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Am J Physiol Lung Cell Mol Physiol 290: L674-L684, 2006. First published November 11, 2005; doi:10.1152/ajplung.00391.2005
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Peroxynitrite mediates TNF-{alpha}-induced endothelial barrier dysfunction and nitration of actin

Paul Neumann,1 Nancy Gertzberg,1 Erin Vaughan,1 Joshua Weisbrot,2 Renee Woodburn,3 William Lambert,3 and Arnold Johnson1

1Research Service of the Stratton Veterans Affairs Medical Center; 2Albany Medical College; and 3University at Albany State University of New York, Albany, New York

Submitted 12 September 2005 ; accepted in final form 8 November 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We tested the hypothesis that tumor necrosis factor (TNF)-{alpha} induces a peroxynitrite (ONOO–)-dependent increase in permeability of pulmonary microvessel endothelial monolayers (PMEM) that is associated with generation of nitrated beta-actin (NO2-beta-actin). The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin. beta-Actin was extracted from PMEM lysate with a DNase-Sepharose column. The extracted beta-actin was quantified in terms of its nitrotyrosine/beta-actin ratio with antinitrotyrosine and anti-beta-actin antibodies, sequentially, by dot-blot assays. The cellular compartmentalization of NO2-beta-actin was displayed by showing confocal localization of nitrotyrosine-immunofluorescence with beta-actin-immunofluorescence but not with F-actin fluorescence. Incubation of PMEM with TNF (100 ng/ml) for 0.5 and 4.0 h resulted in increases in permeability to albumin. There was an increase in the nitrotyrosine/beta-actin ratio at 0.5 h with minimal association of the NO2-beta-actin with F-actin polymers. The TNF-induced increase in the nitrotyrosine/beta-actin ratio and permeability were prevented by the anti-ONOO– agent Urate. The data indicate that TNF induces an ONOO–-dependent barrier dysfunction, which is associated with the generation of NO2-beta-actin.

edema; permeability

NITRIC OXIDE (·NO) rapidly reacts with superoxide anion radical (·O2–), at diffusion limited rates, resulting in generation of peroxynitrite (ONOO–; ·NO +·O2–·ONOO–) (5, 19, 20). Peroxynitrous acid (HONOO) is formed in the range of physiological pH (ONOO– + H+·HONOO) because the pK of ONOO– is ~6.8 (5, 15). ONOO–[H] is capable of inducing significant tissue injury via the reactive intermediates nitrogen dioxide radical (·NO2) and hydroxyl radical (·OH) (5, 15). In addition, ONOO– reacts with carbon dioxide forming the adduct nitrosoperoxocarboxylate, which can produce the reactive intermediates carbonate radical and ·NO2 (7). The suggested mechanisms for ONOO–-directed tissue injury are numerous and still controversial (15, 19, 39). However, a probable pathway for the pathobiology of ONOO– is the oxidation and nitration of amino acids in protein, such as the conversion of cysteine into the oxidized species cystine and nitration of tyrosine into nitrotyrosine (5, 15, 19).

Nitrotyrosine is found in the lungs of animals exposed to either hyperoxia (18) or lung allotransplantation (1) and in patients with sickle cell disease (2), atherosclerosis (6), and respiratory distress syndrome (RDS) (36). Tumor necrosis factor (TNF)-{alpha} is a mediator of RDS and sepsis syndrome (27). Our previous studies indicate that TNF causes the formation of ·O2–, ·NO, and ONOO– in pulmonary microvessel endothelial cells (14, 16, 30). The ·NO and ·O2– are derived from endothelial nitric oxide synthase (eNOS) and NAD(P)H-oxidase, respectively, and mediate the TNF-induced barrier dysfunction (8, 16). The generation of ONOO– is relevant to TNF-induced endothelial injury because we have shown that inhibitors of ONOO– also prevent the increase in nitrotyrosine and oxidation of a 42-kDa protein that we putatively identified as actin (14, 30).

Actin is a protein that exists in three isoforms ({alpha}, beta, and {gamma}) and in various states ranging from monomeric G-actin (43 kDa) to polymeric F-actin (35). beta-Actin is the major isoform in endothelial cells (35). F-actin is a major constituent of the cytoskeleton, particularly in the centralized regions (i.e., the stress fibers) and cortical regions (i.e., the peripheral bands), which modulate barrier function in response to TNF (17, 35, 38, 41). NAD(P)H-oxidase (26) and eNOS (12) are associated with the cortical region of the cell; therefore, a significant proximal target for ONOO– can be actin. Moreover, 5% of the amino acid residues of actin are tyrosine, which is a known substrate for ONOO– (15, 28, 35).

The treatment of endothelium with exogenous ONOO– has been show to nitrate intracellular actin and the intracellular adherence protein beta-catenin and to increase endothelial permeability (23). ONOO– has been shown to nitrate intracellular actin and alter functional responses in neutrophils (9). It has been shown by us (13) and others (23) that ONOO–-treated actin, but not native actin, will cause an increase in endothelial permeability. The direct nitration of actin with tetranitromethane or ONOO– in vitro alters its nucleation and polymerization properties (2, 28, 34), potentially by disrupting the cycling of beta-actin monomers into the growing "barbed" ends of native F-actin polymers (31, 38). Finally, Goldblum et al. (17) demonstrated that TNF-induced barrier dysfunction is prevented by stabilization of actin polymers using phallicidin, supporting a role for actin in barrier dysfunction. Thus we tested the hypothesis that TNF-induced barrier dysfunction is: 1) dependent on ONOO– and 2) associated with nitration of intracellular actin.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents

All reagents were obtained from Sigma (St. Louis, MO) unless otherwise noted.

