Nitric oxide exposure inhibits endothelial NOS activity but not gene expression: a role for superoxide

A. Macduff Sheehy, Michael A. Burson, and Stephen M. Black

Department of Pediatrics, University of California, San Francisco, California 94143-0106

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Recent studies have characterized a rebound pulmonary vasoconstriction with abrupt withdrawal of inhaled nitric oxide (NO) during therapy for pulmonary hypertension, suggesting that inhaled NO may downregulate basal NO production. However, the exact mechanism of this rebound pulmonary hypertension remains unclear. The objectives of these studies were to determine the effect of NO exposure on endothelial NO synthase (eNOS) gene expression, enzyme activity, and posttranslational modification in cultured pulmonary arterial endothelial cells. Sodium nitroprusside (SNP) treatment had no effect on eNOS mRNA or protein levels but did produce a significant decrease in enzyme activity. Furthermore, although SNP treatment induced protein kinase C (PKC)-dependent eNOS phosphorylation, blockade of PKC activity did not protect against the effects of SNP. When the xanthine oxidase inhibitor allopurinol or the superoxide scavenger 4,5-dihydroxy-1-benzene-disulfonic acid were coincubated with SNP, the inhibitory effects on eNOS activity could be partially alleviated. Also, the levels of superoxide were found to be elevated 4.5-fold when cultured pulmonary arterial endothelial cells were exposed to the NO donor spermine/NO. This suggests that NO can stimulate xanthine oxidase to cause an increase in cellular superoxide generation. A reaction between NO and superoxide would produce peroxynitrite, which could then react with the eNOS protein, resulting in enzyme inactivation. This mechanism may explain, at least in part, how NO produces NOS inhibition in vivo and may delineate, in part, the mechanism of rebound pulmonary hypertension after withdrawal of inhaled NO.

enzyme inhibition; protein; phosphorylation; nitric oxide synthase

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

ENDOTHELIAL CELLS synthesize nitric oxide (NO) and L-citrulline from L-arginine; this reaction is catalyzed by the enzyme NO synthase (NOS; see Ref. 30). There are three isoforms of NOS that differ in structure and regulation. One of the isoforms is found only in endothelial cells (eNOS) and is membrane associated due to the presence of a posttranslational modification that results in N-myristoylation of the glycine residue at position two (39). The NO produced by eNOS diffuses from endothelial cells into smooth muscle cells, activates soluble guanylate cyclase, increases the production of cGMP, and initiates a cascade, resulting in smooth muscle relaxation (18). In vivo, NO is labile, with a biological half-life from 111 to 130 ms depending on the surrounding O2 concentration.

Inhaled NO is also now used as a selective pulmonary vasodilator for a number of pulmonary vasculature disease states (23). In animals, inhaled NO (5-80 ppm) reverses pulmonary hypertension induced by alveolar hypoxia, thromboxane-mimetic infusion, or heparin-protamine interactions. The pulmonary vasodilation is rapid, completely reversible, and selective, with no systemic vasodilation or other adverse effects. Inhaled NO selectively decreases pulmonary arterial pressure and pulmonary vascular resistance in patients with congenital heart disease (35) and improves oxygenation in newborns with persistent pulmonary hypertension (36). However, a number of studies have observed an acute and potentially life-threatening increase in pulmonary vascular resistance with acute withdrawal of inhaled NO. In children with congenital heart disease, this is manifested by an increase in pulmonary vascular resistance that may compromise cardiac output (2, 25). In newborns with persistent pulmonary hypertension, this is manifested by a sudden decrease in systemic arterial oxygen saturation. These manifestations of acute NO withdrawal suggest that inhaled NO may suppress endogenous NO production, but the mechanism of NO-induced inhibition of NO generation is unknown. Thus long-term studies needed to be carried out in endothelial cells to determine the effect on eNOS activity, mRNA, and protein levels. The effects on mRNA and protein levels may be important, since it is known that NO can alter transcription rates in a variety of genes (3, 28, 32, 44). Our experiments were designed to determine the effects of the NO donor compound sodium nitroprusside (SNP) on eNOS gene expression and activity in endothelial cells prepared from the main pulmonary artery of late-gestation fetal sheep.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cloning of ovine eNOS cDNA probe and generation of eNOS specific polyclonal antiserum. There is substantial homology between the three isoforms of NOS. Therefore, to ensure sensitivity and specificity for subsequent hybridization experiments, ovine cDNA fragments of the eNOS were cloned. A 680-bp region of minimum homology (encoding the heme binding domain) was identified by comparing the bovine endothelial, murine macrophage, and rat brain NOS cDNAs. Oligonucleotides used were as follows: sense, 5'-CCTCCGGAGGGGCCCAAGTTCCCTCGC-3' and antisense, 5'-CACGTCGAAGCGGCCGTTTCCGGGGGT-3'. RT-PCR was then performed using established procedures. The substrate for the random-primed RT reactions was total RNA prepared by acid-phenol extraction of fetal lamb aorta. Subsequent PCR amplification generated the expected 680-bp fragment, and this was cloned into the pCRII vector (Invitrogen, San Diego, CA). Several clones were sequenced on both strands to verify its origin from the correct NOS mRNA and to ensure that no PCR or cloning artifact had occurred.

