INTRODUCTION

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MYELOPEROXIDASE (MPO) is a heme protein synthesized in granules of neutrophils, monocytes, and macrophages. In response to cell activation, the enzyme is released in phagocytic vacuoles or into the extracellular space (29). Neutrophil activation also initiates the assembly of the enzyme NADPH oxidase that generates the oxidants superoxide anion (O) and H₂O₂. MPO catalyzes the oxidation of chloride by H₂O₂ resulting in the formation of the chlorinating and oxidizing species hypochlorous acid (HOCl) (8, 25). HOCl avidly reacts with a variety of cellular substrates, including thiols, nucleotides, and amines, to result in enhanced vascular permeability, tissue degradation, and DNA fragmentation (9-10, 31). Compared with its parent molecule H₂O_2, HOCl is 10-20 times more effective in oxidizing proteins (9). Because local concentrations of HOCl in inflamed tissues are estimated to be as high as 5 mM, there is significant potential for oxidative tissue injury (13, 34).

Whereas the critical role of HOCl in the host-defense response has been appreciated for some time, recent data suggest that HOCl also contributes to vascular injury associated with acute and chronic inflammatory diseases, including sepsis, atherosclerosis, reperfusion injury, and degenerative neurologic disorders (17, 29). HOCl has been implicated as a mediator of structural injury under these conditions. In this regard, it was shown that HOCl contributes to the degradation of matrix proteins by inhibiting tissue inhibitor of metalloproteinase-I (TIMP-1) and thus increasing the activity of matrix metalloproteinases (7, 32). HOCl also reduces the activity of ₁-antiproteinase, the normal function of which is to inactivate elastase (37). Combined effects of an elevation of elastase and increased degradation of TIMP-I by HOCl would be to enhance the breakdown of extracellular matrix proteins. Data also suggest a role for MPO-derived HOCl in atherogenesis. MPO has been colocalized with macrophages in human atherosclerotic lesions (20-21), and a recent report shows that HOCl modifies the apolipoprotein moiety of low-density lipoprotein, thus enhancing foam cell formation (20, 38). Furthermore, 3-chlorotyrosine, a reaction product of tyrosine and HOCl, has been identified as a marker of MPO-dependent injury in human atheromas (17).

Neutrophil adhesion and/or the elaboration of neutrophil-derived products may also induce functional changes in the vasculature (11-12, 14, 23, 27). Increased tissue MPO activity and HOCl formation have been implicated as mediators of reduced nitric oxide (NO) bioactivity, but the mechanism(s) underlying this inhibitory response is incompletely understood (5, 16, 24). Herein, data are presented showing that HOCl inhibits the endothelium-dependent relaxation of rat aortic ring segments. It is hypothesized that HOCl reduces NO bioavailability by converting endogenous L-arginine into an inactive substrate for the endothelial NO synthase (NOS III) isoform.

	MATERIALS AND METHODS

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Materials. Acetylcholine (ACh), A23187, sodium nitroprusside (SNP), L-methionine, L-arginine, D-arginine, L-NAME, phenylephrine (PE), and sodium hypochlorite were obtained from Sigma Pharmaceuticals; nitrate/nitrite and NOS activity assay kits were from Calbiochem, L-[³H]arginine was obtained from DuPont NEN, and a monoclonal NOS III antibody was from Transduction Laboratories. HOCl concentration was determined by monitoring the absorbance of hypochlorite at 292 nm ( = 350 M¹ · cm¹) in 0.1 N NaOH using a Beckman Diode Array Spectrophotometer model DU 7000.

Animals. Ten-week-old male Sprague-Dawley rats were obtained from Harlan Breeding Laboratories (Indianapolis, IN). All rats were maintained at constant humidity (60 ± 5%), temperature (24 ± 1°C), and light cycle (6 AM to 6 PM) and were fed a standard rat pellet diet (Ralston Purina Diet) ad libitum. All protocols were approved by the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham and were consistent with the Guide for the Care and Use of Laboratory Animals (NIH publication 85-23, Revised 1985).