Pulmonary Microvessel Endothelial Cell Culture

Rat lung microvessel endothelial cells (RLMVEC) and bovine lung microvessel endothelial cells (BLMVEC) were obtained at 4th passage (Vec Technologies, Rensselaer, NY). The preparations were identified by Vec Technologies as pure populations by 1) the characteristic "cobblestone" appearance as assessed by phase contrast microscopy, 2) the presence of factor VIII-related antigen (indirect immunofluorescence), 3) the uptake of acylated low-density lipoproteins, and 4) the absence of smooth muscle actin (indirect immunofluorescence). For all studies, both RLMVEC and BLMVEC were cultured from 4 to 12 passages in culture medium containing either Dulbecco’s modified Eagle’s medium (DMEM; GIBCO, Grand Island, NY) supplemented with 20% fetal bovine serum (Hyclone; Hyclone Laboratories, Logan, UT), 15 µg/ml endothelial cell growth supplement (Upstate Biotechnology, Lake Placid, NY), and 1% nonessential amino acids (GIBCO-BRL) for BLMVEC, and MCDB-131 complete media containing 10% fetal bovine serum (VEC Technologies) for RLMVEC. Both cell lines were maintained in 5% CO2 plus humidified air at 37°C. A confluent pulmonary microvessel endothelial cell monolayer (PMEM) was reached within two to three population doublings, which took 3–4 days.

Treatments

TNF treatment. Highly purified recombinant human TNF-{alpha} from Escherichia coli (Calbiochem-Novabiochem, La Jolla, CA) in a stock solution of 10 µg/ml was used. The endotoxin level was <0.1 ng/µg of TNF-{alpha} as determined by standard limulus assay. We previously showed that boiling TNF-{alpha} for 0.75 h blocks the effect of TNF in our system (14), which indicates no endotoxin contamination. PMEM were treated with TNF-{alpha} at 100 ng/ml, since dose-response studies indicate this dose consistently induces a permeability increase.

Anti-ONOO– agent. The ONOO– inhibitor used was Urate (5 µM). We have previously shown that Urate scavenges TNF-induced ONOO– and has no effect on cell viability in endothelium (30). Cells were either treated with Urate alone or cotreated with Urate and TNF.

Treatment medium. For all studies, incubation of PMEM with TNF, Urate, and all corresponding controls were performed with phenol-free DMEM (GIBCO-BRL) supplemented with 10% FBS to avoid a potential antioxidant effect of phenol.

Assay of Endothelial Permeability

Nucleopore Track-Etch polycarbonate membranes (13 mm diameter, 0.8 mm pore size; Corning Costar, Cambridge, MA) were coated with gelatin (type B from bovine skin) mounted on modified Boyden chemotaxis chambers (9 mm inner diameter; Adaps, Dedham, MA) with MF cement no. 1 (Millipore, Bedford, MA) and sterilized by ultraviolet light for 12–24 h as previously described (8, 16). Either BLMVEC or RLMVEC (1.5 x 105 in 0.50 ml of DMEM) were plated on the gelatinized membranes and allowed to reach confluence within 3–5 days (37°C, 5% CO2).

The experimental apparatus for the study of transendothelial transport in the absence of hydrostatic and oncotic pressure gradients has been described (16). In brief, the system consists of two compartments separated by a microporous polycarbonate membrane lined with the endothelial cell monolayer as described above. The luminal (upper) compartment (0.7 ml) was suspended in the abluminal (lower) compartment (25 ml). The lower compartment was stirred continuously for complete mixing. The entire system was kept in a water bath at a constant temperature of 37°C. The fluid height in both compartments was the same to eliminate convective flux.

Endothelial permeability was characterized by the clearance rate of Evans Blue-labeled albumin using our adaptation (16) of the original technique described by Patterson et al. (29). A buffer solution containing Hanks’ balanced salt solution (GIBCO-BRL) containing 0.5% bovine serum albumin (BSA) and 20 mM HEPES buffer was used on both sides of the monolayer. The luminal compartment buffer was labeled with a final concentration of 0.057% Evans blue dye in a volume of 700 µl. The absorbance of free Evans blue in the luminal and abluminal compartments was always <1% of the total absorbance of Evans blue in the buffer. At the beginning of each study a luminal compartment sample was diluted 1:100 to determine the initial absorbance of that compartment. Abluminal compartment samples (300 ml) were taken every 5 min for 60 min. The absorbance of the samples was measured in a SpectraMax Plus microplate spectrophotometer (Molecular Devices, Sunnyvale, CA) at 620 nm. The clearance rate of Evans blue-labeled albumin was determined by least-squares linear regression between 10 and 60 min for the control and experimental groups.

