990.

10.   Bredt, D, and Snyder S. Nitric oxide: a physiologic messenger molecule. Annu Rev Biochem 63: 175-195, 1994[ISI][Medline].

11.   Brunner, F, Schmidt K, Nielsen EB, and Mayer B. Novel guanylate cyclase inhibitor potently inhibits cyclic GMP accumulation in endothelial cells and relaxation of bovine pulmonary artery. J Pharmacol Exp Ther 277: 48-53, 1996[Abstract].

12.   Chang, J, Rao NV, Markewitz BA, Hoidal JR, and Michael JR. Nitric oxide donor prevents hydrogen peroxide-mediated endothelial cell injury. Am J Physiol Lung Cell Mol Physiol 270: L931-L940, 1996[Abstract/Free Full Text].

13.   Clementi, E. Role of nitric oxide and its intracellular signaling pathways in the control of Ca2+ homeostasis. Biochem Pharmacol 55: 713-718, 1998[ISI][Medline].

14.   Draijer, R, Atsma DE, Van der Laarse A, and Hinsbergh VWM cGMP and nitric oxide modulate thrombin-induced endothelial permeability: regulation via different pathways in human aortic and umbilical vein endothelial cells. Circ Res 76: 199-208, 1995[Abstract/Free Full Text].

15.   Eckly, AE, and Lugnier C. Role of phosphodiesterases III and IV in the modulation of vascular cAMP content by the NO/cyclic GMP pathway. Br J Pharmacol 113: 445-450, 1994[Abstract].

16.   Fouty, B, Komalavilas P, Muramatsu M, Cohen A, McMurtry IF, Lincoln TM, and Rodman DM. Protein kinase G is not essential to NO-cGMP modulation of basal tone in rat pulmonary circulation. Am J Physiol Heart Circ Physiol 274: H672-H678, 1998[Abstract/Free Full Text].

17.   Garat, C, Jayr C, Eddahibi S, Laffon M, Meignan M, and Adnot S. Effects of inhaled nitric oxide or inhibition of endogenous H2O2 formation on hyperoxic lung injury. Am J Respir Crit Care Med 155: 1957-1964, 1997[Abstract].

18.   Garcia, JGN, and Schaphorst KL. Regulation of endothelial cell gap formation and paracellular permeability. J Investig Med 43: 117-126, 1995[ISI][Medline].

19.   Garcia, JGN, Schaphorst KL, Shi S, Verin AD, Hart CM, Callahan KS, and Patterson CE. Mechanisms of ionomycin-induced endothelial cell barrier dysfunction. Am J Physiol Lung Cell Mol Physiol 273: L172-L184, 1997[Abstract/Free Full Text].

20.   Garthwaite, J, Southam E, Boulton CL, Nielsen EB, Schmidt K, and Mayer B. Potent and selective inhibition of nitric oxide-sensitive guanylate cyclase by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Mol Pharmacol 48: 184-188, 1995[Abstract].

21.   Goligorsky, MS, Abedi H, Noiri E, Takhtajan A, Lense S, Romanov V, and Zachary I. Nitric oxide modulation of focal adhesion in endothelial cells. Am J Physiol Cell Physiol 276: C1271-C1281, 1999[Abstract/Free Full Text].

22.   Gudgeon, JR, and Martin W. Modulation of arterial endothelial permeability: studies on an in vitro model. Br J Pharmacol 98: 1267-1274, 1989[Abstract].

23.   Guidot, DM, Repine MJ, Hybertson BM, and Repine JE. Inhaled nitric oxide prevents neutrophil-mediated oxygen radical dependent leak in isolated rat lungs. Am J Physiol Lung Cell Mol Physiol 269: L2-L5, 1995[Abstract/Free Full Text].

24.   Gupta, MP, Evanoff V, Blackburn T, and Hart CM. Nitric oxide donors attenuate hydrogen peroxide-mediated injury to porcine pulmonary artery endothelial cells. Am J Physiol Lung Cell Mol Physiol 272: L1133-L1141, 1997[Abstract/Free Full Text].

25.   Gupta, MP, Steinberg HO, and Hart CM. H2O2 causes endothelial cell barrier dysfunction without disrupting the arginine-nitric oxide pathway. Am J Physiol Lung Cell Mol Physiol 274: L508-L516, 1998[Abstract/Free Full Text].

26.   Hart, CM, Andreoli SP, Patterson CE, and Garcia JGN Oleic acid supplementation reduces oxidant-mediated dysfunction of cultured porcine pulmonary artery endothelial cells. J Cell Physiol 156: 24-34, 1993[ISI][Medline].

27.   Hart, CM, Dominguez JH, and Garcia JGN Fatty acids modulate oxidant stress and intracellular calcium in pulmonary artery endothelial cells (Abstract). Clin Res 41: 721A, 1992.

28.   Hastie, LE, Patton WF, Hechtman HB, and Shepro D. H2O2-induced filamin redistribution in endothelial cells is modulated by the cyclic AMP-dependent protein kinase pathway. J Cell Physiol 172: 373-381, 1997[ISI][Medline].

