INTRODUCTION

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THE FORMATION of peroxynitrite (ONOO) from O₂ and · NO is well recognized, but the bioactivity of ONOO within the vasculature is incompletely understood. The reactivity of ONOO, especially with thiol groups, suggests that the molecule has the ability to react with many target molecules and alter protein function (14). Moreover, nitration of tyrosine and tryptophan residues provides an alternative mechanism by which ONOO might alter protein function (1, 7).

The production of ONOO increases during the reperfusion of ischemic tissue (9, 17, 20). Ischemia-reperfusion represents the underlying mechanism of cerebral artery stroke, and it is possible that ONOO plays a critical role in this process. Several studies suggest that ONOO causes vasodilation (4, 10, 16, 19), and in this regard, ONOO might contribute to the hemorrhagic process that often complicates reperfusion of ischemic brain tissue. On the other hand, vasoconstriction caused by ONOO might contribute to the prolonged phase of rebound ischemia that follows initial reperfusion.

Arteries that supply or are derived from the circle of Willis are frequently involved in intracerebral stroke. The present study set out to investigate the effects of ONOO in this arterial bed and, specifically, in the middle cerebral artery. Because of its reactivity, ONOO is probably nonselective with regard to the cell types that constitute its target. However, this assumption has not been rigorously tested, and it is not clear whether ONOO acts directly on smooth muscle cells to alter vascular tone or whether it acts via other cell types within the vessel wall. To this end, we investigated the contractile activity of ONOO at the single cell level and report that ONOO causes myocyte contraction, an effect that is inhibited by reduced glutathione (GSH). At the whole vessel level, the effect of ONOO is dose dependent. At relatively low concentrations, ONOO causes vasoconstriction, whereas at higher concentrations, ONOO causes vessel injury, evidenced by persistent vasodilation and lack of responsiveness to the endogenous vasoconstrictor 5-hydroxytryptamine.

	MATERIALS AND METHODS

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Vessel preparation. Male Wistar rats (250-350 g; Harlan Sprague Dawley, Indianapolis, IN) were anesthetized via an intraperitoneal injection of pentobarbital sodium. After craniotomy, the brain was removed and the middle cerebral arteries were carefully excised and placed into HEPES-buffered physiological solution (HBS; pH 7.4) maintained at 37°C. Each end of a segment of artery was then carefully secured to a glass pipette. All side branches were tied off, then the bath solution was exchanged twice. The chamber was then transferred to the stage of a Zeiss inverted microscope fitted with a CCD camera (Cohu, San Diego, CA) connected to a video recorder. Arterioles were pressurized to 85 mmHg by adjusting the height of reservoirs connected to the pipettes. By setting both reservoirs to the same height, the vessels were pressurized without flow. Leaks were detected by a decline in intraluminal pressure after the vessel was closed to the reservoirs. Vessels with leaks were excluded from further study. Internal diameters were recorded throughout each experiment. Arteries prepared in this manner developed spontaneous tone.

Cell isolation. To isolate vascular smooth muscle cells, excised cerebral arteries were incubated at 4°C for 15 min in HBS (pH 7.4) that contained papain (1.5 mg/ml) and dithiothreitol (1.0 mg/ml). The solution was then brought to 37°C, and the tissue was incubated for an additional 17 min. After incubation for 25 min at 37°C in HBS containing collagenase (1.5 mg/ml) and trypsin inhibitor (1.0 mg/ml), the tissue was gently triturated with a Pasteur pipette to release single smooth muscle cells. Cells were kept on ice in HBS with 0.1% BSA until use. Cells were isolated on a daily basis and were used within 3 h of isolation. A total of 15 animals were used, resulting in a total of 54 cells. At least five animals were used in each set of experiments.

Measurement of cell contraction. To study cell contractile responses, an aliquot (100 µl) of the cell suspension was placed into a well of a 96-well plate, and the cells were allowed to attach to the surface during an equilibration period of 5 min. An equal volume (100 µl) of HBS containing CaCl₂ (1.8 mM) was added to achieve a working volume of 200 µl and a final concentration of CaCl₂ equal to 0.9 mM. Cells were allowed to equilibrate for an additional 5 min. Images were recorded using an Olympus CK2 inverted microscope connected to a Cohu CCD camera and a personal computer. Images (32 frames) were captured at baseline and 5 min after the addition of each agent. At the conclusion of each experiment, maximum cell contraction was induced by addition of KCl (final concentration 20 mM). Quantitative analysis of cell dimensions was carried out off-line under blinded conditions.