Vessel reactivity studies. Isometric tension was measured in isolated aortic ring segments of Sprague-Dawley rats. After the rat was killed, the aorta was excised and cleansed of fat and adhering tissue. The vessel was cut into individual ring segments (2-3 mm in width) and suspended from a force-displacement transducer in a tissue bath. Ring segments were bathed in Krebs-Henseleit buffer of the following composition (mM): 118 NaCl, 4.6 KCl, 27.2 NaHCO₃, 1.2 KH₂PO₄, 1.2 MgSO₄, 1.75 CaCl₂, 0.03 Na₂EDTA, and 11.1 glucose. Buffer was maintained at 37°C and aerated with 95% O₂-5% CO₂. A passive load of 2 g was applied to all ring segments and maintained at this level throughout the experiment. At the beginning of each experiment, indomethacin-treated ring segments were depolarized with KCl (70 mM) to determine the maximal contractile capacity of the vessel. Rings were then thoroughly washed with Krebs-Henseleit buffer and allowed to equilibrate.

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Cell culture. Bovine aortic endothelial cells (BAECs) were isolated from aortas obtained from a local abattoir. BAECs were maintained in medium 199 containing 5% fetal bovine serum, 5% iron-supplemented calf serum, 10 µM thymidine, and penicillin-streptomycin. Low-passage (subcultures 4-7) BAECs were serum deprived 18 h before the study.

Measurement of endothelial cell NOS III protein and activity. Effects of HOCl pretreatment on NOS III protein were assessed by Western blot. BAECs were preincubated with HOCl (1-50 µM) for 1 h, followed with rinsing. Cells were homogenized in lysis buffer containing 1% Triton X-100 and protease inhibitors in Tris-buffered saline (pH 7.5). The protein was then denatured by boiling. Approximately 100 µg of protein from each sample was separated on a 6% SDS-polyacrylamide gel and transferred to nitrocellulose. The nitrocellulose membrane was blocked for 60 min with 5% dry milk and 0.01% Tween-20 in Tris-buffered saline. The blots were incubated overnight with primary monoclonal NOS III antibody (1:2,000 dilution). Immunoreactive bands were visualized using enhanced chemiluminescence (ECL, Amersham). Autoradiograms exposed in the linear range of film density were scanned and analyzed using a Fluorchem Digital Imaging System (Alpha Innotech).

Measurement of endothelial cell NO synthesis. NO production in BAECs was assessed by monitoring the formation of the NO metabolites nitrate (NO) and nitrite (NO). BAECs were pretreated with HOCl (0-50 µM) in serum-free media for 1 h, followed by washing. In some experiments, the HOCl scavenger L-methionine (50 µM) was concurrently added in the presence of 50 µM HOCl. In other experiments, BAECs were exposed to HOCl (50 µM) for 1 h, followed by rinsing and replacement with media containing 1 mM L-arginine for an additional 30 min. As controls, BAECs were pretreated with either L-arginine or L-methionine in the absence of HOCl. Cells were then exposed to the calcium ionophore A23187 (1 µM) for 2 h. Superoxide dismutase (200 U/ml) was added to the incubation medium to reduce cellular superoxide. Aliquots of media were sampled at the end of this period. NO present in the conditioned media was enzymatically reduced to NO by treatment with Escherichia coli-enriched nitrate reductase. Total NO was used as an index of NO production and was detected using the fluorophore 2,3-diaminonaphthalene (Calbiochem). Under alkaline conditions, NO converts 2,3-diaminonaphthalene to the fluorescent compound 1(H)-naphthotriazole. Nitrite concentration was monitored by the spectrofluorometric excitation of 1(H)-naphthotriazole at 360 nm and emission at 450 nm. A standard curve was constructed for NaNO₂ (0.1-1,000 nM).

Statistical analysis. All results are expressed as means ± SE. Dose-response profiles for different experimental conditions were analyzed and tested to determine differences in relaxation responses using the SigmaStat statistical analysis program. Unpaired observations were assessed by ANOVA and post hoc testing using the Student-Newman-Keuls test.

