INTRODUCTION

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NITRIC OXIDE, released by the vascular endothelium in response to shear stress and pulsatile flow, is a potent vasodilator that performs an important physiological role in the normal regulation of vascular tone (25). Moreover, the vascular endothelium and smooth muscle express the inducible isoform of nitric oxide synthase in response to inflammatory cytokines, leading to the generation of nitric oxide in high concentration (28). Excess nitric oxide produced via the cytokine-mediated activation of the inducible isoform of nitric oxide synthase may be responsible for the hypotension and vasorelaxation characteristic of early septic shock (22). Whether this effect is directly mediated by nitric oxide through activation of vascular smooth muscle soluble guanylate cyclase or is indirectly mediated through the formation of secondary reaction products of nitric oxide has not been established.

Nitric oxide reacts with superoxide anion to form the potent oxidant peroxynitrite (1, 10). The second-order rate constant for the reaction of nitric oxide with superoxide anion is 6.7 × 10⁹ M¹ · s¹ (10), approximately three times greater than the rate of superoxide dismutase-catalyzed dismutation of superoxide anion. Therefore, nitric oxide is capable of outcompeting superoxide dismutase for superoxide anion, making peroxynitrite formation a favored reaction under conditions such as atherosclerosis, ischemia-reperfusion, and sepsis, in which cellular production of nitric oxide and superoxide anion is increased. In vitro cellular production of peroxynitrite has been demonstrated for alveolar macrophages (12), endothelial cells (16), vascular smooth muscle cells (4), and neutrophils (5).

In addition to its role in oxidative reactions, peroxynitrite nitrates free or protein-associated tyrosines and other phenolics either spontaneously or via the low-molecular-mass transition metal-, superoxide dismutase- (2), or carbon dioxide-catalyzed (8) formation of a nitronium ion-like intermediate. With the use of specific antibodies that recognize nitrotyrosine, protein tyrosine nitration has been demonstrated in animal models of endotoxemia (30) and acute lung injury (11) and in human inflammatory diseases, including coronary atherosclerosis (3), myocarditis (15), acute lung injury (18), and sepsis (15, 18). The specificity of tyrosine nitration as a marker for the presence of peroxynitrite has recently come into question (7). Under physiological conditions, however, the nitration of protein tyrosines occurs only in the presence of peroxynitrite or the simultaneous presence of nitric oxide and superoxide anion (8), demonstrating that nitrotyrosine may be a specific in vivo marker for the formation of peroxynitrite.

Peroxynitrite exhibits nitric oxide-like biological activity in vitro, inducing coronary (19, 31) and pulmonary artery (33) relaxation and inhibiting platelet aggregation (23). Moreover, the systemic administration of peroxynitrite produces pronounced hypotensive and vasodilator responses in pentobarbital-anesthetized rats (14, 17). The cellular mechanisms responsible for the acute hypotension and vasodilatation produced by peroxynitrite have not been fully established; however, these effects do not appear to involve the formation of circulating S-nitrosothiols (9).

In the isolated perfused rat heart, rapid tachyphylaxis develops to the coronary artery vasorelaxant effects of peroxynitrite and, after the development of tachyphylaxis to peroxynitrite, vasorelaxation to acetylcholine, isoproterenol, and S-nitroso-N-acetylpenicillamine are also inhibited (31). Moreover, tachyphylaxis to the hemodynamic effects of peroxynitrite develops rapidly after repeated systemic administration of peroxynitrite to pentobarbital-anesthetized rats (14, 17). Furthermore, after the development of tachyphylaxis to peroxynitrite in vivo, a sustained increase in mean arterial blood pressure and substantial increases in hindquarter, renal, and mesenteric vascular resistances occurs over time (14). These peroxynitrite-mediated changes in vascular resistance may result from 1) altered endothelial release of vascular smooth muscle relaxing factors or 2) altered vascular smooth muscle response to endothelium-derived relaxing factors.

Because peroxynitrite has been demonstrated within the vasculature in animal models of endotoxemia (30) and human inflammatory diseases (3, 15, 18), characterization of the vascular responses to peroxynitrite and the subsequent alterations in vascular reactivity in vivo may be relevant for understanding the pathophysiology of altered vascular function in inflammation-mediated disease states.

	MATERIALS AND METHODS

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Rats and surgical procedures. The experimental protocols described in this manuscript were approved by the Institutional Animal Care and Use Committee of The University of Iowa (Iowa City, IA). Sprague-Dawley rats (Madison, WI) weighing between 250 and 350 g were anesthetized with pentobarbital sodium (50 mg/kg ip) and were surgically implanted with femoral arterial and venous catheters (PE-50, Becton Dickinson, Sparks, MD) for the measurement of pulsatile and mean arterial blood pressure and the administration of chemicals, respectively. Immediately after catheterization, a midline laparotomy was performed and miniature pulse Doppler flow probes (Crystal Biotech, Northborough, MA) were placed around the lower abdominal aorta and renal and superior mesenteric arteries for the measurement of hindquarter, renal, and mesenteric blood flow velocities, respectively, and for the determination of hindquarter, renal, and mesenteric vascular resistances. To maintain anesthesia, supplemental doses of pentobarbital (5 mg iv) were given as necessary throughout the experiments.