Synthesis of ONOO. An ice-cold, flowing solution of 1 M NaNO₂ was entrained with an equal volume of acidified H₂O₂ (1.8 M H₂SO₄-0.3 M H₂O₂), and the resultant admixture was dripped into a solution of 1.4 M NaOH. Granular MnO₂ was then added to catalyze the removal of H₂O₂. When effervescence subsided, the solution was filtered (no. 2, Whatman, Kent, UK) to remove MnO₂. The solution was subjected to freeze fractionation, then the uppermost layer containing the yellow ONOO salt was removed. The concentration of this stock solution of ONOO was determined by absorbance spectrophotometry, using the reported extinction coefficient for ONOO ( = 1,670 M¹ · cm¹), and was typically 150-300 mM. Immediately before each experiment, an aliquot of the stock solution was diluted into an ice-cold solution of 1 mM NaOH to achieve a final working concentration of 2 mM ONOO. Control experiments confirmed that the ONOO was stable in this solution through the time period of the experiment. All solutions of ONOO were protected from light and kept on ice or at <4°C until the time of addition to cells or vessels.

Addition of ONOO to cells. To study the effect of ONOO, an aliquot (10 µl) of a 2 mM solution was added to each well. The effective final concentration of ONOO reaching the cells was estimated using dihydrorhodamine 123. Under conditions identical to those used with cells present, the absorbance of dihydrorhodamine 123 at 500 nm was linearly related to 100 µM ONOO. With use of this estimate, addition of ONOO (100 µM) resulted in an effective concentration equal to 15 µM in the extracellular buffer. To test the effect of decomposed ONOO, the stock solution of ONOO was allowed to stand at room temperature for 2 h. The decay of ONOO was confirmed spectrophotometrically. In some experiments the contractile responses of cells to ONOO were tested in the presence of GSH (5 mM) or oxidized glutathione (GSSG, 2.5 mM).

Addition of ONOO to vessels. Aliquots of ONOO were prepared as described above and added to the 37°C chamber in which the artery was mounted. Each artery was obtained from a separate rat and subjected to only one dose of ONOO. The reported vessel diameters represent values recorded 4 min after addition of ONOO.

Reagents and solutions. Papain and collagenase type IV were purchased from Worthington Biochemical (Freehold, NJ). Dithiothreitol, soybean trypsin inhibitor type II, BSA, GSH, and GSSG were obtained from Sigma Chemical (St. Louis, MO). Dihydrorhodamine 123 was purchased from Molecular Probes (Eugene, OR). All buffer salts were of the highest purity available.

Data analysis. Values are means ± SE wherever applicable. Where indicated, n is the number of cells analyzed. The data represent the results obtained from a total of 15 animals. Differences between groups were determined using Student's two-tailed t-test. Statistical significance was assigned when the probability of -error was calculated to be <0.05.

Animal care. This project was reviewed and approved by the Animal Care Committee of the Medical College of Wisconsin. Animal care and use met the regulations and standards published by the Office of Animal Care and Use, National Institutes of Health.

	RESULTS

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Middle cerebral artery responses to ONOO. The contractile responses of middle cerebral arteries to ONOO were investigated. Isolated vessels were first equilibrated to 85 mmHg for 5 min. In response to 10 µM ONOO, arterial internal diameter decreased from 138 ± 7 to 118 ± 8 µm (n = 5). Of six vessels exposed to 25 µM ONOO, three contracted and three underwent dilation. In this respect, 25 µM ONOO appears to represent a watershed between contractile and dilatory responses. When exposed to 50 µM ONOO, vessels dilated from 169 ± 4 to 241 ± 17 µm (n = 3). Similarly, vessels dilated from 175 ± 7 to 241 ± 32 µm (n = 4) on addition of 100 µM ONOO. The dose-dependent responses of middle cerebral arteries to ONOO are depicted in Fig. 1.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Vessel responses of Wistar rat middle cerebral arteries to ONOO. At baseline, vessel internal diameter was 158 ± 4 µm (n = 21). Internal diameters were tracked in real time after addition of ONOO at 5-100 µM. Vessels were maintained at 37°C throughout all experiments. Each vessel was exposed to ONOO once only.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Responses of ONOO-treated vessels to 5-hydroxytryptamine (5-HT). Vessels were exposed to a single dose of ONOO at 5-100 µM before exchange of bath solution and rewarming to 37°C. 5-HT (5 µM) was then added to bath, and responses were recorded. For each concentration of ONOO, n = 3-6. Data represent responses of vessels used to generate Fig. 1. Each vessel was exposed to no more than a single dose of 5-HT.

Effect of ONOO on cell length. The net response of whole vessels to ONOO depends on the various responses contributed by multiple cell types within the vascular wall. The next set of experiments aimed to determine the direct effect of ONOO on smooth muscle cells, in the absence of confounding influences from other cell types. Vascular smooth muscle cells were freshly isolated from circle of Willis arteries. Under baseline conditions, cells were 82 ± 4 µm (n = 12) long and shortened by 30% to 57 ± 5 µm (P < 0.01) on addition of 100 µM ONOO (Fig. 3). Subsequent addition of KCl (20 mM) decreased cell length further to 45 ± 4 µm. The response to KCl was taken as maximal shortening, and thus the change in cell length triggered by ONOO was 60% of maximal shortening.