	RESULTS

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Treatment of rat aortic ring segments with HOCl (1-50 µM) resulted in a concentration-dependent inhibition of ACh-mediated relaxation (Fig. 1). This effect was persistent because the blunted response to ACh was maintained after the removal of HOCl from the tissue bath by extensive washing. The maximum relaxation (R_max) induced by 3 µM ACh decreased progressively with increasing concentration of HOCl (Fig. 1). There was a strong inverse correlation (R = 0.94; P < 0.001) between HOCl concentration and R_max. Concurrent incubation of ring segments with the HOCl scavenger L-methionine (50 µM) completely blocked the inhibitory effect of HOCl (50 µM) on vessel relaxation (Fig. 1). ACh-induced relaxation in vessels treated with L-methionine (50 µM) alone was similar to that of saline vehicle-treated controls. In related experiments, HOCl-treated ring segments were exposed to the receptor-independent vasodilator A23187 (Fig. 2). Relaxation induced by the calcium ionophore A23187 is dependent on endothelial NO production (30). HOCl inhibited the response of ring segments to A23187 in a concentration-dependent manner.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Concentration-dependent effects of hypochlorous acid (HOCl) on endothelium-dependent relaxation. A: rat aortic ring segments were incubated with 1 (, n = 7), 5 (, n = 5), 10 (, n = 7), or 50 µM (, n = 8) HOCl for 60 min. Saline vehicle was added to control ring segments (, n = 11). Tissues were then thoroughly washed to remove unreacted HOCl, and residual effects of the treatment on vessel function were assessed. Ring segments were submaximally contracted with phenylepinephrine (PE) followed by cumulative addition of the endothelium-dependent vasodilator ACh. HOCl inhibited ACh-induced relaxation in a concentration-dependent manner. B: concurrent incubation of ring segments with 50 µM L-methionine (, n = 7) completely blocked the inhibitory effect of 50 µM HOCl () on vessel relaxation. Response to ACh in ring segments treated with 50 µM L-methionine alone (, n = 7) was similar to that of saline vehicle controls. Data are means ± SE. *Significant difference (P < 0.05) compared with saline control.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   HOCl inhibits relaxation induced by A23187. Rat aortic ring segments were incubated with 1 (, n = 6), 5 (, n = 6), 10 (, n = 8), and 50 µM (, n = 5) HOCl for 60 min. Saline vehicle was added to control ring segments (, n = 9). Tissues were then thoroughly washed, and residual effects of HOCl on vessel function were tested. Ring segments were submaximally contracted with PE, followed by cumulative addition of the endothelium-dependent vasodilator A23187. HOCl inhibited A23187-induced relaxation in a concentration-dependent manner. Data are means ± SE. *Significant difference (P < 0.05) compared with saline control.

In other studies, the endothelium-independent vasodilator SNP was added to HOCl-treated ring segments. In contrast to responses observed with ACh and A23187, SNP-induced relaxation, which is mediated by the vascular smooth muscle cell metabolism of SNP and concomitant release of free NO, was unaffected by HOCl treatment (Fig. 3). In additional experiments, the contractile sensitivity of rat aortic ring segments to PE was tested in HOCl-treated vessels to determine whether the diminished vasodilator response to ACh was related to an increased sensitivity of ring segments to vasoconstrictor stimuli. HOCl did not affect the sensitivity of aortic ring segments to PE (not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   HOCl does not inhibit endothelium-independent relaxation. Rat aortic ring segments were incubated with HOCl at concentrations ranging from 0 (, n = 7); 1 (, n = 5); 10 (, n = 6), and 50 µM (, n = 6) for 60 min. Endothelium-independent vasodilator sodium nitroprusside (SNP) was then added to PE-contracted ring segments in a cumulative fashion. Preincubation with HOCl did not affect SNP-induced relaxation. Data are means ± SE.

To gain insight into the mechanism(s) by which endothelial dysfunction is induced, we monitored the effects of HOCl on the NO synthetic pathway. In initial experiments, NOS III protein was quantified in HOCl-treated BAECs. Densitometric analysis of immunoblots showed that prior exposure to HOCl did not result in a loss of NOS III protein (Fig. 4). Effects of HOCl on NOS III activity were assessed using an L-arginine to L-citrulline conversion assay. Because NOS III protein is localized in the cell membrane, we isolated membrane fractions of control and HOCl-treated BAECs. Aliquots of this fraction were added to PBS containing physiological concentrations of calcium, critical NOS III cofactors (NADPH, FAD, FMN, and tetrahydrobiopterin), and L-[³H]arginine (25 µCi/ml). L-[³H]arginine and L-[³H]citrulline were then separated on ion exchange media. In control experiments, L-[³H]arginine was converted to L-[³H]citrulline and inhibited by N^G-nitro-L-arginine methyl ester. Earlier incubation with HOCl did not affect L-[³H]arginine conversion in isolated membrane fractions (Fig. 4).