Immunofluorescence and Confocal Microscopy

Cell preparation and antibody treatment. Either RLMVEC or BLMVEC (1 x 104/0.20 ml of culture medium) were plated on 18-mm coverslips inside a 35-mm culture dish, incubated at 37°C for 2 h to allow attachment, and then grown to confluence in an additional 2 ml of culture medium (16). The PMEM were treated as indicated, washed with Dulbecco’s phosphate-buffered saline (DPBS, GIBCO-BRL), fixed with 3.7% formaldehyde solution at room temperature (RT) for 20 min and then permeabilized with 1% Triton X-100 in DPBS at RT for 5 min. The cells were washed with DPBS and then blocked in 10% normal goat serum (NGS, GIBCO-BRL) at RT for 1 h. PMEM were incubated with mouse monoclonal antinitrotyrosine antibody (clone 1A6, Upstate) at a 1:1,000 dilution in 10% NGS at RT for 1 h then washed sufficiently. The secondary antibody, Alexa Fluor 488-labeled goat anti-mouse IgG (Molecular Probes, Eugene, OR), was added at a 1:1,000 dilution in 10% NGS and incubated at RT for 1 h and then washed sufficiently. F-actin was stained with Alexa Fluor 568 Phalloidin and washed sufficiently, or, in separate studies, total beta-actin was stained with mouse monoclonal anti-beta-actin antibody (clone AC74), followed by Alexa Fluor 568-labeled goat anti-mouse IgG (Molecular Probes).

The quantification strategy for the fluorescent images is as follows. PMEM were visualized and quantified with confocal microscopy using the Leica Confocal System TCS SP2 (Leica Microsystems, Exton, PA). There were four separate studies with four treatment groups and two treatment times per study. All fields were selected by random movement of the microscope stage to another area within an intact endothelial monolayer. Six entire fields per treatment group were analyzed with one image per field. All treatment groups were normalized for fluorescent intensity by initially adjusting the settings for noise, brightness, and contrast, as determined by the slide with the maximum fluorescence (16).

Specificity of the antinitrotyrosine antibody was confirmed by antibody-antigen competition. A 10:1 molar ratio of nitrotyrosine antigen to nitrotyrosine antibody was preincubated in 10% NGS for 30 min at 37°C before application to PMEM. The coverslips were mounted on clean glass slides with Permafluor mounting media (Thermo Shandon, Pittsburgh, PA). The PMEM were visualized with a Spot RT color camera (Diagnostic Instruments, Sterling Heights, MI) mounted on an Olympus IX70 inverted microscope (Olympus America, Melville, NY) equipped for phase, light, and fluorescence detection. Images for illustration were captured at x100 magnification with an exposure time of 8 s and downloaded into Spot RT imaging software (Diagnostic Instruments) (16).

Immunoblot Analysis of Actin

Cell lysate actin extraction. Cellular actin was extracted as per Schafer et al. (32). Cells were lysed in actin extraction buffer (1.0 M Tris·HCl pH 7, 1 mM DTT, 0.6 M KCl, 0.5 mM MgCl2, 0.5 mM ATP, 0.2 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride, 4% Triton X-100, and 2% Tween 20), and then lysates were ultracentrifuged at 110,000 g for 60 min at 4°C to remove DNA and particulates. Lysate supernatant protein concentration was determined by BCA assay (Pierce, Rockford, IL), and proteins in each group were normalized to 2 µg/µl. DNase I, type II, was coupled to CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech, Piscataway, NJ), and 400 µl of a 50% slurry of DNase-Sepharose resin and storage buffer (0.5 M NaCl, 20 mM Tris pH 8, 0.05% Tween 20, and 0.1% sodium azide) were added to Handee Spin Cup Columns (Pierce) and allowed to settle for 15 min. The storage buffer was removed, followed by two sequential washes with 350 µl of G-buffer (1 mM Tris·HCl, 0.2 mM ATP, 0.2 mM CaCl2, 0.1 mM DTT, and 0.005% sodium azide) by centrifugation at 15 g for 2 min at 4°C. The columns were loaded with 350 µl of lysate and centrifuged at 5 g for 10 min at 4°C followed by three washes with 350 µl G-buffer at 15 g, 2 min, 4°C. Column flow-throughs were collected for later SDS-PAGE verification of actin extraction. Actin was eluted from the columns by RT incubation with 100 µl of 6 M urea for 30 min, followed by centrifugation at 5,000 g for 5 min. The average eluate retrieval volume was 240 µl, resulting in an final buffer of 2.5 M urea, 60% G-buffer (U/G-buffer). Eluates were assayed for total protein with the enhanced BCA assay protocol (Pierce) against BSA/IgG (1:1) standards and then normalized to 100 ng/µl with U/G-buffer.

Preparation of nitrated actin standards. Nitrated actin was a gift from the laboratory of James Estes and was prepared from beta,{gamma}-nonmuscle actin (33). The reaction with peroxynitrite was accomplished by slow (1.5 µM/s) infusion of alkaline peroxynitrite into a 23 µM F-actin solution containing 50 mM KHPO4, 0.1 M KCl, 2 mM MgCl2, 0.2 mM ATP, and 0.1 mM EGTA, pH 7.0. The degree of nitration was proportional to the length of infusion time and was determined at pH 9.5 by UV absorbance at 428 nm using an extinction coefficient of 4,200 M–1·cm–1. Further adjustment of the final degree of nitration to 25 mmol NO2-tyrosine/mol actin was made by dilution of the reaction product with nonnitrated actin.