Isolation of ovine main pulmonary arterial endothelial cells. After the death of a 139-day fetal sheep, the main pulmonary artery was dissected free, the adventitia was removed, and the exterior surface was rinsed with 70% ethanol. The vessel was then opened longitudinally, and the interior was rinsed with PBS to remove any blood. The endothelium was then lightly scraped away, placed in DMEM-H-16 supplemented with 10% fetal bovine serum, basic fibroblast growth factor (10 ng/ml), antibiotics (penicillin, streptomycin), and antimycotics (Fungizone), and the cells were incubated in 21% O2-5% CO2 at 37°C. After a few days in culture, moderate-sized aggregates of endothelial cells were transferred using a micropipette, grown to confluence, and maintained in culture.

For experiments, endothelial cells were transferred to serum-free supplemented DMEM-H-16 medium. Cells were then treated for 0-24 h with SNP (1 mM) to examine changes in eNOS mRNA, protein, and activity. For experiments examining the role of protein kinase C (PKC) activation by SNP, cells were pretreated for 2 h with staurosporine (2.5 µM) before the addition of SNP. To determine the role of xanthine/xanthine oxidase activity or superoxide, ovine fetal pulmonary arterial endothelial cells (FPAECs) were exposed to SNP (1 mM) for 12 h in the presence of allopurinol (100 µM; see Ref. 47) or 4,5-dihydroxy-1-benzene-disulfonic acid (Tiron, 10 mM; see Ref. 20), respectively.

Determination of cell viability. An equal volume of trypan blue (0.4%) was added to ovine FPAECs suspended in PBS and mixed thoroughly. The suspension was allowed to stand for 10 min at room temperature, and then viable cells were determined using a hemocytometer.

RNA isolation and analysis. RNA was isolated from SNP-treated ovine FPAECs by using acid-phenol extraction (11) and were analyzed by RNase protection. RNase protections were carried out as described (6) using our specific ovine eNOS cDNA probe. Single-stranded antisense cRNA probes were synthesized, and 20 µg of total RNA were hybridized overnight to 500,000 counts/min probe in 80% formamide-40 mM PIPES-0.4 M NaCl-1 mM EDTA at 42°C. This was followed by digestion with 2.5 units of an RNase A-T1 mixture (Ambion) followed by phenol-CHCl3 extraction and ethanol precipitation. Protected fragments were then separated by electrophoresis on a DNA sequencing gel and exposed to film at -70°C. A 180-bp ovine 18S ribosomal RNA probe was included as a control for input RNA and recovery.

