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(Hypertension. 2001;38:877.)
© 2001 American Heart Association, Inc.

Scientific Contributions

Effects of the Reactive Oxygen Species Hydrogen Peroxide and Hypochlorite on Endothelial Nitric Oxide Production

Edgar A. Jaimes; Charles Sweeney; Leopoldo Raij

From the Nephrology and Hypertension Section, Veterans Administration Medical Center; and University of Minnesota, Minneapolis, Minn.

Correspondence and reprint requests to Leopoldo Raij, MD, Nephrology and Hypertension Section IIIJ, Veterans Administration Medical Center, 1 Veterans Dr, Minneapolis, MN 55417. E-mail raijx001{at}tc.umn.edu

	Abstract

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Abstract—— Reactive oxygen species (ROS) hydrogen peroxide (H₂O₂) and hypochlorite (HOCl) participate in the pathogenesis of ischemia/reperfusion injury, inflammation, and atherosclerosis. Both NO and ROS are important modulators of vascular tone and architecture and of adhesive interactions between leukocytes, platelets, and vascular endothelium. We studied the effect of H₂O₂ and HOCl on receptor-dependent (bradykinin [10^-6 mol/L] and ADP [10^-4 mol/L]) and receptor-independent mechanisms (calcium ionophore A23187 [10^-6 mol/L]) of NO production by porcine aortic endothelial cells (ECs). Changes in the level of EC cGMP (the second messenger of NO) were used as a surrogate of NO production. EC cGMP increased 300% in response to bradykinin and A23187 and 200% in response to ADP. Exposure of ECs to H₂O₂ (50 µmol/L) for 30 minutes significantly impaired cGMP levels in response to ADP, bradykinin, and the receptor-independent NO agonist A23187. In contrast, preincubation with HOCl (50 µmol/L) impaired cGMP production only in response to ADP and bradykinin but not A23187. These concentrations of H₂O₂ and HOCl did not result in increased EC lethality as assessed by lactate dehydrogenase release. Neither H₂O₂ nor HOCl affected EC cGMP production in response to NO donor sodium nitroprusside, which suggests that guanylate cyclase is resistant to these oxidants. We also demonstrated that neither H₂O₂ nor HOCl affects endothelial NO synthase (eNOS) catalytic activity as measured by conversion of L-arginine to L-citrulline in EC homogenates supplemented with eNOS cofactors. The present studies show that H₂O₂ impairs NO production in response to both receptor-dependent and receptor-independent agonists and that these effects are due, at least in part, to inactivation of eNOS cofactors, whereas HOCl inhibits NO production by interfering with receptor-operated mechanisms at the level of the cell membrane. Concentrations of H₂O₂ and HOCl used in the present studies have been shown to be generated in vivo during inflammation and ischemia/reperfusion. Therefore, we infer that these effects of H₂O₂ and HOCl on EC NO production may contribute to disregulated vascular tone and altered leukocyte-EC interactions that occur in vascular injury as a result of those causes in which ROS generation is involved.

Key Words: endothelium • nitric oxide • reactive oxygen species • atherosclerosis • inflammation

	Introduction

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Vascular injury secondary to processes such as ischemia/reperfusion, inflammation, and atherosclerosis is accompanied by synthesis and release of reactive oxygen species (ROS).¹ Among the ROS generated are hydrogen peroxide (H₂O₂), hypochlorite (HOCl), hydroxyl, and superoxide (O₂^-).² These oxidants can induce injury in a variety of mammalian cells, including endothelial cells (ECs).³

Although O₂^- can be directly toxic, it has limited reactivity with most biological molecules.⁴ However, O₂^- can dismutate either spontaneously or through superoxide dismutase to H₂O₂⁵. Therefore, H₂O₂ almost always is formed under any circumstance in which O₂^- is produced.⁵ Acting as an electron donor, O₂^- also can lead to generation of hydroxyl radical through an O₂^--driven Fenton reaction,⁶ and by interaction with NO, O₂^- can generate highly reactive peroxynitrite.⁷ No direct interaction has been described for NO with H₂O₂ or HOCl.

H₂O₂ diffuses freely into cells to produce several biochemical perturbations, including activation of the hexose monophosphate shunt and glutathione redox cycles,⁸ oxidation of intracellular sulfhydryls,⁹ depression of intracellular ATP,¹⁰ DNA damage,¹¹ loss of intracellular ß-nicotinamide adenine dinucleotide,¹² activation of poly(ADP-ribose) polymerase,¹³ fast rise of intracellular calcium,⁸ gross perturbations to the cytoskeleton and plasma membrane,¹⁴ and depression in glycolytic flux.¹⁵ All of these processes occur before loss of membrane integrity as measured by vital stains⁸ or before loss of preloaded Cr⁵¹.¹¹

Myeloperoxidase, a heme protein secreted by phagocytes, amplifies the oxidative potential of H₂O₂ by generating cytotoxic oxidants and diffusible radical species.¹⁶ The major oxidant generated by the myeloperoxidase-H₂O₂-Cl^- system at physiological concentrations of Cl^- is HOCl.¹⁷ Toxic properties of HOCl stem from its chemical reactivity, including oxidative bleaching of heme groups and iron-sulfur centers¹⁶ and chlorination of amines¹⁸ and unsaturated lipids.¹⁹ However, in contrast to H₂O₂, HOCl is less diffusible and interacts mainly with cellular membrane components, oxidizing plasma membrane sulfhydryl groups²⁰ and disturbing cellular functions such as glucose and amino acid transporters and potassium pumping capacity.²¹