SDS-PAGE and Western blotting. To verify actin extraction, proteins of lysate, flow-through samples, and eluate in Laemmli buffer (62.5 mM Tris·HCl pH 6.8, 5% glycerol, 1% 2-mercaptoethanol, 2% SDS, and 0.003% bromphenol blue) were separated on 1.5-mm-thick 8–16% polyacrylamide gradient gels by applying 175 V for 60 min. The gels were transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore, Bedford, MA) in Towbin’s transfer buffer by applying 125 V for 75 min. The blots were stained in Bradford reagent (0.01% Coomassie blue G-250, 9.5% ethanol, 8.5% H3PO4) for ~10 min and then destained (50% methanol, 1.0% acetic acid) until background was reduced, and total proteins were clearly visible. Total destaining in 100% methanol, rehydration, and immunoprobing with anti-beta-actin antibody (clone AC15) was performed on select blots to confirm band identity. Nitrotyrosine immunoblotting control (Upstate) and Cruz Marker molecular mass standards and corresponding Cruz Marker-compatible secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were used for in-image references.

Dot blot. PVDF membranes were hydrated in TBS (10 mM Tris·HCl pH 7.5, 100 mM NaCl), placed in a Bio-Dot Microfiltration Apparatus (Bio-Rad, Hercules, CA), and wells were washed three times with 100 µl of TBS under partial vacuum. Wells were loaded, 100 µl each, with either actin eluate or nitrated beta-actin dilutions in U/G-buffer, allowed to drain by gravity, and then rinsed by gravity with 100 µl of TBS. After removal, the membrane was washed in TBS and blocked overnight in blotto (TBS, 5.0% nonfat dry milk, 0.05% Tween 20) at 4°C. The membrane was then incubated with rabbit polyclonal antinitrotyrosine (Upstate) diluted 1:5,000 in blotto, followed by goat anti-rabbit horseradish peroxidase conjugate diluted in blotto. Both incubations were 45 min at 37°C, with five 5-min RT washes in TTBS (TBS, 0.05% Tween 20) following each antibody. After applying Supersignal West Dura Extended Duration Substrate (Pierce) to the membrane for 3 min, we sealed it in a page protector and imaged it on a Kodak Image Station 440CF (Eastman Kodak, Rochester, NY) for ~40 min. The membrane was then rinsed with water for 5 min and stripped for 15 min at RT with either Re-Blot Plus strong stripping solution (Chemicon, Temecula, CA) or 0.2 M NaOH. After a 5-min rinse in TTBS, the membrane was reblocked 20 min at 37°C and reprobed, using the preceding incubation-wash protocol, with mouse monoclonal anti-beta-actin (clone AC74) diluted 1:15,000, followed by goat anti-mouse alkaline phosphatase conjugate. Imaging followed a 5-min rinse in assay buffer (0.1 M diethanolamine, 1.0 mM MgCl2 pH 9.5) and a 3-min application of CDP-Star (Tropix, Bedford, MA). To confirm antinitrotyrosine signal specificity, selected blots, both dot blots and Westerns, were incubated with a dithionite (sodium hydrosulfite) solution (100 mM dithionite, 100 mM sodium borate, 0.1 mM CaCl2 pH 9.0) for 1 h at RT to reduce nitrotyrosine to aminotyrosine before immunoprobing for nitrotyrosine.

Analysis. Probe intensity units were measured with Kodak 1D image analysis software and the intensity units of nitrated beta-actin standards vs. nitrotyrosine (femtomoles) or beta-actin (ng) were quantified with linear regression analysis. Eluate nitrotyrosine (femtomoles) from 15x diluted samples and beta-actin (ng) from 201x diluted samples, in triplicate, were calculated from these standard curves. Buffer blank values, when measurable, were subtracted from all data values as background. To measure the relative degree of nitration, we calculated a sample’s nitrotyrosine/beta-actin ratio, after correcting for dilution, and data are reported as nitrotyrosine/beta-actin of treatment sample divided by nitrotyrosine/beta-actin of the respective treatment control sample.

Statistics

A one-way analysis of variance was used to compare values among the treatments. If significance among treatments was noted, a post hoc multiple-comparison test was done with a Bonferroni (parametric-equal variance) or a Duncan (nonparametric-unequal variance) test to determine significant differences among the groups (37). A log10 transform was performed to smooth the data when appropriate. Each PMEM well and flask represents a single experiment. All data are reported as means ± SE. Significance was at P < 0.05. There are 5–10 samples per group in all studies.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
TNF Causes Colocalization of Nitrotyrosine With beta-Actin: Effect of Urate

In the representative micrographs of bovine PMEM in Fig. 1, treatments are control ± Urate or TNF ± Urate incubated for either 0.5 or 4 h and assayed for nitrotyrosine immunofluorescence (green) and beta-actin immunofluorescence (red). In PMEM exposed to TNF for either 0.5 or 4 h, there is an increase in nitrotyrosine fluorescence compared with the respective untreated control group. At 0.5-h exposure to TNF, there is also an increase in the colocalization of nitrotyrosine fluorescence with the cortical beta-actin fluorescence, which is seen as an increase in yellow color in the combined green and red images. However, after 4 h, there is no longer an increase in yellow, compared with controls, indicating a reversal in colocalization of nitrotyrosine with beta-actin. In PMEM treated with Urate, with or without TNF, there is neither an increase in nitrotyrosine fluorescence nor colocalization compared with controls. The data shown in Fig. 1 support the hypothesis that TNF causes a reversible increase in NO2-beta-actin in PMEM that is prevented by Urate.