Western blot analysis. SNP-treated ovine pulmonary arterial endothelial cells were scraped into ice-cold PBS supplemented with protease inhibitors, centrifuged, resuspended, and sonicated. Protein estimation was carried out using the Bradford reagent. Total protein (25 µg) was separated on a 7.5% SDS-polyacrylamide gel (Bio-Rad) and electrotransferred to nitrocellulose in 20% methanol, 25 mM Tris · HCl (pH 8.3), and 192 mM glycine. Western blotting using our rabbit anti-ovine eNOS antibody (40) was performed using standard procedures (5). Western blotting results were confirmed using a commercially available anti-eNOS monoclonal antibody (Transduction Laboratories).

eNOS cellular localization. To examine the subcellular localization of the eNOS protein, ovine pulmonary arterial endothelial cells were exposed to SNP (1 mM) for 4 h and then were harvested, and the cells were disrupted by sonication. Debris and unbroken cells were then sedimented at 1,000 g, and the supernatant was recentrifuged at 100,000 g to produce a soluble cytosolic fraction and a membrane-particulate fraction. Extracts (50 µg) were then analyzed by Western blotting, as detailed above.

NO determination. For measurement of NO release, cell culture medium (10 µl) from ovine FPAECs exposed to SNP (1 mM) for 0-24 h was injected into a nitrogen-purge vessel containing vanadium (III) in hydrochloric acid to liberate all of the NO metabolites as gaseous NO. The sample gas was exposed to ozone to form activated nitrogen dioxide, which luminesces in the red and far red spectrum, and was detected by chemiluminescence (NOA 280; Sievers Instruments, Boulder, CO). For each sample, the area under the curve was converted to micromolar NO using a calibration curve from sodium nitrite standards.

Assay for NOS activity. The NOS activity in SNP-treated ovine pulmonary arterial endothelial cells was determined by measuring the formation of [3H]citrulline from [3H]arginine (4, 9). Briefly, subcellular fractions (100 µg) were incubated at 37°C for 45 min in the presence of 1 mM NADPH, 14 µM tetrahydrobiopterin, 5 µM FAD, 1 mM MgCl2, 10 µM unlabeled L-arginine, and 0.1 µCi of [3H]arginine and in the presence or absence of other cofactors (calmodulin and calcium). The reaction was stopped by the addition of iced stop buffer (20 mM sodium acetate, pH 5, 1 mM L-citrulline, 2 mM EDTA, and 0.2 mM EGTA). Samples were applied to columns containing 1 ml of Dowex AG50W-X8 resin, Na+ form, preequilibrated with 1 N NaOH. [3H]citrulline was then quantitated by scintillation counting. For all cell treatments, the effects were compared with untreated cells using ANOVA. P < 0.05 was considered statistically significant (46).

eNOS protein phosphorylation. Ovine pulmomary arterial endothelial cells were incubated for 4 h in phosphate-free growth medium containing 80 µCi of [32P]orthophosphoric acid per milliliter of medium. The cells were then treated with 1 mM SNP or vehicle alone for a further 2 h. The cells were scraped into immunoprecipitation buffer (150 mM NaCl, 50 mM Tris · HCl, pH 7.4, 0.1 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, and 50 mM NaF) plus protease inhibitors. The cells were sonicated and immunoprecipitated using an eNOS antiserum, and the immune complexes were isolated using protein A-Sepharose and eluted in boiling SDS-PAGE loading dye. The phosphorylation status of the eNOS was then analyzed by separation on a 7.5% SDS-polyacrylamide gel followed by autoradiography.

Cytochrome c reduction. Superoxide-induced reduction of ferricytochrome c to ferrocytochrome c was carried out according to the method of Brune et al. (7). Cells (1 × 105) were plated in 10-cm dishes and allowed to adhere, and then the medium was replaced with phenol red-free DMEM containing 0.5% fetal calf serum. Cells were then incubated for 1 h in the presence of 40 µM cytochrome c in the presence or absence of the NO donor spermine/NO (1 mM). Medium with cytochrome c served as a control.

Data analysis. Quantitation of autoradiographic results was performed by scanning (Hewlett-Packard SCAN-Jet IICX; Hewlett-Packard, Palo Alto, CA) the bands of interest into an image-editing software program (Adobe Photoshop; Adobe Systems, Mountain View, CA). Band intensities from RNase protection assays and Western blot analysis were analyzed densitometrically on a Macintosh computer (model 9500; Apple Computer, Cupertino, CA) using the public domain National Institutes of Health (NIH) Image program (developed at the NIH and available on the Internet at http://rsb.info.nih.gov/nih-image; see Ref. 33). ANOVA was used to determine any significant changes in densitometric values. P < 0.05 was considered statistically significant (46).