Endothelial NO synthase (eNOS) is located in small invaginations of the plasma membrane called caveolae²² and produces NO in response to a variety of agonists. Acetylcholine, adenosine nucleotides such as ADP, and bradykinin (BK) stimulate NO synthesis through receptor-operated mechanisms, whereas calcium ionophore A23187 leads to NO synthesis by stimulating calcium fluxes through receptor-independent mechanisms.²³ NO activates guanylate cyclase in an autocrine fashion within the endothelium and in a paracrine fashion in vascular smooth muscle (VSM), which results in increased intracellular cGMP.²³ The amount of cGMP generated is proportional to the amount of NO and, therefore, is an indirect measurement of NO bioactivity.²³ Although increased cGMP in VSM is linked to relaxation, the role of endothelial cGMP has remained more obscure.

Experimentally, ischemia/reperfusion of the heart²⁴ and kidney²⁵ results in increased generation of ROS and decreased endothelial NO bioactivity as evidenced by impaired endothelium-dependent relaxation to acetylcholine and BK, known agonists of NO.²⁵ Furthermore, topical H₂O₂ has been shown to eliminate endothelium-dependent vasodilatory response to acetylcholine in cerebral circulation.²⁶ After ischemia/reperfusion, decreased NO bioactivity is not accompanied by a reduction in eNOS as demonstrated by histochemical methods.²⁵ In addition, infusion of angiotensin (Ang) II into rats results in hypertension accompanied by increased production of O₂^- in the aorta.²⁷

We used several strategies to study the effect of nonlethal concentrations of the ROS H₂O₂ and HOCl on EC NO synthesis. First, we studied the effect of H₂O₂ and HOCl on EC cGMP production. In our laboratory, it has been difficult to obtain consistent measurements of small quantities of NO generated from cultured ECs. Therefore, we used cGMP production within the same ECs as a surrogate of NO generation.²⁸ This approach also permitted us to evaluate the effect of ROS on guanylate cyclase. Second, we studied the effect of H₂O₂ and HOCl on eNOS catalytic activity by measuring the conversion of C¹⁴ L-arginine to C¹⁴ L-citrulline in homogenates of ECs previously exposed to these ROS. Third, we determined the effect of these ROS on eNOS catalytic activity in homogenates of ECs and fourth, the effect of these ROS on individual eNOS cofactors.

	Methods

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EC Culture
ECs were isolated from fresh porcine aorta by incubating the lumen of the vessels in type II collagenase (2 mg/mL; Sigma Chemical Co) at 37°C for 20 minutes. Aortas were rinsed with medium 199 (Cellgro, Mediatech) supplemented with 10% FCS (HyClone) and cells plated directly into 6-well culture dishes (Costar). Cells were identified as endothelial by their morphology and positive uptake of fluorescent acetylated LDL.

Cell Incubation
ECs were washed and preincubated with Krebs-Ringer bicarbonate buffer that contained 3-isobutyl-1-methylxanthine (Sigma; 10^-4 mol/L) for 30 minutes. ECs were preincubated and then incubated for 3 minutes in Krebs buffer that contained the following NO agonists: A23187 (10^-6 mol/L), ADP (10^-4 mol/L), and BK (10^-7 mol/L; Sigma). Sodium nitroprusside (SNP; 10^-3 mol/L) also was tested. At the end of 3 minutes’ incubation, intracellular cGMP was measured by radioimmunoassay as previously described.²⁹ cGMP per well was factored for protein content measured by the method of Lowry et al.³⁰

The following experiments were performed to confirm that changes in EC cGMP in response to agonists of NO were due to autocrine effects of endothelial NO. (1) ECs were incubated for 10 minutes with NOS inhibitor N^G-monomethyl-L-arginine (L-NMMA; 10^-4 mol/L) before incubation with A23187, ADP, or SNP. (2) ECs were preincubated for 24 hours in cell-culture medium free of L-arginine (Selectamine, Gibco). A23187 (10^-6 mol/L) and SNP (10^-3 mol/L), were added to cells preincubated in standard and L-arginine-depleted medium; cGMP was measured as described above.

Effect of Oxidant Injury on EC cGMP Synthesis
To induce oxidant injury, ECs were preincubated with either H₂O₂ (50 µmol/L) or HOCl (50 µmol/L) (Sigma) for 30 minutes before incubation with NO agonist or corresponding vehicle. After incubation with NO agonist, cGMP was measured as described above.

Effect of Oxidant Injury on NOS Activity
To determine the direct effect of oxidant injury on eNOS, ECs were preincubated with either H₂O₂ (50 µmol/L) or HOCl (50 µmol/L) for 30 minutes, and eNOS activity was determined in vitro in homogenates of ECs. In separate experiments, homogenates of ECs were exposed to H₂O₂ (50 µmol/L) or HOCl (50 µmol/L) for 30 minutes and eNOS activity determined in the presence of optimal amounts of NOS cofactors (Figure 1). In other experiments, individual NOS cofactors were exposed to H₂O₂ (50 µmol/L) or HOCl (50 µmol/L) for 30 minutes and then added to homogenates of ECs for eNOS activity determination. NOS activity was measured as previously described³¹ (Figure 1).

