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Differential regulation of xanthine and NAD(P)H oxidase by hypoxia in human umbilical vein endothelial cells. Role of nitric oxide and adenosine

Hae-Young Sohn, Florian Krotz, Torsten Gloe, Matthias Keller, Karl Theisen, Volker Klauss, Ulrich Pohl
DOI: http://dx.doi.org/10.1016/S0008-6363(03)00262-1 638-646 First published online: 1 June 2003

Abstract

Objectives: Although in tissue injury following hypoxia/reoxygenation (H/R) an increased endothelial formation of superoxide anions (O2) plays an important role, it is still not fully understood which of the potential enzymatic sources of endothelial O2 are crucially involved. In this study, we particularly examined the activities of NAD(P)H oxidase and xanthine oxidase (XO) after 8 h of exposure to mild hypoxia. We further studied whether enzyme activities can be modified by NO and adenosine during hypoxic treatment. Methods and results: In human umbilical vein endothelial cells O2 production was measured immediately after exposure to hypoxia (‘early reoxygenation’) or after 2 h of reoxygenation at normoxic conditions (‘late reoxygenation’). In the early reoxygenation phase the O2 production was attenuated by 28.5% while it was enhanced by 58.2% after late reoxygenation. Using specific inhibitors of NAD(P)H oxidase and XO, gp91ds-tat and oxypurinol, respectively, we show that the constitutively active NAD(P)H oxidase was blocked following hypoxia while XO was activated. The presence of NO during hypoxia had no effect on NAD(P)H oxidase activity but it significantly inhibited the activation of XO. Inhibition of XO activation was, at least in part, caused by the release of adenosine from endothelial cells which induces an increased formation of NO by its A1 and A2 receptors. Conclusion: Our results indicate that during exposure to mild hypoxia for 8 h, a change in the enzymatic source of endothelial O2 occurs: a prolonged inhibition of NAD(P)H oxidase was found while an enhanced activity of XO occurs in the reoxygenation phase. These results suggest that different strategies of antioxidant therapy should be taken into consideration in oxidative stress related to chronic hypoxia when compared to normoxic atherosclerotic tissues with an activated vascular NAD(P)H oxidase as the main source of O2.

Keywords
  • Endothelial function
  • Hypoxia/anoxia
  • Free radicals
  • Nitric oxide
  • Adenosine

Time for primary review 27 days.

1 Introduction

Both hypoxia and reoxygenation (H/R) are important in human cardiovascular pathophysiology since they occur in a wide variety of ischemic cardiovascular disorders such as myocardial ischemia or stroke. Previous studies have suggested that in the pathogenesis of H/R-induced tissue injury, an increased formation of reactive oxygen species (ROS) plays a pivotal role [1]. Neutrophils are important sources of ROS, but during reoxygenation they are not essential for vascular damage since injury in cultured endothelial cells or isolated re-perfused organs also occurs in the absence of activated neutrophils [2,3].

Vascular cells display a constitutive release of superoxide anions (O2) in the nanomolar range which is mainly dependent on a NAD(P)H dependent oxidase [4]. In vessels isolated from hypertensive or hyperlipidemic animals, the activity of this vascular NAD(P)H oxidase has been found to be increased [4]. In the endothelium, the enzyme seems to be an isoform of the neutrophil-type NADPH oxidase including its subunit gp91phox as site of the electron transfer which exists as a pre-assembled intracellular complex associated with the cytoskeleton [5]. We and others have previously shown that the activity of the endothelial NAD(P)H oxidase strongly depends on the small G-protein rac1 [6,7]. Inhibition of rac1 by over-expression of its dominant negative isoform protects from hypoxia/reoxygenation (H/R) induced endothelial cell death indicating a role for NAD(P)H oxidase in this process [8]. However, whether the endothelial NAD(P)H oxidase plays a crucial role in H/R associated O2 formation has not been studied yet in detail.

Xanthine oxidase (XO) also represents a potential candidate enzyme for O2 formation although the vast majority of recent studies failed to demonstrate a significant role for its contribution, at least in normoxic endothelial cells [4]. In conditions of H/R, however, an increased XO activity as reflected by an oxypurinol sensitive O2 formation can be detected in the same cell-type. It is probably due to a posttranslational conversion to XO from the constitutively active xanthine dehydrogenase (XD) rather than to a novel synthesis of the enzyme [9,10]. On the other hand, the conversion of XD to XO does not seem to be mandatory for post-ischemic O2 generation since both XD and XO can oxidize NADH at their FAD/FAD+ domain to form O2 [11,12].