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Reduction in eNOS activity by SNP in ovine FPAECs. Preliminary experiments were undertaken with ovine FPAECs to determine the effect of SNP on the activity of eNOS. To determine the time course of NO release from SNP (1 mM), we measured the levels of NO released into the culture medium. We measured a time-dependent increase in NO accumulation in the culture medium, although the rate of NO release was significantly higher at 2-4 h than at 8-24 h (P < 0.05; Fig. 1A). Subsequently, the effect of SNP treatment on the activity of eNOS in ovine FPAECs was determined (Fig. 1B). SNP treatment significantly inhibited NOS activity in ovine FPAECs by 2 h of exposure (P < 0.05). There was a further significant drop in activity at 4 h (P < 0.05) and no further significant reduction for the duration of the experiment (24 h). These results confirm that SNP decreases eNOS activity in pulmonary arterial endothelial cells and that the reduction in NOS activity correlates with the rate of NO release. Thus the higher rate of NO released at 2-4 h produces a significantly greater inhibition of eNOS than does the lower rate of NO release at 4-24 h. To determine if the reduction in eNOS activity was due to a cytotoxic effect of SNP treatment, we determined the number of viable ovine FPAECs over the course of a 24-h SNP treatment. The results obtained (Fig. 1C) indicated that there was no significant difference in the numbers of viable cells over the SNP time course. Thus a reduction in cell viability could not account for the reduction in eNOS activity induced by SNP treatment.


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Fig. 1.   Nitric oxide synthase (NOS) activity in ovine pulmonary arterial endothelial cells treated with sodium nitroprusside (SNP). Cells were treated for varying times with SNP (1 mM). Culture medium from SNP-treated cells was analyzed to determine nitric oxide (NO) levels (A). Cells were also harvested, and the NOS activity was determined using the conversion of [3H]arginine to [3H]citrulline (B), or cell viability was determined using trypan blue exclusion (C). A: NO release from cells treated with 1 mM SNP was measured in the culture medium using a gas-phase chemiluminescent reaction between NO and ozone and detected using a model 280 NO analyzer. Rate of NO release is presented as µM/h and is corrected for the background of nitrites present in the culture medium. Data represent means ± SD of 3 separate experiments carried out in triplicate. There is a time-dependent increase in NO detected in the culture medium but a decrease in the rate of NO release. * P < 0.05 vs. 2-h treatment. # P < 0.05 vs. 4-h treatment. B: NOS activities are presented as a percentage ± SD of untreated cells (0 h). Data represent means of 4 separate experiments carried out in triplicate. * P < 0.05 vs. untreated cells. # P < 0.05, 2-h vs. 4-h SNP treatment. C: cell counts are expressed as a percentage ± SD of viable cells compared with untreated cells (0 h). Data represent means of 3 separate experiments carried out in duplicate. SNP treatment caused no significant change in the number of viable pulmonary arterial endothelial cells, P > 0.05.

Effect of SNP treatment on eNOS mRNA and protein expression. It is known that NO can repress the expression of gonadotropin-releasing hormone (GnRH; see Ref. 3) and the genes involved in iron homeostasis (44). Thus the effect of SNP on eNOS activity could be due to a repression of eNOS gene expression, resulting in an overall decrease in eNOS protein. To examine this possibility, ovine FPAECs were treated for up to 24 h with SNP, and eNOS mRNA and protein expression were evaluated by RNase protection and Western blotting, respectively. Results obtained demonstrated that SNP had no effect on either eNOS mRNA (Fig. 2A) or protein expression (Fig. 2B). Densitometric analysis of the bands representing eNOS mRNA and protein (Fig. 2C) from five independent experiments allowed quantitation of the relative band intensities. This analysis indicated that SNP treatment left eNOS mRNA and protein levels unaffected. Thus SNP does not alter eNOS mRNA or protein expression in ovine FPAECs, and thus an effect on eNOS gene expression is not responsible for the loss of NOS activity.