View larger version (15K): [in this window] [in a new window]   Fig. 4.   HOCl does not influence nitric oxide synthase (NOS) III protein or activity. Bovine aortic endothelial cells (BAECs) were treated with 0, 10, and 50 µM HOCl for 1 h, followed by rinsing with serum-free medium 199. Western blot analysis (A, top) shows no loss of NOS III protein in BAECs exposed to HOCl at concentrations up to 50 µM. Densitometric analysis of bands from 3 gels are depicted below the blot. B: NOS III enzymatic activity under these HOCl incubation conditions. Membrane fraction containing NOS III protein was isolated from BAECs that were previously treated with 1, 10, or 50 µM HOCl. In control experiments, an equivalent volume of saline vehicle or 1 mM NG-nitro-L-arginine methyl ester (L-NAME) was added to BAEC monolayers. Whereas control experiments showed that L-NAME inhibited NOS III activity by 70%, no effect of HOCl was noted. Data are means ± SE (n = 5 for each treatment group). *Significant difference (P < 0.05) compared with the L-NAME control group.

The residual effects of HOCl on total NO formation were monitored in A23187-stimulated BAECs. Over a 2-h treatment period, 1 µM A23187 stimulated an 85% increase in total NO above baseline. Pretreatment of BAECs with HOCl resulted in a concentration-dependent decrease in A23187-stimulated NO formation (Fig. 5). The maximum inhibitory effect of HOCl on total NO formation was blocked by concurrent incubation with L-methionine (50 µM). In some experiments, BAECs were treated with HOCl for 1 h and then rinsed with fresh media containing 1 mM L-arginine. Addition of L-arginine also prevented the inhibitory effect of HOCl on NO formation in A23187-stimulated cells. In the absence of HOCl, NO formation in BAECs pretreated with either L-methionine or L-arginine was similar to that of saline vehicle controls. In a final series of experiments, the effects of supplemental L-arginine on endothelium-dependent relaxation were tested in HOCl-treated aortic ring segments. Addition of L-arginine completely reversed the inhibitory effect of HOCl on ACh-induced relaxation. Under the same treatment conditions, D-arginine was without effect (Fig. 6).

View larger version (16K): [in this window] [in a new window]   Fig. 5.   HOCl inhibits the formation of endothelial cell NO metabolites. NO production in BAECs was assessed by monitoring the formation of the NO metabolites nitrate (NO) and nitrite (NO). Data are expressed as total NO. BAECs were pretreated with HOCl (0-50 µM) in serum-free media for 1 h, followed by washout. NO formation was stimulated by exposure of BAECs to the calcium ionophore A23187 (1 µM) for 2 h. Total NO formation was monitored in media samples using the fluorophore 2,3-diaminonaphthalene. HOCl inhibited NO formation in a concentration-dependent manner. The effect of 50 µM HOCl was prevented by concurrent inhibition with 50 µM L-methionine (L-meth). Addition of L-arginine (L-arg) after treatment with HOCl also reversed the inhibitory effect of 50 µM HOCl. Control experiments showed no effect of either L-arginine or L-methionine on A23187-stimulated NO formation in the absence of HOCl. Data are means ± SE (n = 12-18 for each treatment group). *Significant difference (P < 0.05) compared with saline vehicle treatment. #Significant difference (P < 0.05) compared with 50 µM HOCl treatment.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   L-Arginine reverses the inhibitory effect of HOCl on endothelium-independent relaxation. A: rat aortic ring segments were incubated with 10 µM HOCl for 1 h and washed. Tissues were then incubated with saline vehicle (, n = 7), 1 mM L-arginine (, n = 9), or 1 mM D-arginine (, n = 7) for 30 min. Tissues were then contracted with PE, and ACh dose-response experiments were performed. L-Arginine, but not D-arginine, completely reversed the inhibitory effect of HOCl on ACh-induced relaxation. The vasodilator response to ACh in control rats (, n = 11) is included for comparison. B: in the absence of HOCl, ACh-induced relaxation in ring segments pretreated with 1 mM L-arginine (, n = 7) or 1 mM D-arginine (, n = 9) was similar to that of saline vehicle controls (, n = 9). Data are means ± SE. *Significant difference (P < 0.05) compared with saline vehicle- and L-arginine-treated ring segments.