Although it has been clearly demonstrated that a longer time of reoxygenation increases O2 formation in endothelial cells, it is not fully understood which enzymes are involved and whether their activity can be modified. In this context, Cote et al. have shown that treatment of endothelial cells with a blocker of the NO synthase (eNOS) prior to exposure to H/R results in an increased O2 formation by activation of intracellular XO [13]. These data suggest that the presence of increased amounts of NO during hypoxia might control subsequent O2 production, an effect that could also involve NAD(P)H oxidase activation. Under hypoxic conditions, the production of NO should be stimulated by adenosine, which is released by hypoxic endothelial cells. Beside its cAMP-dependent vasodilatory effects on vascular smooth muscle cells, adenosine opens potassium channels leading to endothelial hyperpolarisation which has been shown to promote activation of eNOS [14].

In this study, we examined whether endothelial O2 formation is altered during reoxygenation after exposure to hypoxia for 8 h. Using specific inhibitors and direct measurements of enzymatic activities, we particularly investigated the role of endothelial NAD(P)H oxidase and XO. We further studied whether the enzyme activities are modulated by endothelial NO during hypoxia. Since adenosine has been shown to be released under hypoxic conditions from endothelial cells, we examined whether adenosine affects O2 production and whether this is mediated by NO.

2 Methods

2.1 Cell culture

Endothelial cells (HUVEC) were isolated from human umbilical veins as described [15]. The cells were maintained in Medium 199 supplemented with 16% fetal calf serum and 20% endothelial growth medium (Promocell, Germany). The cells used for experiments were in the 1-2 subpassage.

2.2 Experimental hypoxia

Confluent endothelial cells were placed in an airtight chamber and exposed to a water saturated hypoxic gas mixture (5% CO2, 95% N2) for 8 h. The oxygen tension in the cell supernatant was monitored by an O2-analyzer (Chiron Diagnostics, RapidLab 248). The experiments were carried out either immediately after reoxygenation termed as ‘early reoxygenation’ or after 2 h after return to normoxia (‘late reoxygenation’). Control cells were maintained at normoxia throughout.

2.3 O2 production

The O2 formation was determined by the cytochrome c assay as described before [6]. Endothelial cells were incubated in a modified Tyrode's buffer containing 40 μmol/l cytochrome c with or without SOD (200 U/ml). After 20 min, the supernatant was removed and the reduction of cytochrome c was measured photometrically at 550 nm (Ultrospec 2000, Pharmacia). The O2 dependent part of cytochrome c reduction was calculated from the difference in absorbance between samples incubated with or without SOD (ε550, reduced-oxidised=21.1 nM−1 cm−1). All measurements of O2 formation were carried out in the presence of the eNOS inhibitor NG-nitro-l-arginine (L-NA, 30 μmol/l) to prevent scavenging of O2 by NO.

2.4 Intracellular reactive oxygen species

The intracellular formation of ROS was determined using the 2,7-dihydrodichlorofluoresceine (DCFH-DA) fluorescence assay. The cells were washed twice with PBS and incubated in a modified Tyrode's buffer (135 mmol/l NaCl, 2.7 mmol/l KCl, 1.8 mmol/l CaCl2, 0.49 mmol/l MgCl2, 0.28 mmol/l NaH2PO4, 5.5 mmol/l glucose, 20 mmol/l HEPES) containing 10 μmol/l DCFH and the eNOS inhibitor L-NA (30 μmol/l). The fluorescence intensity (excitation 488 nm, emission >515 nm) was recorded after 15 min using a confocal microscope (Zeiss LSM 410).

2.5 NAD(P)H oxidase activity

In separate experiments, the NADH-dependent O2 production was measured in cell lysates. The cells were suspended in lysis buffer (20 mmol/l potassium phosphate buffer containing 1 mmol/l EDTA, 5 μg/ml aprotinin, 2 μg/ml pepstatin, 2 μg/ml leupeptin, pH 7.0). In 400 μl phosphate-buffered saline (PBS, 160 mmol/l NaCl, 2.7 mmol/l KCl, 8 mmol/l Na2HPO4, 1.5 mmol/l KH2PO4, pH 7.40) containing aliquots of the samples (5 μg protein content)±SOD (200 U/ml), NADH (100 μmol/l) was added and the SOD sensitive reduction of cytochrome c (40 μmol/l) determined for 15 min.