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Fig. 2.   Endothelial NOS (eNOS) mRNA and protein expression in ovine pulmonary arterial endothelial cells treated with SNP. Cells were treated with SNP (1 mM) for 0-24 h, and either RNA or protein was isolated. Representative RNase protection (A) and Western blot (B) are shown. For RNase protection analysis, a cRNA probe was prepared for ovine eNOS and hybridized overnight to total RNA (20 µg) prepared from ovine fetal pulmonary artery endothelial cells (FPAECs) treated for 0-24 h with 1 mM SNP. There is no change in eNOS expression on SNP treatment (A). No protected fragments are detected in the lane in which the probe was hybridized without RNA (PD) or in the presence of yeast tRNA (20 µg). The 18S ribosomal RNA band indicates that there was even loading in each lane. Protein extracts (25 µg) from the SNP-treated cells were separated on a 7.5% SDS-polyacrylamide gel and analyzed by Western blotting using our polyclonal eNOS antiserum (B). These experiments were carried out 5 times, and the autoradiographic bands from each experiment were analyzed using National Institutes of Health (NIH) Image 1.6 (C). Results obtained confirm that SNP treatment does not significantly reduce either eNOS mRNA or protein levels (P > 0.05).

Effect of SNP treatment on posttranslational modifications of eNOS. The eNOS protein can be posttranslationally modified in at least two ways; alterations can be induced in its subcellular localization or phosphorylation status. Thus we determined if SNP treatment alterations in either the subcellular localization or phosphorylation of the eNOS protein in ovine FPAECs could account for the decrease in enzyme activity.

Treatment of endothelial cells with certain agonists can cause the eNOS enzyme to be translocated from the membrane to the cytosol (37). This effect is believed to be due to depalmitoylation of the enzyme (37), although the effect on eNOS activity remains unclear. To determine if SNP had this effect, ovine FPAECs were treated for 4 h in the presence of 1 mM SNP before producing a soluble cytosolic fraction and a membrane-particulate fraction by differential centrifugation. Results of Western blotting (Fig. 3A) and densitometric analysis (Fig. 3B) from four independent experiments demonstrated that SNP did not significantly alter the subcellular localization of eNOS in ovine FPAECs.


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Fig. 3.   Posttranslational modification of eNOS protein in ovine pulmonary arterial endothelial cells treated with SNP. To determine the effect of SNP exposure on the subcellular localization of eNOS protein, cytosolic and membrane fractions (50 µg) prepared from ovine FPAECs treated (SNP) or not (basal) with SNP (1 mM, 2 h) were separated on a 7.5% SDS-polyacrylamide gel and subjected to Western blot analysis using our polyclonal eNOS antiserum (A). This experiment was carried out 4 times, and the autoradiographic bands from each experiment were analyzed using NIH Image 1.6 (B), confirming that SNP treatment does not significantly alter the subcellular localization of the eNOS protein (P > 0.05). To determine the effect of SNP exposure on the phosphorylation status of eNOS protein, ovine FPAECs were labeled for 4 h with [32P]orthophosphoric acid and then treated for 2 h with either vehicle alone (untreated), with 1 mM SNP, or with a combination of 1 mM SNP and 2.5 µM staurosporine (SNP/Stau). Cells were then either used for immunoprecipitation (C) with our eNOS-specific antiserum or harvested, and NOS activity was determined using the conversion of [3H]arginine to [3H]citrulline (D). Immunoprecipitation data are representative of an experiment that was carried out 5 times with the same result. Activity data represent means ± SD of 4 separate experiments carried out in triplicate. Presence of staurosporine does not significantly alter the inhibition of eNOS caused by SNP (P > 0.05).

Because SNP did not affect eNOS subcellular localization, we then determined whether SNP-induced phosphorylation was responsible for the SNP inhibitory effect on eNOS activity. Ovine FPAECs grown for 4 h in the presence of [32P]orthophosphoric acid were treated with SNP, and cellular extracts were immunoprecipitated with our specific eNOS antiserum. The results obtained (Fig. 3C) demonstrated that the eNOS protein in ovine FPAECs is phosphorylated in the presence of SNP, whereas phosphorylated eNOS is absent in untreated cells. Also, this SNP-induced phosphorylation is PKC dependent, since the PKC inhibitor staurosporine blocks the phosphorylation event. We then determined if the phosphorylation of eNOS induced by SNP treatment was responsible for the observed decrease in NOS enzyme activity in ovine FPAECs. NOS activity was determined in the presence or absence of staurosporine and compared with untreated control cells. The results obtained from four independent experiments showed that staurosporine treatment did not alter the ability of SNP to inhibit eNOS activity (Fig. 3D), although it did prevent phosphorylation of the protein itself (Fig. 3C). Thus, although SNP treatment induces eNOS phosphorylation, this does not appear to be responsible for the decrease in eNOS activity.