View larger version (17K): [in this window] [in a new window]   Figure 1. Schematic of experimental design. Intact ECs, eNOS from EC homogenates, or eNOS cofactors were exposed to H2O2 or HOCl (50 µmol/L) before cGMP or eNOS activity determinations.

Statistical Analysis
Data are expressed as mean±SEM. For statistical comparison involving 2 groups, an unpaired Student’s t test was used, whereas for comparison involving >2 groups, ANOVA by means of Statview 512 statistical program was used (Abacus Concepts Inc). Significance was considered present when P<0.05.

An expanded Methods section can be found in an online data supplement available at http://www.hypertensionaha.org.

	Results

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NO Agonists Increase EC cGMP Synthesis
ECs exposed for 30 minutes to the NO agonists A23187, ADP, or BK showed increased cGMP compared with that of control (Figure 2). NOS inhibitor L-NMMA (10^-4 mol/L) significantly inhibited cGMP production stimulated by these agonists (Figure 2).

View larger version (22K): [in this window] [in a new window]   Figure 2. L-NMMA inhibits EC cGMP production stimulated by NO agonists. ECs were preincubated for 10 minutes with Krebs buffer or NOS inhibitor L-NMMA (10-4 mol/L) and stimulated for 3 minutes with A23187 (10-6 mol/L; n=7), ADP (10-4 mol/L; n=9), or BK (10-7 mol/L; n=6). *P<0.05 vs control; #P<0.05 vs agonist alone.

Arginine Depletion Diminishes EC cGMP Production in Response to NO Agonists
Twenty-four hours of L-arginine depletion lowered basal levels of cGMP slightly but not significantly from 8.5±2.5 to 5.8±0.5 fmol/µg of protein (n=3; P=NS). L-arginine depletion significantly blunted cGMP production stimulated by A23187: for cells in standard medium with A23187, cGMP level was 27.4±2.0 fmol/µg of protein (n=3); in L-arginine-depleted medium with A23187, cGMP level was 11.8±0.40 fmol/µg of protein (n=3, P<0.05).

Thus, elimination of L-arginine, the substrate of eNOS, from the culture medium or inhibition of NO synthesis with L-NMMA significantly prevented increases in EC cGMP induced by NO agonists, thereby establishing that observed increases in EC cGMP are a result of increased EC NO.

H₂O₂ Impairs EC cGMP Production in Response to NO Agonists
As shown in Figure 3, H₂O₂ preexposure for 30 minutes significantly impaired increases in EC cGMP synthesis in response to all NO agonists tested. These changes in cGMP production were not accompanied by increased EC lethality as assessed by LDH release (0% release). However, H₂O₂ preexposure did not significantly affect EC cGMP production stimulated by the exogenous NO donor SNP (Figure 3). This occurrence suggests that H₂O₂ did not affect EC guanylate cyclase levels.

View larger version (24K): [in this window] [in a new window]   Figure 3. H2O2 preexposure impairs EC cGMP production in response to NO agonists. ECs were preexposed to Krebs buffer or H2O2 (50 µmol/L) for 30 minutes and stimulated for 3 minutes with A23187 (10-6 mol/L; n=3), ADP (10-4 mol/L; n=5), BK (10-7 mol/L; n=3), or SNP (10-3 mol/L; n=3). *P<0.05 vs control; #P<0.05 vs agonist alone.

HOCl Impairs EC cGMP Production in Response to NO Receptor-Dependent Agonists ADP and BK But Not Calcium Ionophore A23187
Preincubation with HOCl (50 µmol/L) inhibited EC cGMP synthesis induced by ADP and BK (Figure 4). These changes in cGMP production were not accompanied by increased EC lethality as assessed by LDH release (0% release). However, in contrast to H₂O₂, A23187-stimulated production of cGMP was not significantly affected by preexposure of EC to HOCl (Figure 4). In a manner similar to H₂O₂, HOCl did not affect SNP-stimulated cGMP production (Figure 4).

View larger version (22K): [in this window] [in a new window]   Figure 4. HOCl preexposure impairs EC cGMP production in response to ADP and BK but not A23187. EC were preexposed to Krebs buffer or HOCl (50 µmol/L) for 30 minutes and stimulated for 3 minutes with A23187 (10-6 mol/L; n=3), ADP (10-4 mol/L; n=4), BK (10--7 mol/L; n=3) or SNP (10-3 mol/L; n=3). *P<0.05 vs control; #P<0.05 vs agonist alone.

Exposure of ECs to H₂O₂ or HOCl Does Not Affect Intrinsic Catalytic Activity of eNOS
To explore whether observed effects of H₂O₂ and HOCl on NO production were due to oxidative damage of eNOS or oxidation of the necessary cofactors for optimal eNOS activity, we determined eNOS activity in cell homogenates from ECs preexposed to H₂O₂ or HOCl in which all eNOS cofactors were added from exogenous sources. As shown in Figure 5, eNOS activity was similar in control ECs and ECs preexposed to H₂O₂ (50 µmol/L) for 30 minutes. These findings suggest that effects of H₂O₂ on NO production are because of a direct effect of H₂O₂ on eNOS cofactors and not on total eNOS catalytic activity. As expected, eNOS catalytic activity in ECs pretreated with HOCl was also preserved, consistent with the notion that effects of HOCl are at a relatively early point in signal transduction (cell membrane), whereas eNOS and its cofactors are spared from HOCl-induced oxidative damage (Figure 5).