2.6 Xanthine oxidase activity

The activity of XO/XD was measured according to Ichimori et al. [16]. Endothelial cells were harvested using ice cold lysis buffer (50 mmol/l Tris, 1% NP-40, 1 mmol/l EDTA, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 10 μg/ml aprotinin, 1 mmol/l PMSF, pH 8.0) and disrupted by passing them five times through a 29-gauge needle. The lysates were centrifuged (10 000×g/5 min) and the supernatant was used for the experiments. Aliquots (40 μg protein) were incubated with 100 μmol/l 2,6-dichlorophenolindophenol (DCPIP) in the assay buffer (50 mmol/l Tris, pH 8.8, including 1 mmol/l EDTA, 1000 U/ml catalase, 10% DMSO (v/v)) and the reaction started by addition of 150 μmol/l hypoxanthine. The oxypurinol sensitive part of the DCPIP absorbance was measured at 600 nm (Ultrospec 2000, Pharmacia).

2.7 Nitrite/nitrate measurements

Estimations of the NO produced by endothelial cells during hypoxic conditions were carried out following the nitrite/nitrate measurements method as described previously [17]. The calculated concentrations of NOx were normalized to milligrams of total cellular protein and are expressed as nmol/h.

2.8 NO synthase expression

The expression of the eNOS protein was determined as described before [17]. HUVEC were harvested using ice cold lysis buffer (PBS, 1% Triton X-100, 1 mmol EDTA, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 10 μmol/l PMSF, 2 mmol/l orthovanadate, pH 7.0) and disrupted by passing them 10 times through a 29-gauge needle. The lysates were centrifuged (10 000×g/5 min) to remove debris. Equal amounts of proteins were then separated via SDS–PAGE following standard procedures and transferred onto nitrocellulose membrane (Sartorius, Germany). The membrane was probed with eNOS antibody (Dianova, Germany) and a second antibody (Sigma, Germany) detection was carried out with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate staining for alkaline phosphatase. Detected bands were recorded and quantified with a commercially available videodensitometric system from Bio-Rad (Germany).

2.9 NAD(P)H oxidase expression

The expression of the NAD(P)H oxidase subunits p22phox was determined by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) [6]. Total RNA was isolated using TRIZOL reagent according to the manufacturer's instructions (Gibco, Germany); 1 μg of total RNA was used for the RT-PCR which was carried out using the Titan one-step RT-PCR system (Boehringer-Mannheim, Germany). The primer sequences used in this study for the p22phox are described by Jones et al. [18] (sense-GTT TGT GTG CCT GCT GGA GT; antisense-TGG GCG GCT GCT TGA TGG T). As housekeeping gene β-actin was used.

2.10 MTT assay

The cell survival was tested by the metabolism of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as described previously [19]. After a 2 h co-incubation with anhydrous MTT (0.5 mg/ml), HUVEC were solubilized with isopropanol (100%) and the OD570nm was measured.

2.11 Materials

Endothelial growth medium was purchased from PromoCell (Heidelberg, Germany), DCFH-DA from Molecular Probes (Leiden, Netherlands). The A1 receptor antagonist 1,3-diptopyl-8-cyclopentylxanthine (DPCPX), and the A2 receptor antagonist 3,7-dimethyl-1-1propagylxanthine (DMPX) were obtained from RBI International (USA) and SOD from Roche (Mannheim, Germany). gp91ds-tat peptide was kindly provided by Dr P. Pagano (Detroit, USA), and HOE234 was obtained from (Aventis, Germany). All other substances were from Sigma (Germany).

2.12 Statistical analysis

Statistical comparisons within the same experimental group with and without treatment were carried out using the Wilcoxon signed rank test for paired observations. Differences were considered significant at an error probability of P<0.05. All results are expressed as means±S.E.M.