Recovery of eNOS activity on SNP removal. We wished to determine the long-term effect of SNP treatment on eNOS activity. Thus ovine FPAECs were treated for 8 h, and the medium was changed to remove the SNP. The cells were then allowed to recover for 0-24 h, and the cellular NOS activity was determined and compared with untreated cells. The results obtained (Fig. 4) from three independent experiments demonstrated that SNP treatment had a long-lasting inhibitory effect on eNOS activity. After 24 h, the NOS activity present within the ovine FPAECs was only at 81.4 ± 7.9% of untreated levels. This long recovery time after SNP removal suggests that the effect of SNP on eNOS activity is irreversible.


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Fig. 4.   Recovery of eNOS activity in SNP-treated ovine pulmonary arterial endothelial cells. Cells were treated for 8 h with SNP (1 mM) and then were allowed to recover for up to 24 h before harvest. NOS activity was then estimated using [3H]arginine to [3H]citrulline conversion. Activities are presented as a percentage of untreated cells (0 h). Data represent means ± SD of 3 separate experiments carried out in triplicate. * P < 0.05 vs. no recovery.

Effect of reducing cellular superoxide levels on eNOS activity in the presence of SNP. Previous work has shown that cellular superoxide levels can be induced by NO (27). This superoxide is then able to react with NO to form peroxynitrite, a powerful oxidizing agent. Peroxynitrite can react with a variety of cellular macromolecules, including proteins, and can produce enzyme inactivation. Thus, to determine if the effect of NO on eNOS activity was via a reaction with superoxide, we treated ovine FPAECs with SNP in the presence of the superoxide scavenger Tiron (20). Also, since the xanthine/xanthine oxidase system can produce superoxide, we evaluated its potential role using the specific inhibitor allopurinol (47). The results obtained (Fig. 5) indicated that either a reduction in superoxide generation or an increase in superoxide removal resulted in less SNP-mediated inhibition of eNOS activity, although there was still a significant reduction in eNOS activity compared with untreated cells. This indicates that, at least in part, the inhibitory effect of SNP appears to be mediated through superoxide generation by the xanthine/xanthine oxidase pathway and perhaps peroxynitrite formation. Finally, we determined if NO exposure increased the levels of superoxide within ovine FPAECs. Cells were treated with the NO donor spermine/NO, and the superoxide levels were estimated using the reduction of ferricytochrome c to ferrocytochrome c. After 1 h of exposure to spermine/NO the superoxide levels in the ovine FPAECs was 4.5-fold higher (P < 0.05) compared with untreated control cells (Fig. 6). This indicates that NO can stimulate enzyme systems within the cell to increase the production of superoxide, at least one of which appears to be the xanthine/xanthine oxidase system.


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Fig. 5.   Effect of superoxide scavenging and xanthine oxidase inhibition on eNOS activity in the presence of SNP. Cells were exposed to SNP (1 mM) for 12 h in the presence or absence of allopurinol (100 µM) or 4,5-dihydroxy-1-benzene-disulfonic acid (Tiron; 10 mM) and then harvested. NOS activity was then estimated using [3H]arginine to [3H]citrulline conversion. Activities are presented as a percentage of untreated cells (0 h). Data represent means + SD of 4 separate experiments carried out in triplicate. * P < 0.05 vs. SNP treated. # P < 0.05 vs. untreated.