View larger version (9K): [in this window] [in a new window]   Figure 5. eNOS catalytic activity in ECs preexposed to H2O2 or HOCl. ECs were exposed to H2O2 (50 µmol/L) or HOCl (50 µmol/L) for 30 minutes. eNOS activity was determined in EC homogenates in which eNOS cofactors were added from exogenous sources (n=3, P=NS).

To demonstrate a direct effect of H₂O₂ on eNOS cofactors, individual eNOS cofactors were exposed to H₂O₂ (50 µmol/L) and then added to the purified enzyme before eNOS catalytic activity was measured. Prior exposure of individual eNOS cofactors tetrahydrobiopterin (BH4), NADPH, and flavin adenine dinucleotide (FAD) to H₂O₂ did not significantly modify eNOS activity in homogenates of ECs: control, 36.6±1.4 pmol · mg^-1 · min^-1; cells treated with BH4 plus H₂O₂, 32.4±1.9 pmol · mg^-1 · min^-1; with NADPH plus H₂O₂, 37.8±2.8 pmol · mg^-1 · min^-1; with FAD plus H₂O₂, 34.7±1.7 pmol · mg^-1 · min^-1 (n=3 per duplicate; P=NS versus control). However, when flavin mononucleotide (FMN) or all eNOS cofactors combined were exposed to H₂O₂, a significant decrease occurred in NOS enzymatic activity, which suggests that oxidative modification of eNOS cofactors and FMN in particular impairs eNOS activity: cells treated with FMN plus H₂O₂, 30.3±1.8 pmol · mg^-1 · min^-1 (n=3 per duplicate; P<0.05 versus control) and with all cofactors plus H₂O₂, 26.5±1.4 pmol · mg^-1 · min^-1 (n=3 per duplicate; P<0.05 versus control). In contrast, exposure of eNOS from EC homogenates to H₂O₂ (50 µmol/L) did not impair eNOS activity, which demonstrated that eNOS is resistant to oxidative effects of H₂O₂, at least at the concentrations used in the present studies: control, 34.4±1.3 pmol · mg^-1 · min^-1 and cells treated with H₂O₂, 37.2±2.7 pmol · mg^-1 · min^-1 (n=3 per duplicate; P=NS). Similar results were found when EC homogenates were prepared in the absence of dithiothreitol, which suggests that the lack of effect of H₂O₂ on isolated eNOS was not due to the protective effect of dithiothreitol (not shown).

Similar experiments were performed in which the eNOS cofactors or EC homogenates were exposed to HOCl. With the exception of NADPH (and in contrast to our results with H₂O₂), exposure of individual eNOS cofactors to HOCl significantly impaired eNOS activity, which suggests that these cofactors are highly sensitive to the oxidative effects of HOCl: control, 36.6±1.4 pmol · mg^-1 · min^-1; cells treated with BH4 plus HOCl, 16.1±1.1 pmol · mg^-1 · min^-1, with flavin adenine dinucleotide plus HOCl, 27.2±1.4 pmol · mg^-1 · min^-1; with FMN plus HOCl, 22.4±3.0 pmol · mg^-1 · min^-1 (n=3 per duplicate; P<0.05 versus control); and with NADPH plus HOCl, 38.5±1.8 pmol · mg^-1 · min^-1 (n=3 per duplicate; P=NS versus control). In addition, when all eNOS cofactors were exposed to HOCl, eNOS activity was almost completely abolished (4.34±1.2 pmol · mg^-1 · min^-1; n=3; P<0.05 versus control). Furthermore, exposure of eNOS from EC homogenates to HOCl resulted in impaired eNOS activity: control, 34.4±1.3 pmol · mg^-1 · min^-1 versus HOCl, 17.1±1.1 pmol · mg^-1 · min^-1 (n=3 per duplicate; P<0.05 versus control).

	Discussion

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A growing body of evidence implicates NO as an important modulator of vascular tone and architecture as well as of adhesive interactions between leukocytes, platelets, and vascular endothelium.³² Processes of ischemia/reperfusion and atherosclerosis are characterized by increased adhesion of leukocytes and platelets to the vascular endothelium³³ as a result of increased adhesion-molecule expression.³⁴ The latter has been attributed to diminished bioactivity of NO and increased production of ROS such as H₂O₂ and O₂^-.³⁵ O₂^- in the extracellular milieu, given its great affinity for NO, rapidly interacts with this molecule and results in NO inactivation and/or production of toxic peroxynitrite.⁷ No direct interaction between H₂O₂ and NO has been described.