3 Results

3.1 Endothelial O2/ROS formation is attenuated in early reoxygenation

Endothelial O2 production was measured immediately after exposure to hypoxia (‘early reoxygenation’, 5% CO2, 95% N2 for 8±2 h) or after 2 h of reoxygenation at normoxic conditions (‘late reoxygenation’). The Po2 measured in cell supernatant immediately after hypoxia was 30–35 mmHg which rapidly increased within 10 min up to control values (Po2: 150 mmHg). In order to rule out scavenging effects of NO, the assessments of O2 after hypoxia were carried out in the presence of the eNOS inhibitor L-NA, which was added immediately after re-exposure to normoxia. In early reoxygenation, the O2 production was significantly attenuated by 28.5% (Fig. 1A, n = 16). This was not observed when NO formation in HUVEC was blocked by L-NA (30 μmol/l) during hypoxia treatment (Fig. 1A, n = 16). Similar results were obtained using the DCF fluorescence assay: the attenuation of the DCF fluorescence in the early reoxygenation was again less pronounced when the cells were pre-treated with L-NA during hypoxia (Fig. 1B, n = 12). Cell viability, assessed by the MTT assay, was not significantly different between normoxic and hypoxic cells (OD570nm: 0.511±0.03 vs. 0.498±0.04, n = 8, n.s.).

Fig. 1

Endothelial O2/ROS formation is attenuated in the early reoxygenation phase. (A) When O2 production (cytochrome c assay) was measured immediately after treatment with hypoxia for 8 h (‘early reoxygenation’, E.R.), it was found to be significantly attenuated (n = 16, **P<0.01 vs. control). Pre-treatment with the NO synthase inhibitor L-NA (30 μmol/l) during hypoxia prevented the hypoxia-related reduction of endothelial O2 production (n = 16, #P<0.05 vs. E.R.). (B) Similar results were obtained using the DCF fluorescence assay (n = 12, **P<0.01 vs. control, #P<0.05 vs. E.R.).

3.2 Role of endothelial NAD(P)H oxidase

In normoxic HUVEC, the NAD(P)H oxidase inhibitor diphenyleneiodonium chloride (DPI, 30 μmol/l, n = 16) as well as the more specific blocker gp91ds-tat (100 μmol/l, n = 8) significantly attenuated basal O2 formation indicating a constitutively active enzyme (Fig. 2A). The same inhibitors failed to have an effect in untreated cells after early reoxygenation (data not shown). Interestingly, in cells pre-treated with L-NA, DPI but not gp91ds-tat inhibited O2 formation in early reoxygenation (Fig. 2B, n = 8 each). Direct measurement of the activity of endothelial NAD(P)H oxidase revealed no significant attenuation (3.4±0.61 vs. 3.0±0.42 nmol O2/min per mg protein, n = 8). Hypoxia also did not alter mRNA expression of the NAD(P)H oxidase subunit p22phox (Fig. 2C, n = 5).

Fig. 2

Role of endothelial NAD(P)H oxidase and xanthine oxidase. (A) Under normoxic conditions, the NAD(P)H oxidase inhibitor diphenyleneiodonium chloride (DPI, 30 μmol/l, n = 16, **P<0.01 vs. normoxic control) as well as the more specific blocker gp91ds-tat (100 μmol/l, n = 8, **P<0.01 vs. normoxic control) significantly attenuated the basal endothelial O2 formation while oxypurinol (100 μmol/l, n = 15) was without any effect. (B) In early reoxygenation (E.R.), the hypoxia associated reduction of endothelial O2 formation (0.32±0.03 nmol/min per mg protein) was restored when L-NA was present during hypoxic treatment. This O2 formation could be inhibited by DPI (30 μmol/l, n = 8,**P<0.01 vs. E.R. +L-NA) and oxypurinol (100 μmol/l, n = 8,*P<0.05 vs. E.R. +L-NA) but not by gp91ds-tat (100 μmol/l, n = 8). (C) After hypoxia for 8 h, the mRNA expression of the NAD(P)H oxidase subunit p22phox was not significantly altered. A representative blot of five independent experiments is shown.

3.3 Role of endothelial xanthine oxidase

In normoxic cells, the XO inhibitor oxypurinol (100 μmol/l) failed to have an inhibitory effect on O2 formation (Fig. 2A, n = 15). The same was true in untreated cells in the early reoxygenation phase (data not shown) while in cells pre-treated with L-NA, oxypurinol significantly inhibited O2 formation during early oxygenation (Fig. 2B, n = 8). Accordingly, a detectable activity of XO also was found only in cell lysates obtained from endothelial cells which had been pre-treated with L-NA during hypoxia (Fig. 3, n = 5, **P<0.01 vs. normoxic control cells).