29.   He, P, Zeng M, and Curry FE. Effect of nitric oxide synthase inhibitors on basal microvessel permeability and endothelial cell [Ca2+]i. Am J Physiol Heart Circ Physiol 273: H747-H755, 1997[Abstract/Free Full Text].

30.   Hobbs, AJ, and Ignarro LJ. Nitric oxide-cyclic GMP signal transduction system. Methods Enzymol 269: 134-148, 1996[ISI][Medline].

31.   Holschermann, H, Noll T, Hempel A, and Piper M. Dual role of cGMP in modulation of macromolecule permeability of aortic endothelial cells. Am J Physiol Heart Circ Physiol 272: H91-H98, 1997[Abstract/Free Full Text].

32.   Kavanagh, BP, Mouchawar A, Goldsmith J, and Pearl RG. Effects of inhaled NO and inhibition of endogenous NO synthesis in oxidant-induced acute lung injury. J Appl Physiol 76: 1324-1329, 1994[Abstract/Free Full Text].

33.   Kubes, P. Nitric oxide-induced microvascular permeability alterations: a regulatory role for cGMP. Am J Physiol Heart Circ Physiol 265: H1909-H1915, 1993[Abstract/Free Full Text].

34.   Kurose, I, Kubes P, Wolf R, Anderson DC, Paulson J, Miyasaka M, and Granger DN. Inhibition of nitric oxide production: mechanisms of vascular albumin leakage. Circ Res 73: 164-171, 1993[Abstract].

35.   Lofton, CE, Newman WH, and Currie MG. Atrial natriuretic peptide regulation of endothelial permeability is mediated by cGMP. Biochem Biophys Res Commun 172: 793-799, 1990[ISI][Medline].

36.   MacMilian-Crow, LA, Murphy-Ullrich JE, and Lincoln TM. Identification and possible localization of cGMP-dependent protein kinase in bovine aortic endothelial cells. Biochem Biophys Res Commun 201: 531-537, 1994[ISI][Medline].

37.   Malinski, T, Taha Z, Grunfeld S, Patton S, Kapturczak M, and Tomboulian P. Diffusion of nitric oxide in the aortic wall monitored in situ by porphyrinic microsensors. Biochem Biophys Res Commun 193: 1076-1082, 1993[ISI][Medline].

38.   McElroy, M, Wiener-Kronish J, Miyazaki H, Sawa T, Modelska K, Dobbs L, and Pittet J. Nitric oxide attenuates lung endothelial injury caused by sublethal hyperoxia in rats. Am J Physiol Lung Cell Mol Physiol 272: L631-L638, 1997[Abstract/Free Full Text].

39.   McQuaid, KE, Smyth EM, and Keenan AK. Evidence for modulation of H2O2-induced endothelial cell barrier dysfunction by nitric oxide in vitro. Eur J Pharmacol 307: 233-241, 1996[ISI][Medline].

40.   Meyer, DJ, and Huxley VH. Capillary hydraulic conductivity is elevated by cGMP-dependent vasodilators. Circ Res 70: 382-391, 1992[Abstract].

41.   Mittal, CK, and Murad F. Formation of adenosine 3':5'-monophosphate by preparations of guanylate cyclase from rat liver and other tissues. J Biol Chem 252: 3136-3140, 1977[ISI][Medline].

42.   Nishio, E, Fikushima K, Shiozaki M, and Watanabe Y. Nitric oxide donor compound SNAP induces apoptosis in smooth muscle cells through cGMP-dependent mechanism. Biochem Biophys Res Commun 221: 163-168, 1996[ISI][Medline].

43.   Nicholson, CD, Raj C, and Shahid M. Differential modulations of tissue functions and therapeutic potential of selective inhibitors of cyclic nucleotide phosphodiesterase isoenzymes. Trends Pharmacol Sci 11: 19-27, 1991.

44.   O'Donnel, VB, Chumley PH, Hogg N, Bloodsworth A, Darley-Usmar VM, and Freeman BA. Nitric oxide inhibition of lipid peroxidation: kinetics of reaction with lipid peroxyl radicals and comparison with alpha -tocopherol. Biochemistry 36: 15216-15223, 1997[ISI][Medline].

45.   Oliver, JA. Endothelium-derived relaxing factor contributes to the regulation of endothelial permeability. J Cell Physiol 151: 506-511, 1992[ISI][Medline].

46.   Patterson, C, Rhoades R, and Garcia JGN Evans blue dye as a marker of albumin clearance in cultured endothelial monolayer and isolated lung. J Appl Physiol 72: 865-873, 1992[Abstract/Free Full Text].

47.   Philips, PG, and Tsan MF. Hyperoxia causes increased albumin permeability of cultured endothelial monolayers. J Appl Physiol 64: 1196-1202, 1989[Abstract/Free Full Text].

48.   Polte, T, and Schroder H. Cyclic AMP mediates endothelial protection by nitric oxide. Biochem Biophys Res Commun 251: 460-465, 1998[ISI][Medline].

49.   Poss, WB, Timmons OD, Farrukh IS, Hoidal JR, and Michael JR. Inhaled nitric oxide prevents the increases in pulmonary vascular permeability caused by hydrogen peroxide. J Appl Physiol 79: 886-891, 1995[Abstract/Free Full Text].