View larger version (79K): [in this window] [in a new window]   Fig. 3.   ONOO and vascular smooth muscle cell contraction. A: a typical cell at its resting length. B and C: its response to sequential additions of 100 µM ONOO and 20 mM KCl, respectively. In this and subsequent figures, effective concentration of ONOO was estimated to be 15 µM. Scale bars, 10 µm.

View larger version (77K): [in this window] [in a new window]   Fig. 4.   Decomposed ONOO and cell length. A: a typical cell at its resting length; B and C: its response to addition of decomposed ONOO and 20 mM KCl, respectively. Scale bars, 10 µm.

Extracellular glutathione and the contractile activity of ONOO. The activity of ONOO in whole vessels has been reported to be influenced by ambient glutathione. Therefore, we tested whether extracellular GSH influences the effect of ONOO on single cells. Extracellular GSH (5 mM) significantly inhibited the response of cells to ONOO (Figs. 5 and 6). In the presence of GSH, ONOO caused only a marginal change in cell length, from 72 ± 4 to 66 ± 4 µm (n = 14). Thus, as with the addition of decomposed ONOO or vehicle, cells retained ~90% of their original length. To determine whether the inhibitory effect of GSH is specific to its redox state, contractile responses were measured in the presence of GSSG. Extracellular GSSG (2.5 mM) had no effect on cell responses to ONOO (Figs. 5 and 7).

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Cumulative data for contractile responses of cerebrovascular smooth muscle cells to ONOO. Change in cell length (percent decrease from initial length) is plotted for each condition. Values are means ± SE for responses to ONOO (n = 12), ONOO in presence of glutathione (GSH, n = 14), and ONOO in presence of oxidized glutathione (GSSG, n = 12).

View larger version (79K): [in this window] [in a new window]   Fig. 6.   Effect of GSH on contractile action of ONOO. A: a typical cell at its resting length. B and C: its response to sequential additions of ONOO and 20 mM KCl, respectively, in presence of 5 mM GSH. Scale bars, 10 µm.

View larger version (84K): [in this window] [in a new window]   Fig. 7.   Effect of GSSG on contractile action of ONOO. A: a typical cell at its resting length. B and C: its response to sequential additions of ONOO and 20 mM KCl, respectively, in presence of 2.5 mM GSSG. Scale bars, 10 µm.

	DISCUSSION

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ONOO is emerging as a molecule of substantial biologic importance within the vasculature. As the reaction product of O₂ and · NO, ONOO probably is formed in all vascular beds. The modulation of vascular tone by ONOO appears to comprise several components. First, the reaction by which ONOO is formed consumes the endogenous vasodilator · NO. Second, ONOO itself consumes · NO (5). Third, the vasoactivity of ONOO might also comprise the S-nitrosation of GSH to S-nitrosoglutathione (GSNO) with the subsequent release of · NO (2, 11). This latter possibility is suggested by the observation that ONOO stimulates guanylyl cyclase in a GSH-dependent manner (15, 19). However, the yield of GSNO from ONOO is <1% (11, 12), and ONOO-induced dilation of cat pial vessels is unaffected by LY-83583, an inhibitor of guanylyl cyclase (18). Thus the contribution of GSNO in the vascular biology of ONOO is uncertain. A fourth component to the vasoactivity of ONOO is suggested by studies performed in acellular conditions, which indicate that, in the presence of glucose, organic buffers facilitate the formation of · NO from ONOO (13). Whereas this chemical mechanism might have contributed to the dilating effect of ONOO observed in some in vitro studies, the extent to which it contributes in vivo is unknown.

The data presented in this report indicate that ONOO causes middle cerebral arteries to constrict. This effect of ONOO is reversible, in that exchange of the bath solution causes the vessel to relax (data not shown). Moreover, the vessels remained sensitive to 5-hydroxytryptamine (Fig. 2). Indeed, after exposure of vessels to 10 µM ONOO, addition of 5 µM 5-hydroxytryptamine caused vessels to contract by 46%. This value is consistent with the 37% contraction responses observed in rat basilar arteries exposed to a similar concentration (1 µM) of 5-hydroxytryptamine (8). In contrast, exposure of arteries to vasodilatory concentrations of ONOO attenuated contractile responses to 5-hydroxytryptamine, suggesting that dilatory responses to ONOO reflect an injurious process that results in vascular dysfunction. Interestingly, decomposed ONOO elicited relaxation, indicating that at least some of the relaxant effect is due not to ONOO but to one or more of its breakdown products. At least one previous study reported a relaxant response to decomposed ONOO (4).