Fig. 3

Effect of hypoxia on xanthine oxidase activity. The activity of xanthine oxidase was determined in endothelial cell lysates using the oxypurinol sensitive oxidation of DCPIP (100 μmol/l). Measurable activity of xanthine oxidase could only be found in lysates from hypoxic endothelial cells which had been pre-treated with the NO synthase blocker L-NA (n = 5, **P<0.01 vs. normoxia).

3.4 Endothelial O2/ROS formation is increased in late reoxygenation

In the late reoxygenation phase, endothelial O2 formation was increased compared to normoxic cells as assessed by cytochrome c (Fig. 4A, n = 13, P<0.01) and DCF fluorescence assay (n = 11, P<0.01) by 58.2 and 56.9%, respectively. The enhancement was more pronounced when L-NA was present during hypoxic treatment. In the latter case, O2 formation was attenuated by DPI (30 μmol/l) and oxypurinol (100 μmol/l) but not by gp91ds-tat (100 μmol/l, Fig. 4B, n = 8 each).

Fig. 4

Endothelial O2/ROS formation is augmented in late reoxygenation. (A) In late reoxygenation (L.R.), the O2 formation was enhanced, particularly when L-NA (30 μmol/l) was present during hypoxia (n = 11, **P<0.01 vs. normoxia, #P<0.05 vs. L.R.). (B) In the latter cells (L.R. in the presence of L-NA), the O2 formation was inhibited by DPI (30 μmol/l, n = 8,**P<0.01 vs. control) and oxypurinol (100 μmol/l, n = 8,*P<0.05 vs. control) but not by gp91ds-tat (100 μmol/l, n = 8).

3.5 Adenosine mediates attenuation of O2 production in early reoxygenation

Adenosine (0.1–1 μmol/l, n = 14) significantly attenuated basal O2 formation in a concentration-dependent manner (Fig. 5A). The effect of adenosine (1 μmol/l) was mediated by A1 and A2 receptors since only the presence of blockers of both receptor subtypes (DPCPX and DMPX, 100 nmol/l each, n = 12) abolished the inhibitory effect of adenosine on endothelial O2 formation (Fig. 5B, n = 12, n.s. vs. normoxic control cells). The same receptor antagonists also significantly attenuated the hypoxia induced inhibition of endothelial O2 formation (Fig. 6, n = 9). Under normoxic conditions, blockade of NO production (L-NA, 30 μmol/l) abolished the inhibitory effects of adenosine (1 μmol/l) on O2 formation while reduction of O2 formation induced by the KATP-channel opener HOE234 (1 μmol/l) was not affected by L-NA (30 μmol/l, Fig. 7, n = 9–13).

Fig. 7

Role of NO in adenosine and KATP channel mediated inhibition of O2 formation. Adenosine (0.1 μmol/l, n = 9) as well as the KATP channel opener HOE234 (0.1 μmol/l, n = 13) significantly inhibited endothelial O2 formation (**P<0.01 vs. normoxic control cells). Blockade of endothelial NO production (L-NA, 30 μmol/l) abolished the inhibitory effects of adenosine (not significant vs. normoxic control cells with L-NA) while the reduction in O2 formation by HOE234 was not affected by L-NA (n = 9, **P<0.01 vs. normoxic control cells with L-NA).

Fig. 6

Adenosine receptor blockers restore inhibition of endothelial O2 production in early reoxygenation. Only the presence of both adenosine receptor blockers, DPCPX and DMPX (100 nmol/l each) during hypoxia attenuated the inhibition of endothelial O2 formation in early reoxygenation phase (E.R., n = 9, *P<0.05, **P<0.01 vs. control, #P<0.05 vs. E.R.). O2 formation in cells subjected to hypoxia in the presence of both adenosine receptor (A1 and A2) blockers was not significantly different to values obtained from normoxic control cells (n.s.).

Fig. 5

Adenosine inhibits endothelial O2 production. (A) Adenosine significantly attenuated basal O2 formation determined by the cytochrome c method (n = 14, *P<0.05, **P<0.01 vs. normoxic control cells). (B) The effect of adenosine was prevented in the presence of blockers of the adenosine receptor subtypes A1 and A2 (DPCPX and DMPX, 100 nmol/l each, n = 12, *P<0.05, **P<0.01 vs. normoxic control cells, #P<0.05 vs. adenosine).