Figure 1
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  		Fig. 1. Urate prevents the TNF-induced colocalization of nitrotyrosine with beta-actin in pulmonary microvessel endothelial cell monolayers (PMEM). Representative confocal micrographs (bovine cells) of control, TNF-, Urate alone-, and TNF+Urate-treated PMEM after 0.5 and 4 h. Nitrotyrosine has been immunostained with antinitrotyrosine and appears as green fluorescence. beta-Actin has been immunostained with anti-beta-actin and appears as red fluorescence. The resultant color change of the combined red and green micrographs appears yellow where colocalization occurs (inset: arrows). A total of 4 preparations were generated for each treatment and time point from both rat and bovine PMEM.

 
TNF Does Not Alter Colocalization of Nitrotyrosine With F-Actin in PMEM: Effect of Urate

In the representative micrographs of bovine PMEM in Fig. 2, treatments are control ± Urate or TNF ± Urate incubated for either 0.5 or 4 h, then assayed for nitrotyrosine immunofluorescence (green) and polymeric F-actin fluorescence (red). In PMEM treated with TNF alone, there is an increase in nitrotyrosine fluorescence compared with the untreated control groups. However, there is no significant increase in yellow color in the combined green and red images compared with the untreated control groups, indicating a lack of colocalization of nitrotyrosine with F-actin. In PMEM treated with Urate, with or without TNF, there is no significant increase in nitrotyrosine fluorescence compared with the respective control groups.


Figure 2
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  		Fig. 2. TNF causes minimal colocalization of nitrotyrosine with F-actin in PMEM. Representative confocal micrographs (bovine cells) of control, TNF-, Urate alone- and TNF+Urate-treated PMEM after 0.5 and 4 h. Nitrotyrosine has been immunostained with antinitrotyrosine and appears as green fluorescence. F-actin fibers have been stained with phalloidin and appear as red fluorescence. The resultant color change of the combined red and green micrographs appears yellow where colocalization occurs. A total of 4 preparations were generated for each treatment and time point from both rat and bovine PMEM.

 
Figure 3A is an experimental control study done to establish the specificity of antinitrotyrosine binding to PMEM. Panel 1 demonstrates the characteristic appearance of nitrotyrosine immunofluorescence, whereas in panel 2, the nitrotyrosine signal is blocked by antibody preincubation with the antigen, 3-nitrotyrosine. Bovine PMEM are represented. Figure 3B illustrates confocal histogram analysis of nitrotyrosine immunofluorescence from digitized micrographs of rat and bovine PMEM control ± Urate or TNF ± Urate treatments. The rat and bovine data have been combined since the effects of TNF are similar between the two species. In PMEM exposed to TNF alone for 0.5 or 4 h, there is an increase in nitrotyrosine fluorescence compared with the respective control groups. In PMEM treated with Urate, with or without TNF, there is no increase in nitrotyrosine fluorescence compared with the respective control. The data of Figs. 1, 2, and 3 indicate that TNF induces an increase in nitrotyrosine that is inhibited by Urate and not associated with F-actin polymers.


Figure 3
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  		Fig. 3. Antinitrotyrosine immunocytochemical specificity and the effect of Urate on the TNF-induced increase in nitrotyrosine. A-1: representative micrograph (bovine cells) of nitrotyrosine fluorescence of PMEM immunostained with monoclonal antinitrotyrosine. A-2: micrograph of PMEM immunostained with the same antibody after preincubation of the antibody with 3-nitrotyrosine for 30 min at a 10:1 antigen-antibody molar ratio. B: confocal histogram analysis of nitrotyrosine fluorescence obtained from both rat and bovine control, TNF-, Urate alone-, or TNF+Urate-treated PMEM after 0.5 or 4 h (n = 4; 6 samplings each per treatment). Statistical difference is determined with Kruskal-Wallis 1-way ANOVA on ranks followed by multiple comparisons using Dunn’s method. *Different from control group; #different from respective TNF group.

 
Extraction of beta-Actin

To extract endothelial actin, we used the specific actin binding characteristic of DNase, because most or all of the beta-actin-specific antibodies are unsuitable for immunoprecipitation. Figure 4A shows a representative Western blot of DNase-Sepharose column fractions retrieved during the purification of actin from PMEM lysate. In Fig. 4A, panel 1, the blot has been stained for total proteins. In the column flow-through (lane 3) the disappearance of a ~43-kDa protein indicates binding of the ~43-kDa protein to the DNase column. The bulk of the 43-kDa protein then reappears following elution with urea buffer (lane 7). The molecular mass of 43 kDa is consistent with the molecular mass of monomeric beta-actin (35). Figure 4A, panel 2, is the same blot as that in Fig. 4A, panel 1, after destaining and immunoprobing for beta-actin. The beta-actin immunoreactivity disappears in the column flow-through fraction (lane 3), which indicates retention in the column, and reappears primarily in the elution fraction (lane 7). The molecular mass of the only immunoreactive band is ~43 kDa, which is consistent with the beta-actin standard (lane 1), confirming the purity of the extracted beta-actin.