ROS generation may be a common pathway for many types of pathological processes. Several ROS including H₂O₂ and O₂^- are produced by leukocytes after perturbation of their membranes as part of the respiratory burst³⁶ or by vascular endothelium during the reperfusion period after ischemic injury.² In addition, O₂^- is generated by Ang II-mediated activation of NADH/NADPH oxidase by the AT₁ receptor in VSM cells,^27,37 vascular ECs,³⁸ and glomerular mesangial cells.³⁹ H₂O₂ can be generated through dismutation of O₂^- either spontaneously or by superoxide dismutase.⁵ Both O₂^- and H₂O₂ have been shown to activate intracellular pathways in VSM cells and mesangial cells, which culminate in cellular hypertrophy^39,40 and proliferation.⁴¹ These actions of Ang II are believed to be important to explain the role of Ang II in pathological vascular remodeling in hypertension^42,43 and in atherosclerosis.⁴⁴ A study recently reported that antioxidants restore endothelial function and decrease blood pressure in animal models of hypertension.⁴⁵ Hyperlipidemia in rabbits is associated with increased AT₁ receptor density and increased O₂^- production derived from NADH/NADPH oxidase.^46,47 In humans with atherosclerosis, AT₁ receptor blockade improves endothelium-dependent relaxation mediated by NO.⁴⁸ On the other hand, NO has been shown to have an inhibitory effect on NADH/NADPH oxidase⁴⁹ and to downregulate Ang II receptors in VSM cells.⁵⁰

H₂O₂ generation is followed by generation of other toxic molecules, including HOCl.¹⁷ When H₂O₂ is formed by polymorphonuclear cells in vivo, up to 40% is halogenated by myeloperoxidase into HOCl and in acidic conditions into Cl₂,⁵¹ which also has oxidant activity and may be implicated in several oxidative and cytotoxic reactions at sites of inflammation and vascular disease.⁵¹ Furthermore, the myeloperoxidase-H₂O₂-chloride system of phagocytes⁵² promotes the formation of advanced glycation end products,⁵³ which play an active role in the pathogenesis of atherosclerosis.⁵⁴

ADP and BK are 2 agonists of NO that bind to receptors located on the EC membrane, whereas A23187 is a calcium ionophore that induces NO synthesis and release, directly promoting a calcium influx into ECs, which activates eNOS.²³ The present studies show that exposure of ECs to nonlethal concentrations of H₂O₂ and HOCl impairs endothelial NO production in response to several NO agonists, as evidenced by decreased production of cGMP, the intracellular NO second messenger. H₂O₂ impairs NO synthesis in response to receptor-dependent (BK and ADP) and receptor-independent agonists (A23187), which suggests that H₂O₂ may affect eNOS catalytic action or eNOS cofactors. However, we found that the catalytic activity of eNOS in extracts from ECs exposed to H₂O₂ and activated in the presence of exogenously added cofactors is similar to that in control ECs not exposed to this oxidant. On the other hand, exposure of eNOS cofactors to H₂O₂ before such cofactors are added to EC homogenates resulted in impaired eNOS activity. These findings suggest that H₂O₂ affects oxidant-sensitive eNOS cofactors but not the intrinsic catalytic activity of eNOS. Among the eNOS cofactors, FMN was the most sensitive to oxidation by H₂O₂, whereas other eNOS cofactors, including BH4, were less sensitive. Similar findings with BH4 have been previously reported by Milstien and Katusik.⁵⁵ In the aggregate, these findings support our previous studies in a rat model of ischemia/reperfusion acute renal failure that demonstrated preserved eNOS activity in the renal vasculature despite decreased NO bioactivity.²⁵ Similar findings also have been reported in a model of lead-induced hypertension.⁵⁶

In contrast to H₂O₂, HOCl impaired responses only to the receptor-dependent agonists BK and ADP. The response was preserved to the calcium ionophore and the eNOS catalytic activity in homogenates of ECs obtained after HOCl exposure. These results with intact cells are consistent with the notion that HOCl affects specific plasma membrane targets and impairs EC production of NO at a relatively early point in receptor-operated signal transduction.¹⁶ eNOS is located in small invaginations of the plasma membrane called caveolae.²² We speculate that HOCl may be affecting eNOS-caveolae interaction, resulting in impaired receptor-dependent NO production.⁵⁷

On the other hand, exposure of eNOS cofactors or EC homogenates to HOCl resulted in impaired eNOS catalytic activity. Because of the high reactivity of HOCl, its reactions are dependent on the relative concentration and reactivities of compounds in the immediate vicinity of where it is produced.¹⁶ In intact ECs, the closest targets for HOCl to react are the sulfhydryl groups of the cell membrane; however, in the absence of cell membranes, HOCl can react with multiple targets, including eNOS cofactors or eNOS itself, resulting in impaired eNOS activity.

Neither HOCl nor H₂O₂ blunted SNP-stimulated cGMP production by ECs, which suggests that guanylate cyclase can withstand the oxidative stress imposed by these agents. Moreover, the preserved response to SNP and the lack of LDH release by ECs exposed to H₂O₂ or HOCl provide strong evidence that cell viability was not affected by exposure to these oxidants.

The present studies suggest that ROS such as H₂O₂ and HOCl contribute to decreased NO bioactivity observed in vascular ischemia/reperfusion processes and, perhaps, in atherosclerosis⁵⁸ by interfering with the mechanisms that participate in NO synthesis and release without affecting the intrinsic eNOS catalytic activity. We speculate that this novel effect of H₂O₂ and HOCl on NO production contributes to altered adhesive interactions between leukocytes and endothelium as well as disregulated vascular tone, which occurs during ischemia/reperfusion, vascular inflammation, and atherosclerosis.