3.6 Hypoxia increases endothelial NO production

Exposure of HUVEC to mild hypoxia for 8±2 h significantly enhanced expression of eNOS protein (n = 3, Fig. 8A). This was accompanied by an increased production of NO as measured by the NOx concentration in the cell culture supernatants (Fig. 8B, n = 8).

Fig. 8

Hypoxia increases endothelial NO production. (A) Exposure of HUVEC to hypoxia (5% CO2, 95% N2 for 8±2 h) significantly enhanced the expression of endothelial NO synthase protein (a typical blot of three independent experiments is shown). (B) Hypoxia also increased the production of NO as measured by the NOx concentration in the cell culture supernatants (n = 8, **P<0.01 vs. normoxic control cells).

4 Discussion

The present study demonstrates that during early reoxygenation after 8 h of hypoxia, endothelial O2 production is significantly attenuated while it is increased 2 h after reoxygenation is initiated. The early attenuation of O2 formation is elicited by an enhanced production of endothelial NO during hypoxia which, at least in part, seems to be mediated by adenosine.

This reduction of endothelial O2/ROS formation in the early reoxygenation phase is in accordance with the observation by Lum et al.: after hypoxia of 24 h reoxygenation for 5 or 20 min did not increase the release O2, whereas reoxygenation for 40–60 min augmented O2 formation nearly twofold over normoxic cells [20]. Likewise, Zulueta et al. showed that intracellular ROS formation, as detected by DCF fluorescence, declined by 40% during early reoxygenation after 6 h of anoxia while it was increased to 148% after 6 h of reoxygenation [21]. This attenuation seems to occur before reoxygenation since Mohazzab et al. demonstrated a decreased formation of O2 in intact coronary segments during hypoxia [22]. These results together strongly suggest that in the early stage of reoxygenation endothelial ROS formation is attenuated. The apparent discrepancy between these results and that of other groups who demonstrated an enhanced formation of ROS during reoxygenation is likely due to measurements at later time points as most of the studies analyzed a long-term reoxygenation period (0.45–24 h) [13,20,21,23–25]. Accordingly, after a reoxygenation period of >2 h termed as ‘late reoxygenation’ in the present study, we also observed an enhanced ROS formation. In this context, it should be considered that the variability of the studies dealing with hypoxia is not only due to differences in the species and vascular bed but also to the use of different ranges of oxygen tension [26]. In the present study, the endothelial cells were exposed to a mild hypoxia of 30–35 mmHg Po2.

The origin of H/R-induced vascular O2 formation is still controversially discussed. We have previously shown that under normoxic conditions, a NAD(P)H oxidase is the main source of basal and angiotensin II mediated O2 formation in HUVEC [27]. We failed to determine a significant role of XO for basal endothelial O2 formation, which is in accordance with the majority of studies dealing with vascular O2 formation [4]. Inhibitors of NAD(P)H oxidase such as DPI and the highly specific gp91ds-tat attenuated O2 formation in normoxic endothelial cells in the present study. Rey et al. showed recently that the chimeric peptide gp91ds-tat specifically inhibits the association of p47phox with gp91phox in NAD(P)H oxidase in isolated aortic rings from mice treated with angiotensin II [28]. Very recently, we showed that platelet O2 formation could be inhibited by the same peptide as well [29]. In the present study, we for the first time demonstrate that the same peptide also attenuates O2 formation in human endothelial cells.

After 8 h of hypoxia, however, gp91ds-tat failed to have any effects on endothelial O2formation. This suggests that chronic hypoxia arrests the constitutively active NAD(P)H oxidase by an unknown mechanism. Our results further suggest that there was no restoration of the NAD(P)H oxidase activity during the 2-h reoxygenation period since gp91ds-tat was still not effective. These results suggest that XO but not NAD(P)H oxidase is the major source of O2 at least up to 2 h after return to normoxia.

In lysates of cells, we did not observe any inhibition of NADH-dependent O2 formation by 8 h of hypoxia. It is concluded that this hypoxia-related effect on NAD(P)H oxidase can be detected in intact cells only. This is consistent with our previous observation that the attenuation of endothelial O2 formation mediated by the angiotensin II type 2 receptor could only be measured in intact cells but not in cell lysates and suggests that during hypoxia, an inhibitory component was formed that may be lost during the lysis procedure [27]. However, it remains to be established by which mechanism the activity of endothelial NAD(P)H oxidase is blocked during exposure to hypoxia.