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Fig. 6.   Effect of NO on cellular superoxide levels. Cells (1 × 105) were allowed to adhere and then were switched to phenol red-free DMEM containing 0.5% fetal calf serum. Subsequently, the cells were either exposed (SPER/NO) or not (untreated) to the NO donor spermine/NO (1 mM) in the presence of ferricytochrome c (40 µM). Superoxide levels were measured using the cytochrome c reduction assay. Superoxide levels are expressed as nmol · min-1 · 105 cells-1. Data represent means ± SD of 6 separate experiments. * P < 0.05 vs. untreated.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

The purpose of this study was to investigate the effects of the NO donor SNP in cultured ovine FPAECs. SNP was found to be a potent inhibitor of eNOS activity. SNP could reduce eNOS activity in these cells to a significant level within 2 h of treatment. We wished to determine how this effect was occurring. NO has been shown to affect the transcriptional regulation of a number of genes, including induction of c-fos (28) and c-jun (32), and repression of GnRH (3) and the genes involved in iron homeostasis (44). Thus we determined if NO was causing the reduction in eNOS enzyme activity by inhibiting the expression of the eNOS gene. However, when eNOS expression was examined, using RNase protections and Western blotting to determine RNA and protein levels, respectively, no changes were detected. Densitometric scanning indicated that a 24-h treatment with 1 mM SNP left eNOS mRNA and protein levels unchanged. Thus our results demonstrate that NO acts, in cultured endothelial cells, to reduce the catalytic activity of the eNOS protein without altering the transcription or translation of the eNOS gene.

One possible mechanism by which SNP treatment could have resulted in an inhibition of eNOS activity was by inducing phosphorylation of the enzyme. Previously, it has been shown that eNOS activity can be reduced by 30% by PKC-dependent phosphorylation (15). This has been shown to occur in bovine aortic endothelial cells and with purified eNOS protein (15). Immunoprecipitation experiments on our ovine pulmonary arterial endothelial cells exposed to [32P]orthophosphoric acid and SNP did indicate a PKC-dependent phosphorylation of eNOS. This phosphorylation could be blocked by the specific PKC inhibitor staurosporine. However, when enzyme activity was determined, no significant difference between cells exposed to SNP alone and those exposed to SNP and staurosoporine could be detected. Thus we concluded that PKC-induced phosphorylation of eNOS was not responsible for the reduction in eNOS activity.

Because SNP-induced PKC phosphorylation of eNOS did not account for the reduction in eNOS activity, we further determined recovery time required for eNOS activity after SNP-induced enzyme inhibition. The data demonstrated that eNOS activity is slow to recover in ovine FPAECs. Significant recovery of NOS activity required 24 h. This suggests that NOS activity returns only after new protein is synthesized. Because NOS is a hemoprotein and NO can react with metal-containing proteins (especially hemoproteins) at rates limited only by diffusion (10, 12), the heme domain of eNOS may represent a target for NO-induced inhibition. It has been speculated that NOS may be inhibited by the NO produced during catalysis of L-arginine, and a few reports support this hypothesis. Exogenously added NO or NO-releasing compounds have been shown to inhibit the formation of citrulline from crude preparations of NOS from bovine cerebellum (38), rat alveolar macrophages (14), and bovine aortic endothelial cells (8). This inhibition has more recently been shown to occur with purified rat cerebellar NOS (13). These inhibitory effects may be due to the formation of a tight complex between NO and the heme moiety (17). NO has also been demonstrated to inhibit cytochrome P-450-catalyzed reactions (21, 41, 45). Similar studies on the neuronal and inducible NOS isoforms have indicated that the inhibition of these enzymes with NO donors is reversible for the neuronal enzyme (38), whereas the inhibition is irreversible for the inducible isoform (1). The exact effect of NO donors on eNOS has not been determined, but the long lag time between SNP removal and recovery of NOS activity suggests that NO has an irreversible inhibitory effect on the eNOS protein.