	Acknowledgments

 
The present work was supported by research funds from the Department of Veterans Affairs. We are thankful for the secretarial assistance of Barb Devereaux and Betty Mart.

Received July 26, 2000; first decision September 19, 2000; accepted March 15, 2001.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. McCord JM. Oxygen-derived free radicals: a link between reperfusion injury and inflammation. Fed Proc. 1987; 46: 2402–2406.[Medline] [Order article via Infotrieve]
2. Halliwell B. Reactive oxygen species in living systems: source, biochemistry, and role in human disease. Am J Med. 1991; 91 (suppl 3C): 14S–22S.[Medline] [Order article via Infotrieve]
3. Farber JL, Kyle ME, Coleman JB. Mechanisms of cell injury by activated oxygen species. Lab Invest. 1990; 62: 670–675.[Medline] [Order article via Infotrieve]
4. Bast A, Haenen GRMM, Doelman CJA. Oxidants and antioxidants: state of the art. Am J Med. 1991; 91 (suppl 3C): 2S–13S.[Medline] [Order article via Infotrieve]
5. Revak SD, Rice CL, Schraufstatter IU, Halsey WAJ, Bohl BP, Clancy RM, Cochrane CG. Experimental pulmonary inflammatory injury in the monkey. J Clin Invest. 1984; 76: 1182–1192.
6. McCord J, Day EJ. Superoxide-dependent production of hydroxyl radical catalyzed by iron-EDTA complex. FEBS Lett. 1978; 86: 139–142.[Medline] [Order article via Infotrieve]
7. Beckman ST, Beckman TW, Chen J, Marshall PM, Freeman BA. Apparent hydroxyl radical production from peroxynitrite: implications for endothelial injury by nitric oxide and superoxide. Proc Natl Acad Sci U S A. 1990; 87: 1620–1624.[Abstract/Free Full Text]
8. Hyslop PA, Hinshaw DB, Schraufstatter IU, Sklar LA, Spragg RG, Cochrane CG. Intracellular calcium homeostasis during hydrogen peroxide injury to cultured P388D1 cells. J Cell Physiol. 1986; 129: 356–366.[Medline] [Order article via Infotrieve]
9. Harlan JM, Levine JD, Callahan KS, Schwartz BR, Harker LA. Glutathione redox cycle protects cultured endothelial cells against lysis by extracellularly generated hydrogen peroxide. J Clin Invest. 1984; 73: 706–713.
10. Spragg RH, Hinshaw DB, Hyslop PA, Schraufstatter IU, Cochrane CG. Alterations in adenosine triphosphate and energy charge in cultured endothelial and PD388D1 cells after oxidant injury. J Clin Invest. 1985; 76: 1471–1476.
11. Schraufstatter IU, Hinshaw DB, Hyslop PA, Spragg RG, Cochrane CG. Oxidant injury of cells. DNA strand-breaks activate polyadenosine diphosphate-ribose polymerase and lead to depletion of nicotinamide adenine dinucleotide. J Clin Invest. 1986; 77: 1312–1320.
12. Schraufstatter IU, Hinshaw DB, Hyslop PA, Spragg RG, Cochrane CG. Glutathione cycle activity and pyridine nucleotide levels in oxidant-induced injury of cells. J Clin Invest. 1985; 76: 1131–1139.
13. Schraufstatter IU, Hyslop PA, Hinshaw DB, Spragg RG, Sklar LA, Cochrane CG. Hydrogen peroxide-induced injury of cells and its prevention by inhibitors of poly(ADP-ribose) polymerase. Proc Natl Acad Sci U S A. 1986; 83: 4908–4912.[Abstract/Free Full Text]
14. Hinshaw DB, Sklar LA, Bohl B, Schraufstatter IU, Hyslop PA, Rossi MW, Spragg RG, Cochrane CG. Cytoskeletal and morphologic impact of cellular oxidant injury. Am J Pathol. 1986; 123: 454–464.[Abstract]
15. Hyslop PA, Hinshaw DB, Halsey WA, Schraufstatter IU, Sauerheber RD, Spragg RG, Jackson JH, Cochrane CG. Mechanisms of oxidant-mediated cell injury: the glycolytic and mitochondrial pathways of ADP phosphorylation are major intracellular targets inactivated by hydrogen peroxide. J Biol Chem. 1988; 263: 1665–1675.[Abstract/Free Full Text]
16. Schraufstatter I, Browne K, Harris A, Hyslop P, Jackson J, Quehenberger O, Cochrane C. Mechanisms of hypochlorite injury of target cells. J Clin Invest. 1990; 85: 554–562.
17. Foote CS, Goyne TE, Lehrer RI. Assessment of chlorination by human neutrophils. Nature (Lond). 1981; 301: 715–716.
18. Thomas EL, Jefferson MM, Grisham MB. Myeloperoxidase-catalyzed incorporation of amines into proteins: role of hypochlorous acid and dichloramines. Biochemistry. 1982; 21: 6299–6308.[Medline] [Order article via Infotrieve]
19. Winterbourn CC, VandenBerg JJM, Ritman E, Kuypers FA. Chlorohydrin formation from unsaturated fatty acids reacted with hypochlorous acid. Arch Biochem Biophys. 1992; 296: 547–555.[Medline] [Order article via Infotrieve]