A substantial activity of XO was only detectable when eNOS had been blocked during hypoxia which is in accordance with previous studies [13]. This was observed in cell lysates as well as in intact cells as O2 formation was inhibited by oxypurinol. These results indicate that the L-NA mediated ‘restoration’ of O2 formation in the early reoxygenation is due to an increased activity of XO rather than of NAD(P)H oxidase. In late reoxygenation, going along with an enhanced O2 formation, similar results could be observed: oxypurinol and DPI but not the more specific inhibitor of NAD(P)H oxidase gp91ds-tat inhibited O2 formation suggesting that the predominant source of O2 was again XO. The considerable inhibition obtained by the non-specific flavoprotein blocker DPI might be due to a blockade of the FAD/FAD+ catalytic domain of XO/XD. It has been shown that in HUVECs both XD and XO can efficiently oxidize NADH to yield O2 after hypoxia, a process which can be blocked by DPI [11,12]. Our results substantially underline the fact that DPI cannot be used as a specific inhibitor of NAD(P)H oxidase while the gp91ds-tat peptide seems to be a favorable tool to study the role of NAD(P)H oxidases in intact vascular cells.

In support of our findings, NO generating compounds were found to reduce XO activity [16,30–32]. Cote et al. reported in rat microvascular pulmonary endothelial cells that hypoxia increases XO activity, an effect which was markedly enhanced in the presence of eNOS inhibitor but reduced after l-arginine supplementation, the substrate of eNOS [13]. The mechanism of this NO-mediated effect on XO is, however, not understood. It has been suggested that inhibition of XD/XO activity by NO might be mediated through direct binding of NO to the flavin and iron–sulfur moiety or to its sulfhydryl groups [30,32], while others reported that NO inhibits XO/XD at their Mo(V) domain [16].

In the present study, only the activity of XO/XD but not of NAD(P)H oxidase was altered by NO. Clancy et al. reported that NO inhibits neutrophil O2 production via direct effects on membrane components of the NADPH oxidase—an event which must occur before the assembly of the activated complex to be effective [33,34]. Although the activity of endothelial NAD(P)H oxidase was also blocked by hypoxic treatment, we have no evidence that NO was involved in this process. Similar results were obtained in normoxic cells using NO donors (unpublished observations). These results suggest that NO scavenges O2, but it does not alter the activity of endothelial NAD(P)H oxidase.

Our data indicate that the hypoxia associated attenuation of O2 generation in the early reoxygenation phase seems to be, at least in part, mediated by the release of adenosine from endothelial cells. Adenosine is known to be released under hypoxic conditions [35–38] and enhances NO production via its A1 [35,37,39] and/or A2a [35,36,38,40,41] receptor subtypes. In our study, only a blockade of both adenosine receptor subtypes A1 and A2 prevented the hypoxia-mediated ROS formation. It has been supposed that adenosine modulates endothelial potassium channels such as the KATP channel [35,37,39] resulting in membrane hyperpolarization and activation of the NO synthase. Since we have previously shown that opening of endothelial KATP channels and the following membrane hyperpolarization per se reduces O2 formation, we tested whether the effect of adenosine could be due to the latter mechanism. Since only the adenosine mediated but not the HOE234 induced inhibition of O2 formation was sensitive to the eNOS blocker L-NA, we conclude that the adenosine mediated reduction of O2 was due to a stimulation of NO formation rather than to changes in endothelial membrane potential [6].

Taken together, our data indicate that endothelial cells exposed to mild hypoxia for 8 h produce an increased amount of NO. This results in a significant attenuation of the reoxygenation related O2 formation by down-regulating the activation of XO. Moreover, during hypoxia a change in the enzymatic source of endothelial O2 occurs: while the constitutively active NAD(P)H, oxidase was blocked in a NO-independent manner, an enhanced activation of endothelial XO was responsible for O2 formation after reoxygenation. These results suggest that different strategies in antioxidant therapy should be taken into consideration in oxidative stress related to chronic hypoxia when compared to normoxic tissues such as in atherosclerotic lesion when an increased activated NAD(P)H oxidase is the main source of O2.

Acknowledgements

This study was supported by a grant from the Friedrich-Baur Foundation of the Ludwigs-Maximilians-University.

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View Abstract