Although these results demonstrated that SNP could inhibit eNOS activity, the mechanism remained unclear. Recent studies in rabbits chronically exposed to organic nitrates have shown an attenuated vasodilator response and an associated increase in superoxide production (27). It was suggested that the NO released from the organic nitrate treatment affects eNOS activity by increasing cellular concentrations of superoxide anion (27). To determine if this mechanism could account for the effect of SNP in our ovine FPAECs, we treated the cells with SNP in the presence of either a xanthine oxidase inhibitor or a superoxide scavenger. The results obtained showed that the presence of either of these agents in the culture medium produced a significant protection against the inhibitory effects of SNP on eNOS activity. Neither agent gave a significantly greater protective effect than the other. The superoxide anion can be directly toxic, but its oxidant reactivity is limited compared with other free radicals (43). However, NO contains an unpaired electron, is paramagnetic, and can react rapidly with superoxide to form peroxynitrite. Peroxynitrite is a strong oxidizing agent and can react readily with biological molecules, including protein and nonprotein sulfhydryls, DNA, and membrane phospholipids (22). Peroxynitrite is capable of nitrating free or protein-associated tyrosines and other phenolics (19). This suggests a mechanism in which the inactivation of eNOS by SNP in cultured ovine FPAECs is via peroxynitrite formation due to the reaction of NO with superoxide. This increase in peroxynitrite can then lead to the oxidation of amino acids within the eNOS protein that are critical for enzyme activity. When we measured the superoxide levels in ovine FPAECs exposed to NO, there was a 4.5-fold increase in superoxide production compared with untreated cells.

Vascular endothelial cells have been shown to generate superoxide both under basal (29) and stimulated (24, 42) conditions, although the sources of this superoxide remain unclear. There are a variety of potential sources for the incresed superoxide production detected in SNP-treated ovine FPAECs. We have demonstrated that the xanthine/xanthine oxidase system appears to be one such pathway. However, although both the inhibition of xanthine oxidase and scavenging of superoxide anions reduced the inhibitory effect of SNP on eNOS activity, there was still a significant level of NOS inhibition compared with untreated cells. This indicates that the inhibitory effect of NO on eNOS appears to be via multiple mechanisms. Other potential sources of superoxide in the endothelium include NADH oxidoreductase, cyclooxygenase, lipoxygenase, NADPH oxidase, and the autoxidation of certain tissue metabolites. In the endothelium, cycloxygenase (16), NADH oxidoreductase (26), and xanthine oxidase (34) have been identified as significant producers of superoxide. Also, recent studies on the effect of NO donors on eNOS activity have suggested that NO can induce the activity of the enzyme NADPH oxidase (27). The activation of this enzyme can result in elevated superoxide generation and peroxynitrite levels and can have a direct inhibitory effect on the activity of eNOS (31). The direct NO effect can be reduced by the action of the thioredoxin/thioredoxin reductase system (31). Because NOS is a hemoprotein containing several cysteinyl residues (including thiolate as its proximal heme ligand), exposure to NO can induce S-nitrosylation of protein thiols. This S-nitrosylation of vicinal thiols promotes disulfide formation. Thus the disulfide reduction catalyzed by thioredoxin/thioredoxin reductase suggests that an oxidative conformational change in vicinal thiols, resulting in the formation of intramolecular or intermolecular disulfides or both, by the direct action of NO is also involved in the inhibitory mechanism.

In conclusion, it appears that NO inhibits eNOS activity without altering eNOS gene expression. Furthermore, at least part of the inhibitory effect of NO on eNOS activity can be explained by the reaction of NO with superoxide formed by the action of the xanthine/xanthine oxidase pathway. This reaction could lead to an increase in cellular peroxynitrite levels and produce an irreversible inhibition of eNOS protein via nitrosation of critical amino acid residues. This mechanism may also explain, in part, how acute withdrawal from inhaled NO results in rebound pulmonary hypertension.

    ACKNOWLEDGEMENTS

We thank Jim Bristow and Jeff Fineman, Department of Pediatrics, University of California (UC), San Francisco, for helpful discussions and critical reading of the many drafts of this manuscript. We also thank Scott Soifer, Department of Pediatrics, UC, San Francisco, for help with the statistical analyses.

    FOOTNOTES

This work was supported, in part, by grants from the American Heart Association (to S. M. Black) and UC, San Francisco, Research Evaluation and Allocation Committee---Harris Fund (to S. M. Black).

Address for reprint requests: S. M. Black, M-680, Dept. of Pediatrics, Univ. of California, San Francisco, CA 94143-0106.

Received 21 August 1997; accepted in final form 14 January 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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