Figure 4
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  		Fig. 4. Actin extraction and antinitrotyrosine specificity. A-1: representative Western blot of DNase column fractions retrieved during the purification of actin from PMEM lysate, stained for total protein with Bradford reagent. The lanes are 1) beta-actin standard, 2) lysate, 3) 1st column flow-through, 4–6) column washes, 7) eluate, and 8) Cruz Marker (molecular mass marker). A-2: blot from A-1, destained and immunoprobed with anti-beta-actin antibody (clone AC15). B: matched pair of Western blots (left and right) of DNase column eluates of PMEM lysates. B-1: the blots immunoprobed with polyclonal antinitrotyrosine. Lanes 1 and 7 are Cruz Markers, lanes 3 and 4 are 0.5 and 4 h controls, and lanes 4 and 6 are 0.5 and 4 h TNF treated, respectively. Lanes 8 and 9 are nitrated and native beta-actin standards, respectively, and lane 10 is a nitrotyrosine immunoblotting positive control (Upstate), consisting of peroxynitrite-treated myosin, BSA, and SOD. Lane 2 is blank. B-2: both blots were stripped of probe; the left was reduced in dithionite for 1 h at RT, while the right incubated in the corresponding buffer, after which both were reprobed for nitrotyrosine. B-3: both blots were stripped of probe and then reprobed for beta-actin (clone AC15). The blot pair images were acquired and adjusted for presentation in each panel section simultaneously. The samples for both A and B were obtained from rat PMEM.

 
Eluate Antinitrotyrosine Signal Specificity

To confirm the polyclonal nitrotyrosine antibody signal specificity, we generated a matched pair of Western blots containing DNase column eluates from lysates of control and TNF-treated PMEM, nitrated, and native beta-actin standards, and a nitrotyrosine immunoblotting control (Fig. 4B). One of the pair served as a procedural control, whereas the other was exposed to dithionite. Dithionite causes the reduction of nitrotyrosine to aminotyrosine and thus can be used to demonstrate the specificity of an antinitrotyrosine antibody for nitrotyrosine (5, 6, 11, 15).

Figure 4B, panel 1, shows the result of the initial nitrotyrosine immunoprobe; the primary eluate nitrotyrosine signal is a 43-kDa band, which is consistent with the nitrated beta-actin standard at 43 kDa (lane 8). The nitrated beta-actin (lane 8) and nitrotyrosine immunoblotting control (lane 10) react with the nitrotyrosine antibody, but the native beta-actin (lane 9) does not. The signal from the molecular mass standard (lanes 1 and 7) is entirely dependent on the secondary antibody, not the primary.

Figure 4B, panel 2 (left), shows the effect of dithionite treatment of the blot on the immunoreactivity to nitrotyrosine antibody. Both left and right blots were stripped before treatment; the left blot was incubated in dithionite, and the right blot was incubated in buffer only, after which both blots were reprobed for nitrotyrosine. All of the nitrotyrosine-dependent signal was attenuated in the reduced blot, including the overall background noise from the 5% milk-blocking proteins. The sham-treated blot (right) exhibited an identical pattern and intensity of signal as in its initial image, except for the molecular mass markers, which appear to degrade in both blots with successive strip/reprobes.

To confirm blot integrity and to demonstrate that dithionite affected only the nitrotyrosine signal, both blots were again stripped and then reprobed for beta-actin. Figure 4B, panel 3, reveals that there is little to no difference in the signal intensity or pattern of the eluates or the native beta-actin standard (lane 9) between the left and right blots. Identical results were obtained for the procedures of Fig. 4 with both rat and bovine PMEM lysates; results from rat PMEM lysates are illustrated here.

Nitrotyrosine Quantification

As illustrated in Fig. 4, the DNase eluate is essentially pure beta-actin which is shown to have antigen-specific binding of anti-nitrotyrosine. When a purified protein solution is obtained, the 8 x 12 dot-blot array format becomes much more suitable than a Western blot for immunodetection and quantification against known standards.

Figure 5A is a representative dot-blot assay containing DNase eluate samples from all treatment groups (two dilutions each, in triplicate) as well as a series of dilutions of the nitrated beta-actin standard. The treatments are control ± Urate or TNF ± Urate for 0.5 and 4 h, and the samples were obtained from bovine PMEM. The top panel is the initial antinitrotyrosine probe, and the bottom panel is the same blot, stripped, and then reprobed with anti-beta-actin. The femtomoles of nitrotyrosine and the nanograms of beta-actin in each dot were calculated for the respective immunoprobe by linear regression analysis against the standards. Figure 5B represents the relative degree of nitration of beta-actin derived from dot-blot analysis of DNase eluates from all treatments in both rat and bovine PMEM combined. The units of beta-actin nitration are femtomoles of nitrotyrosine per nanogram of beta-actin, expressed as an experimental/baseline ratio of TNF treatment over the respective treatment control. The baseline levels of beta-actin nitration for the control and Urate control are similar among the 0.5 h and 4 h groups (not shown). After TNF treatment, the degree of beta-actin nitration increases significantly at 0.5 h but not at 4 h. In the Urate ± TNF groups, there is no increase in the degree of beta-actin nitration. The data of Fig. 5 verify that TNF increases the degree of nitration of beta-actin in PMEM that can be prevented by the anti-ONOO– agent Urate.