20. Cochrane CG. Cellular injury by oxidants. Am J Med. 1991; 91 (suppl 3C): 23S–30S.[Medline] [Order article via Infotrieve]
21. Schraufstatter IU, Browne K, Harris A, et al. Mechanisms of hypochlorite (HOCl) injury of target cells. J Clin Invest. 1990; 85: 554–562.
22. Shaul PW, Smart EJ, Robinson LJ, German Z, Yuhanna IS, Ying Y, Anderson RG, Michel T. Acylation targets endothelial nitric-oxide synthase to plasmalemmal caveolae. J Biol Chem. 1996; 271: 6518–6522.[Abstract/Free Full Text]
23. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev. 1991; 43: 109–141.[Medline] [Order article via Infotrieve]
24. Ihnken K, Morita K, Buckberg G, Winkelman B, Schmitt M, Ignarro L, Sherman M, Nitric-oxide-induced reoxygenation injury in the cyanotic immature heart is prevented by controlling oxygen content during initial reoxygenation. Angiology. 1997; 48: 189–202.
25. Conger J, Robinette J, Villar A, Raij L, Shultz P. Increased nitric oxide synthase activity despite lack of response to endothelium-dependent vasodilators in postischemic acute renal failure in rats. J Clin Invest. 1995; 96: 631–638.
26. Wei E, Kontos H. H₂O₂and endothelium-dependent cerebral arterial dilation: implications for the identity of endothelium-derived relaxing factor generated by acetylcholine. Hypertension. 1990; 16: 162–169.[Abstract/Free Full Text]
27. Rajagopalan S, Kurz S, Munzel T, Tarpey M, Freeman BA, Griendling KK, Harrison DG. Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation: contribution to alterations of vasomotor tone. J Clin Invest. 1996; 97: 1916–1923.[Medline] [Order article via Infotrieve]
28. Fournet-Bourguignon MP, Castedo-Delrieu M, Bidouard JP, Leonce S, Saboureau D, Delescluse I, Vilaine JP, Vanhoutte PM. Phenotypic and functional changes in regenerated porcine coronary endothelial cells: increased uptake of modified LDL and reduced production of NO. Circ Res. 2000; 86: 854–861.[Abstract/Free Full Text]
29. Shultz PJ, Tayeh MA, Marletta MA, Raij L. Synthesis and action of nitric oxide in rat glomerular mesangial cells. Am J Phsyiol. 1991; 30: F600–F606.
30. Lowry O, Rosenbrough N, Farr A, Randall R. Protein measurement with the protein phenol method. J Biol Chem. 1951; 193: 265–275.[Free Full Text]
31. Jaimes EA, Nath KA, Raij L. Hydrogen peroxide downregulates IL-1 stimulated iNOS activity in mesangial cells: implications for glomerulonephritis. Am J Physiol. 1997; 272: F721–F728.[Abstract/Free Full Text]
32. Kurose I, Wolf R, Grisham MB, Granger DN. Modulation of ischemia/reperfusion-induced microvascular dysfunction by nitric oxide. Circ Res. 1994; 74: 376–382.[Abstract/Free Full Text]
33. Sullivan GW, Sarembock IJ, Linden J. The role of inflammation in vascular diseases. J Leukoc Biol. 2000; 67: 591–602.[Abstract]
34. Kokura S, Wolf RE, Yoshikawa T, Granger DN, Aw TY. T-lymphocyte-derived tumor necrosis factor exacerbates anoxia- reoxygenation-induced neutrophil-endothelial cell adhesion. Circ Res. 2000; 86: 205–213.[Abstract/Free Full Text]
35. Kurose I, Wolf R, Grisham MB, Aw TY, Specian RD, Granger DN. Microvascular responses to inhibition of nitric oxide: role of active oxidants. Circ Res. 1995; 76: 30–39.[Abstract/Free Full Text]
36. Zweier JL, Broderick R, Kuppusamy P, Thompson-Gorman S, Lutty GA. Determination of the mechanism of free radical generation in human aortic endothelial cells exposed to anoxia and reoxygenation. J Biol Chem. 1994; 269: 24156–24162.[Abstract/Free Full Text]
37. Griendling KK, Minieri CA, Ollerenshaw JD, Alexander RW. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res. 1994; 74: 1141–1148.[Abstract/Free Full Text]
38. Zhang H, Schmeisser A, Garlichs CD, Plotze K, Damme U, Mugge A, Daniel WG. Angiotensin II-induced superoxide anion generation in human vascular endothelial cells: role of membrane-bound NADH-/NADPH-oxidases. Cardiovasc Res. 1999; 44: 215–222.[Abstract/Free Full Text]
39. Jaimes EA, Galceran JM, Raij L. Angiotensin II induces superoxide anion production by mesangial cells. Kidney Int. 1998; 54: 775–784.[Medline] [Order article via Infotrieve]
40. Zafari AM, Ushio-Fukai M, Akers M, Yin Q, Shah A, Harrison DG, Taylor WR, Griendling KK. Role of NADH/NADPH oxidase-derived H₂O₂in angiotensin II-induced vascular hypertrophy. Hypertension. 1998; 32: 488–495.[Abstract/Free Full Text]