	DISCUSSION

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MPO-derived HOCl plays an important role in structural tissue injury under conditions of inflammation and ischemia-reperfusion (7, 9-10, 31-32). Previous studies also suggest a correlation between vascular MPO-HOCl content and a reduction of NO bioavailability (24, 35-36). In this regard, it was shown that infusion of HOCl into the guinea pig coronary circulation significantly reduces basal blood flow (24). Under these conditions, coronary vasodilation in response to ACh, bradykinin, and adenosine was abolished in vivo (24). Results of the current studies show that HOCl also impairs in vitro functional responses of rat arterial ring segments by inhibiting ACh-mediated relaxation. Additionally, the impairment of NO function was prevented by concurrent incubation of HOCl-treated vessels with the scavenger L-methionine. The inhibitory response to HOCl was persistent because it was maintained after the oxidant was removed from the tissue bath. Vasodilation elicited by the calcium ionophore A23187 was similarly inhibited by HOCl. These data suggest that HOCl treatment did not modify binding interactions between ACh and muscarinic receptors, but rather interfered with the signaling processes involved in the calcium-dependent synthesis of NO. In contrast, SNP-mediated relaxation was not altered by HOCl, suggesting that the "machinery" required for vessel relaxation was fully intact. Collectively, these data point to the endothelium as a critical site of HOCl action.

To gain insight into mechanism(s) underlying HOCl-dependent endothelial dysfunction, we assessed interactions between the oxidant and the NOS III synthetic pathway. A recent report suggests that the HOCl-dependent chlorination of NADPH alters the ability of the cofactor to support NADPH-dependent enzyme activity (2). Because NOS III activity requires NADPH, it is possible that the HOCl-dependent modification of this cofactor may limit the activity of NOS III. In the current studies, treatment of BAECs with HOCl did not result in loss of NOS III protein or activity. In these experiments, enzyme activity was measured in isolated membrane fractions in buffer that was replete with NOS III cofactors and substrates. HOCl treatment, however, significantly reduced formation of the NO metabolites NO and NO in A23187-stimulated BAECs. This response was reversed by exposure of BAECs to L-arginine. Results of these cellular studies are consistent with other mechanisms, including the possibility that HOCl limits the availability of L-arginine, the substrate for NOS III.

Addition of L-arginine to ring segments or cultured BAECs in the absence of HOCl did not enhance ACh-induced relaxation or NO formation, respectively. This suggests that supplemental L-arginine does not enhance NO production in substrate replete blood vessels or endothelial cells. Results of previous studies suggest that L-arginine depletion/modification contributes to the development of endothelial dysfunction in models of inflammatory vascular disease (26, 28). The impaired endothelium-dependent relaxation, characteristic of isolated blood vessels from hypercholesterolemic rabbits, can be reversed by the addition of exogenous L-arginine to tissues in vitro (4, 6). L-Arginine similarly protects vascular function in acute inflammatory models that are characterized by neutrophil adhesion and infiltration in the vessel wall (15, 33). Results of the present studies also point to endogenous L-arginine as a potential target of HOCl action. We found that addition of L-arginine to aortic ring segments after prior exposure to HOCl restored endothelium-dependent relaxation. In contrast, addition of D-arginine under these conditions was without effect. Whereas L-arginine is clearly a substrate for NO production in endothelial cells, D-arginine does not react with NOS III. These results suggest that the ability of endogenous L-arginine to serve as a substrate for NOS III is compromised by prior exposure to HOCl. The restoration of ACh-induced relaxation, which occurs with L-arginine supplementation, is likely due to an increase in functional levels of L-arginine in the endothelial cell.

Reactions of HOCl with -amino acids are well documented (18, 19). Specifically, activated neutrophils use MPO-derived HOCl to convert -amino acids into reactive aldehydes (19). This proceeds through a series of reactions in which the -amino acid is first converted to an -amino-monochloramine. A reactive carbonyl intermediate is formed which then undergoes molecular rearrangement to form the corresponding aldehyde. This reaction pathway can be blocked by catalase demonstrating a dependence on HOCl formation (19). Reactive aldehydes play an important role in tissue injury by covalently modifying proteins (1). Previous data support the biochemical modification of L-arginine as a component of endothelial dysfunction. In this respect, it was shown that methylation of L-arginine compromises NO production and contributes to the pathogenesis of inflammatory cardiovascular disease (3, 22, 28). It is hypothesized that the defective relaxation induced by HOCl in the current studies is also related to a modification of endogenous L-arginine. We recently found that HOCl reacts with L-arginine to form chlorinated metabolites that possess similar pharmacological properties as traditional NOS inhibitors (unpublished observation). HOCl may thus convert endothelial L-arginine into a new product that binds to NOS III in a reversible manner and acts as a competitive inhibitor of the enzyme. Clearly, additional studies are required to identify mechanisms underlying the HOCl-dependent inhibition of endothelial cell function.