Figure 5
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  		Fig. 5. Dot-blot assay of PMEM-derived beta-actin and the effect of Urate on the TNF-induced increase in NO2-beta-actin in PMEM. A: representative dot blot of DNase-extracted PMEM beta-actin immunoprobed with polyclonal antinitrotyrosine (top), then stripped and reprobed with monoclonal beta-actin (clone AC74, bottom). The top 2 rows on the blot are nitrated beta-actin standards (25 mmol NO2-tyrosine/mol actin), from 2.5 to 80 ng/dot, as well as buffer blanks. Rows 3–6 contain beta-actin from control, TNF-, Urate alone-, and TNF+Urate-treated PMEM (bovine), respectively, in triplicate at 2 different dilutions. B: nitrotyrosine (fmol)/beta-actin (ng) values were quantified from dot-blot assays of PMEM beta-actin fractions. The relative change with treatment of beta-actin nitration is expressed as treatment (fmol/ng)/treatment control (fmol/ng). The treatments are control ± Urate or TNF ± Urate at 0.5 and 4 h (n = 5–7). Data from both rat and bovine PMEM are included in all treatment groups. A log10 transform was performed on the absolute data followed by an ANOVA and Bonferroni post hoc multiple-comparison test. *Different from respective control group.

 
TNF and Urate Have No Effect on Cellular beta-Actin Concentration

The mean protein concentration of the eluted beta-actin for each treatment group is represented in Fig. 6A. The mean nanograms of beta-actin for each treatment group, measured from the dot-blots and corrected for dilution, are represented in Fig. 6B. The data from both rat and bovine PMEM combined are presented, as no difference was found between the two. Urate and TNF cause no significant change in beta-actin concentration compared with their respective controls. The data of Fig. 6 indicate that TNF and Urate have no effect on the cellular levels of beta-actin in PMEM. The data of Figs. 3–6 support the hypothesis that TNF treatment of PMEM causes an ONOO–-mediated increase in nitrated beta-actin that is independent of the total beta-actin concentration.


Figure 6
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  		Fig. 6. TNF and/or Urate do not affect beta-actin concentration or anti-beta-actin binding in PMEM. A: bar graph representation of beta-actin protein concentration (ng/µl) of DNase-Sepharose column eluates of PMEM lysates for all treatment groups and times posttreatment (n = 5). Proteins were measured in 3x diluted sample using the enhanced BCA assay protocol against BSA/IgG (1:1) standards. B: bar graph representation of nanograms of beta-actin derived from dot-blot immunoprobe analysis (monoclonal anti-beta-actin, clone AC-74) of DNase eluates from all treatment groups and times posttreatment (n = 5–7). Combined data from both rat and bovine PMEM were used.

 
Urate Prevents TNF-Induced Increases in Albumin Clearance Rate

Figure 7 shows the permeability response of PMEM control ± Urate or TNF ± Urate for 0.5 and 4 h. The rat and bovine data are combined because the effects of TNF are similar between the two groups. In the group exposed to TNF for 0.5 h, there is a significant increase in albumin clearance rate compared with the 0.5-h control group. In the group exposed to TNF + Urate for 0.5 h, there is no change in albumin clearance rate compared with the respective Urate-alone group. Similarly, in the group exposed to TNF for 4 h, there is a significant increase in albumin clearance rate compared with the 4-h control group. In the group exposed to TNF + Urate for 4 h, there is no change in albumin clearance rate compared with the respective Urate-alone group. The data of Fig. 7 support the notion that ONOO– mediates the increase in permeability in response to TNF in PMEM.


Figure 7
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  		Fig. 7. Urate prevents TNF-induced increases in albumin clearance rate in PMEM. The albumin clearance response of combined data obtained from rat and bovine PMEM. The treatments are control ± Urate or TNF ± Urate for 0.5 and 4 h. Statistical difference is determined with Kruskal-Wallis 1-way ANOVA on ranks followed by multiple comparisons using Dunn’s method. *Different from control group; #different from TNF group.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, TNF induces an increase in endothelial permeability and the generation of nitrotyrosine, which is prevented by the anti-ONOO– agent Urate. Thus the present data support the hypothesis that TNF induces generation of ONOO–, which mediates the increase in protein permeability of PMEM. Urate is a classic scavenger of ONOO– (15, 39) that we have previously shown prevents the TNF-induced activity of ONOO– in PMEM and in isolated lungs (30, 21). The issue of specificity of scavengers such as Urate for quenching the effects of reactive nitrogen/oxygen species (i.e., ·O2–, ·OH, and ·NO) is noted; however, a role for ONOO– is indicated because ·O2– is a primary reactant for generation of ONOO– (5, 6, 15) and ·OH is a metabolite of ONOO– (5, 6, 15). In addition, our previous studies indicate that inhibition of ·O2– and ·NO activity has similar protective effects on barrier function, which supports the notion that the reaction ·O2– + ·NO -> ONOO– participates in the effect of TNF (8, 14, 16, 30). Finally, the responses to TNF and Urate are not due to changes in cell viability because we have previously shown that Urate, either in the absence or presence of TNF, does not affect trypan blue exclusion or cell density (30).