41. Sundaresan M, Yu ZX, Ferrans VJ, Irani K, Finkel T. Requirement for generation of H₂O₂for platelet-derived growth factor signal transduction. Science. 1995; 270: 296–299.[Abstract/Free Full Text]
42. Reckelhoff JF, Zhang H, Srivastava K, Roberts LJ2nd, Morrow JD, Romero JC. Subpressor doses of angiotensin II increase plasma F(2)-isoprostanes in rats. Hypertension. 2000; 35: 476–479.[Abstract/Free Full Text]
43. Pagano PJ, Clark JK, Cifuentes-Pagano ME, Clark SM, Callis GM, Quinn MT. Localization of a constitutively active, phagocyte-like NADPH oxidase in rabbit aortic adventitia: enhancement by angiotensin II. Proc Natl Acad Sci U S A. 1997; 94: 14483–14488.[Abstract/Free Full Text]
44. Mombouli JV, Vanhoutte PM. Endothelial dysfunction: from physiology to therapy. J Mol Cell Cardiol. 1999; 31: 61–74.[Medline] [Order article via Infotrieve]
45. Schnackenberg CG, Welch WJ, Wilcox CS. Normalization of blood pressure and renal vascular resistance in SHR with a membrane-permeable superoxide dismutase mimetic: role of nitric oxide. Hypertension. 1998; 32: 59–64.[Abstract/Free Full Text]
46. Warnholtz A, Nickenig G, Schulz E, Macharzina R, Brasen JH, Skatchkov M, Heitzer T, Stasch JP, Griendling KK, Harrison DG, Bohm M, Meinertz T, Munzel T. Increased NADH-oxidase-mediated superoxide production in the early stages of atherosclerosis: evidence for involvement of the renin-angiotensin system. Circulation. 1999; 99: 2027–2033.[Abstract/Free Full Text]
47. Yang BC, Phillips MI, Mohuczy D, Meng H, Shen L, Mehta P, Mehta JL. Increased angiotensin II type 1 receptor expression in hypercholesterolemic atherosclerosis in rabbits. Arterioscler Thromb Vasc Biol. 1998; 18: 1433–1439.[Abstract/Free Full Text]
48. Prasad A, Tupas-Habib T, Schenke WH, Mincemoyer R, Panza JA, Waclawin MA, Ellahham S, Quyyumi AA. Acute and chronic angiotensin-1 receptor antagonism reverses endothelial dysfunction in atherosclerosis. Circulation. 2000; 101: 2349–2354.[Abstract/Free Full Text]
49. Fujii H, Ichimori K, Hoshiai K, Nakazawa H. Nitric oxide inactivates NADPH oxidase in pig neutrophils by inhibiting its assembling process. J Biol Chem. 1997; 272: 32773–32778.[Abstract/Free Full Text]
50. Cahill PA, Redmond EM, Foster C, Sitzmann JV. Nitric oxide regulates angiotensin II receptors in vascular smooth muscle cells. Eur J Pharmacol. 1995; 288: 219–229.[Medline] [Order article via Infotrieve]
51. Hazen SL, Hsu FF, Mueller DM, Crowley JR, Heinecke JW. Human neutrophils employ chlorine gas as an oxidant during phagocytosis. J Clin Invest. 1996; 98: 1283–1289.[Medline] [Order article via Infotrieve]
52. Anderson MM, Hazen SL, Hsu FF, Heinecke JW. Human neutrophils employ the myeloperoxidase-hydrogen peroxide-chloride system to convert hydroxy-amino acids into glycolaldehyde, 2-hydroxypropanal, and acrolein: a mechanism for the generation of highly reactive alpha-hydroxy and alpha,beta-unsaturated aldehydes by phagocytes at sites of inflammation. J Clin Invest. 1997; 99: 424–432.[Medline] [Order article via Infotrieve]
53. Glomb MA, Monnier VM. Mechanism of protein modification by glyoxal and glycolaldehyde, reactive intermediates of the Maillard reaction. J Biol Chem. 1995; 270: 10017–10026.[Abstract/Free Full Text]
54. Haberland ME, Fong D, Cheng L. Malondialdehyde-altered protein occurs in atheroma of Watanabe heritable hyperlipidemic rabbits. Science. 1988; 241: 215–218.[Abstract/Free Full Text]
55. Milstien S, Katusic Z. Oxidation of tetrahydrobiopterin by peroxynitrite: implications for vascular endothelial function. Biochem Biophys Res Commun. 1999; 263: 681–684.[Medline] [Order article via Infotrieve]
56. Vaziri ND, Ding Y, Ni Z. Nitric oxide synthase expression in the course of lead-induced hypertension. Hypertension. 1999; 34: 558–562.[Abstract/Free Full Text]
57. Peterson TE, Poppa V, Ueba H, Wu A, Yan C, Berk BC. Opposing effects of reactive oxygen species and cholesterol on endothelial nitric oxide synthase and endothelial cell caveolae. Circ Res. 1999; 85: 29–37.[Abstract/Free Full Text]
58. Raij L, Nagy J, Coffee K, DeMaster EG. Hypercholesterolemia promotes endothelial dysfunction in vitamin E- and selenium-deficient rats. Hypertension. 1993; 22: 56–61.[Abstract/Free Full Text]

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