The present findings are consistent with those of Chabot et al. (4), who reported that isolated pulmonary artery rings from rats initially contracted in response to ONOO. These investigators found that ONOO, at concentrations as high as 100 µM, had no relaxant effect on quiescent rings. In the present experiments we found that the responses to ONOO were essentially unaffected by equilibration pressure and vessel diameter.

Although characterization of the bioactivity of ONOO in tissues and organs provides important information, more reduced systems are required if the effects of the oxidant are to be defined at the cellular level. To this end, the present study aimed to define the direct effects of ONOO on vascular smooth muscle cells. ONOO, when added to isolated cerebrovascular smooth muscle cells, caused the cells to contract in a robust manner. On application of ONOO, cells contracted by 30%. This degree of contraction was ~60% of the maximal response to KCl, indicating that the contraction triggered by ONOO is quantitatively and physiologically significant. Moreover, the effect of ONOO could not be replicated by decomposed ONOO or by vehicle.

In regard to the response of single myocytes to ONOO, two considerations emerge. First, a concentration of ONOO (100 µM) that causes dilation of whole vessels causes single smooth muscle cells to contract. This finding appears to confirm that multiple cell types are involved in the overall response of whole vessels to ONOO. At this time, it is not known which cell type is responsible for the vasodilation observed when relatively high doses of ONOO are employed.

The response of single cells to 10 µM ONOO provides the basis for a second conclusion to be drawn. On the basis of the known reaction kinetics and half-life of ONOO, a dose of 10 µM ONOO is exceedingly small. This is further supported by the dihydrorhodamine assay, which, in the present study, indicates that the effective concentration of ONOO is ~15% of that which is added. On addition of 10 µM ONOO, the degree of cell contraction was 42% of the maximal contraction to KCl. The contractile response to this tiny dose of ONOO suggests that, at least under the present experimental conditions, cerebrovascular smooth muscle cells are extremely sensitive to ONOO.

It is important to note, however, that the concentration of ONOO in vivo is not known. ONOO is a transient intermediate in free radical chemistry and is highly reactive at physiological pH. For this reason, ONOO cannot be considered in the same light as a pharmacological drug that has steady-state concentrations and measurable on and off binding rates. As with other reactive molecules and free radicals, the biologic effects of ONOO are determined by the flux of molecules through reaction pathways rather than by steady-state concentrations. However, in contrast to other reactive oxidants and free radicals, which have to be generated in situ in experimental paradigms, ONOO is unique, in that stable solutions can be prepared. Addition of a particular concentration of ONOO from a stable solution is not meant to imply that ONOO exists in vivo at that concentration in a steady state.

We hypothesized that GSH, a soluble thiol that is abundantly and endogenously present in vascular tissue, might modify the effect of ONOO. In the presence of GSH, ONOO had no contractile effect on single cells. The most reasonable explanation for this result is that GSH represents a surrogate target for ONOO, thereby preventing the oxidant from reaching cells. Consistent with this interpretation, GSSG failed to exert any effect. Thus the reduced sulfhydryl on glutathione is the moiety that provides GSH with its protective property. In the biological milieu, GSH likely serves as a key protector against the bioactivity of ONOO. This role for GSH is probably applicable not only to the extracellular compartment, but also to the cytosolic compartment, where ONOO can be formed as a result of the reaction between O₂ and · NO and where the ambient GSH concentration is typically in the millimolar range.

The mechanism underlying the contractile effect of ONOO on cerebrovascular cells is uncertain. Preliminary data from our own laboratory indicate that ONOO inhibits K⁺ currents in smooth muscle cells isolated from circle of Willis arteries (3). Inhibition of K⁺ current exerts a depolarizing influence on vascular smooth muscle cells, leading to the opening of L-type Ca²⁺ channels and activation of force-developing proteins. It is possible, therefore, that the effect of ONOO on membrane K⁺ permeability predominates in the absence of secondary influences from other cell types.

The bioactivity of ONOO with the vasculature ultimately depends on the oxidant's net effect on multiple types of cells, including cells of the vessel wall and cells that circulate within the bloodstream. For example, ONOO inhibits bradykinin-activated Ca²⁺ signaling in vascular endothelial cells (6), an effect that would be expected to inhibit Ca²⁺-dependent activation of endothelial cell nitric oxide synthase. The contractile agonist properties of ONOO, as reported here, suggest that any ONOO that is formed in a location abluminal to the endothelium has the potential to cause smooth muscle cell contraction and vasoconstriction. In this regard, ONOO might represent an oxidant that is mechanistically involved in many forms of pathological vasoconstriction, including the sustained phase of vasoconstriction that follows the initial reperfusion of ischemic tissue.