Our previous studies using bovine pulmonary artery endothelium indicate that Urate and the ·O2– scavengers superoxide dismutase and tiron prevent the generation of ONOO– that should occur in response to TNF (14, 30). In the in situ isolated guinea pig lung, we have previously shown that Urate prevents the edemagenic effect of ONOO– (21). Other laboratories clearly demonstrate evidence for ONOO–-mediated lung injury, endothelial dysfunction, and the protective effects of Urate and other anti-·O2– agents on the responses to ONOO–-generating systems (6, 9, 11, 15). The present data support the finding of Knepler et al. (23), indicating that direct application of exogenous ONOO– will increase endothelial permeability; however, the role of endogenously produced intracellular ONOO– on barrier function during a pathophysiological model of endothelial injury is not known. Thus this study importantly demonstrates ONOO– as a likely mediator for TNF-induced barrier dysfunction.

In the present study, TNF increases the degree of nitrotyrosine associated with beta-actin. Our previous data, using bovine endothelium, indicated that TNF caused generation of 1) ONOO–, 2) nitrotyrosine, and 3) oxidized reactants (i.e., p42-carbonyls and GSSG) (14, 30). The TNF-induced nitrotyrosine, p42-carbonyls, and GSSG are not formed following treatment with anti-ONOO–, ·NO, and/or ·O2– agents (14, 30). Presently, Urate prevents both the generation of nitrotyrosine and its association with beta-actin; thus the present study demonstrates significant nitration of beta-actin is likely mediated by ONOO–. The role of peroxidase-mediated nitration is not apparent in the present study because peroxidase-mediated nitration should be independent of ONOO– (40). The present data, shown by the extracted beta-actin and fluorescent imaging of beta-actin, indicate a transient increase in the nitrated beta-actin 0.5 h after TNF. The turnover of NO2-beta-actin (i.e., return to baseline NO2-beta-actin levels by 4 h of TNF) indicates possible denitrification (i.e., NO2-beta-actin + H2O -> H-beta-actin + H+ + NO3–) of the NO2-beta-actin and/or increased ubiquitin-proteasome degradation of NO2-beta-actin (24, 25).

Although not addressed in the present study, the literature indicates at least three potential target residues of actin, tyrosine-69, tyrosine-198, and tyrosine-302, that could influence nucleotide binding and myosin activation (22, 28, 35). Importantly, our research, now in progress, indicates that we can use a mutant enhanced yellow fluorescent protein (EYFP)-beta-actin to study the actin’s response to TNF. We expressed an EYFP-beta-actin that had a base mutation from tyrosine (Y-198) to phenylalanine (F-198). In preliminary studies, after 0.5 h of TNF exposure, the albumin clearance rate was less in the MUTANTF-198 EYFP-beta-actin group compared with the untransfected group (0.24 ± 0.03 ml/min < 0.31 ± 0.04 ml/min, P < 0.05, respectively) but similar in the wild-type EYFP-beta-actin to the untransfected group (0.29 ± 0.04 µl/min {cong} 0.31 ± 0.04 µl/min, respectively). Thus our present studies are actively investigating the role of actin-tyrosine residues in the TNF-mediated barrier response.

The literature indicates compelling mechanisms for the barrier dysfunction that occurs in response to ONOO-mediated nitration of tyrosine in beta-actin. Importantly, the present data indicate that the NO2-beta-actin does not incorporate into beta-actin polymers because we detect minimal colocalization of the nitrotyrosine with the F-actin. Thus the potential exists for the NO2-beta-actin to disrupt the cycling of the beta-actin monomers into the growing barbed ends of the native F-actin polymers, which alters actin-polymerization dynamics and ultimately barrier function (31, 38). There may be altered binding of the NO2-beta-actin with the zonular adherence (e.g., a-catenin) and/or zonula occludin (e.g., ZO-1) proteins resulting in decreased cell-cell adherence (23). The nitration of tyrosine in the beta-actin can alter phosphorylation events for the same target tyrosine which regulate beta-actin polymerization (3). Finally, the activity of nitrated actin is integrated with the effects of nitrotyrosination of other proteins including a-tubulin, which may cause altered cytoskeletal homeostasis (11). Finally, the present study indicates that other ONOO–-mediated events may contribute to the increased endothelial permeability because there is increased permeability 4 h after TNF despite the return to the control levels for nitrated beta-actin. ONOO– is implicated in cell injury via pathways involved with glycolysis, mitochondrial enzyme function, DNA injury and repair, and transcription factor activity (15, 39).

In summary, our study indicates that TNF induces the dysfunction of the endothelial barrier, which is dependent on ONOO– generation. The response to TNF is associated with nitration of beta-actin. The development of strategies that target on-going ONOO– activity and associated NO2-beta-actin may provide novel directions for therapy of vascular injury.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the Department of Veterans Affairs Research Incentive Fund Program (A. Johnson), Charitable Leadership Foundation Medical Technology Acceleration Program, Inc. (A. Johnson), National Heart, Lung, and Blood Institute Grant RO1-HL-59901 (A. Johnson), and the Ronald McNair Program of the University at Albany State University of New York (R. Woodburn and W. Lambert).


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Johnson, 151, 113 Holland Ave., Dept. of Veterans Affairs Medical Center, Albany, NY 12208 (e-mail: jmurd{at}msn.com, arnold.johnson{at}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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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