50.   Radi, R, Beckman JS, Bush KM, and Freeman BA. Peroxynitrite induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide. Arch Biochem Biophys 288: 481-487, 1991[ISI][Medline].

51.   Rubbo, H, Radi R, Trujillo M, Telleri R, Kalayanaraman B, Barnes S, Kirk M, and Freeman BA. Nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation. J Biol Chem 269: 26066-26075, 1994[Abstract/Free Full Text].

52.   Schmidt, K, Werner ER, Mayer B, Wachter H, and Kukovetz WR. Tetrahydrobiopterin-dependent formation of endothelium-derived relaxing factor (nitric oxide) in aortic endothelial cells. Biochem J 281: 297-300, 1992[ISI][Medline].

53.   Shasby, DM, Lind EE, Shasby SS, Goldsmith JC, and Hunninghake GW. Reversible oxidant-induced increases in albumin transfer across cultured endothelium: alterations in cell shape and calcium homeostasis. Blood 65: 605-614, 1985[Abstract].

54.   Shasby, DM, Shasby SS, and Peach MJ. Granulocytes and phorbol myristate acetate increase permeability to albumin of cultured monolayers and isolated perfused lungs. Am Rev Respir Dis 127: 72-76, 1983[ISI][Medline].

55.   Siflinger-Birnboim, A, Bode DC, and Malik AB. Adenosine 3',5'-cyclic monophosphate attenuates neutrophil-mediated increase in endothelial permeability. Am J Physiol Heart Circ Physiol 264: H370-H375, 1993[Abstract/Free Full Text].

56.   Siflinger-Birnboim, A, Goligorsky MS, Del Vecchio PJ, and Malik AB. Activation of protein kinase C pathway contributes to hydrogen peroxide-induced increase in endothelial permeability. Lab Invest 67: 24-30, 1992[ISI][Medline].

57.   Suttorp, N, Hippenstiel S, Fuhrmann M, Krull M, and Podzuweit T. Role of nitric oxide and phosphodiesterase isoenzyme II for reduction of endothelial hyperpermeability. Am J Physiol Cell Physiol 270: C778-C785, 1996.

58.   Suttorp, N, Weber U, Welsch T, and Schudt C. Role of phosphodiesterases in the regulation of endothelial permeability in vitro. J Clin Invest 91: 1421-1428, 1993[ISI][Medline].

59.   Suzuki, YJ, Forman HJ, and Sevanian A. Oxidants as stimulators of signal transduction. Free Radic Biol Med 22: 269-285, 1997[ISI][Medline].

60.   Taher, MM, Garcia JGN, and Natarajan V. Hydroperoxide-induced acylglycerol formation and protein kinase C activation in vascular endothelial cells. Arch Biochem Biophys 303: 260-266, 1993[ISI][Medline].

61.   Vepa, S, Scribner WM, Parinandi NL, English D, Garcia JGN, and Natarajan V. Hydrogen peroxide stimulates tyrosine phosphorylation of focal adhesion kinase in vascular endothelial cells. Am J Physiol Lung Cell Mol Physiol 277: L150-L158, 1999[Abstract/Free Full Text].

62.   Vigne, P, Lund L, and Frelin C. Cross talk among cyclic AMP, cyclic GMP, and Ca+2-dependent intracellular signaling mechanisms in brain capillary endothelial cells. J Neurochem 62: 2269-2274, 1994[ISI][Medline].

63.   Vila-Petroff, MG, Younes A, Egan J, Lakatta EG, and Sollott SJ. Activation of distinct cAMP-dependent and cGMP-dependent pathways by nitric oxide in cardiac myocytes. Circ Res 84: 1020-1031, 1999[Abstract/Free Full Text].

64.   Wang, DL, Wung B, Shy Y, Lin C, Chao Y, Usami S, and Chien S. Mechanical strain induces monocyte chemotactic protein-1 gene expression in endothelial cells: effects of mechanical strain on monocyte adhesion to endothelial cells. Circ Res 77: 294-302, 1995[Abstract/Free Full Text].

65.   Westendorp, RGJ, Draijer R, Meinders AE, and van Hinsbergh VWM Cyclic-GMP-mediated decrease in permeability of human umbilical and pulmonary artery endothelial cell monolayers. J Vasc Res 31: 42-51, 1994[ISI][Medline].

66.   Wink, DA, and Mitchell JB. Chemical biology of nitric oxide: insights into regulatory, cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic Biol Med 25: 434-456, 1998[ISI][Medline].

67.   Yonemaru, M, Ishii K, Murad F, and Raffin TA. Atriopeptin-induced increases in endothelial cell permeability are associated with elevated cGMP levels. Am J Physiol Lung Cell Mol Physiol 263: L363-L369, 1992[Abstract/Free Full Text].

68.   Yuan, Y, Granger HJ, Zawieja DC, DeFily DV, and Chilian WM. Histamine increases venular permeability via a phospholipase C-NO synthase-guanylate cyclase cascade. Am J Physiol Heart Circ Physiol 264: H1734-H1739, 1993[Abstract/Free Full Text].


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