Experimental protocols. For each of the studies, stock peroxynitrite was injected into the femoral venous catheter and immediately infused into the animal using 0.15 ml of sterile saline. In the first group of studies, peroxynitrite (1-10 µmol/kg iv; n = 5) was systemically administered and the changes in mean arterial pressure and hindquarter, renal, and mesenteric blood flow were recorded. In a second group of studies, 10 successive doses of peroxynitrite (10 µmol/kg iv; n = 8) were systemically administered and the changes in mean arterial pressure and hindquarter, renal, and mesenteric blood flow were recorded for each dose.

Materials. Peroxynitrite was synthesized in a quench flow reactor as previously described (1). Briefly, solutions of 0.6 M NaNO₂ and 0.6 M HCl/0.7 M H₂O₂ were vacuum suctioned into a T junction and mixed in glass tubing. The acid-catalyzed reaction of nitrous acid with hydrogen peroxide to form peroxynitrous acid was quenched by adding 1.5 M NaOH into a second T junction at the end of the glass tubing. Excess hydrogen peroxide was removed by the addition of hydrated manganese dioxide, which was subsequently removed by filtration. The peroxynitrite solution was stored at 70°C. Before each study, the concentration of peroxynitrite was determined spectrophotometrically (₃₀₂ = 1,670 M¹ · cm¹) to be 120-130 mM. To decompose peroxynitrite, a sample of peroxynitrite was left at 20°C for 2-3 wk, after which no further absorbance was noted at 302 nm. Acetylcholine and bradykinin were from Sigma Chemical (St. Louis, MO). MAHMA NONOate was from Alexis Biochemicals (San Diego, CA). Prostacyclin was from Cayman Chemical (Ann Arbor, MI). Pentobarbital and sterile saline, for administration and dilution of chemicals, respectively, were from Abbott Laboratories (North Chicago, IL).

Statistics. The data were analyzed by repeated-measures ANOVA followed by Student's modified t-test with the Bonferroni correction for multiple comparisons. The SE were derived from the formula (EMS/n)^1/2, where EMS is the error mean square term from the ANOVA and n is the number of rats. A value of P < 0.05 was taken to denote statistical significance.

	RESULTS

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Effects of peroxynitrite on hemodynamic variables. A typical example of the dose-dependent changes in mean arterial pressure and vascular flows produced by the systemic administration of peroxynitrite is demonstrated in Fig. 1. The systemic administration of peroxynitrite produced dose-dependent decreases in mean arterial pressure and hindquarter and mesenteric vascular resistances but relatively little change in renal vascular resistance (Fig. 2).

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Hemodynamic effects of peroxynitrite. Increasing doses of peroxynitrite (1.0-10 µmol/kg) were systemically administered to pentobarbital-anesthetized rats, and changes in mean arterial pressure (MAP), hindquarter blood flow (HQF), renal blood flow (RF), and mesenteric blood flow (MF) were recorded. A typical hemodynamic dose response to peroxynitrite is demonstrated.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Hemodynamic effects of peroxynitrite. Increasing doses of peroxynitrite (1.0-10 µmol/kg) were systemically administered to pentobarbital-anesthetized rats. Changes in MAP and hindquarter, renal, and mesenteric vascular resistances produced by peroxynitrite are expressed as means ± SE (n = 5) of %changes (%) from baseline.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Tachyphylaxis to hemodynamic effects of peroxynitrite. Ten consecutive bolus doses of peroxynitrite (10 µmol/kg) were systemically administered to pentobarbital-anesthetized rats, and changes in MAP, HQF, RF, and MF were recorded. Typical hemodynamic responses produced by 1st, 5th, and 10th doses of peroxynitrite are demonstrated (indicated by arrows).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Tachyphylaxis to hemodynamic effects of peroxynitrite. Ten consecutive bolus doses of peroxynitrite (10 µmol/kg) were systemically administered to pentobarbital-anesthetized rats. Changes in MAP and hindquarter, renal, and mesenteric vascular resistances are expressed as means ± SE (n = 8) of %changes from baseline for each of the 10 doses. Changes in duration of hypotensive effect are expressed as means ± SE (n = 8) in seconds for each of the 10 doses. * P < 0.05, subsequent doses vs. 1st dose.

Effects of peroxynitrite on hemodynamic actions of acetylcholine, bradykinin, MAHMA NONOate, and prostacyclin. The systemic administration of acetylcholine (100-800 ng/kg) produced dose-dependent hypotensive and vasodilator responses in each of the vascular beds (Fig. 5). The hypotensive and vasodilator effects on the renal and mesenteric vasculature were significantly diminished after tachyphylaxis to peroxynitrite, whereas the vasodilator response in the hindquarter bed was not significantly altered (Fig. 5).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Effects of peroxynitrite on hemodynamic changes produced by acetylcholine. Acetylcholine (100-800 ng/kg) was systemically administered to pentobarbital-anesthetized rats before (pre-ONOO) and after (post-ONOO) systemic administration of peroxynitrite (100 µmol/kg total). Changes in MAP and hindquarter, renal, and mesenteric vascular resistances produced by acetylcholine are expressed as means ± SE (n = 6) of %changes from baseline. * P < 0.05, post-ONOO vs. pre-ONOO.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Effects of peroxynitrite on hemodynamic changes produced by bradykinin. Bradykinin (1-8 µg/kg) was systemically administered to pentobarbital-anesthetized rats pre- and post-ONOO (100 µmol/kg total). Changes in MAP and hindquarter, renal, and mesenteric vascular resistances produced by bradykinin are expressed as means ± SE (n = 6) of %changes from baseline. Note that there are no significant differences between pre- and post-ONOO.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Effects of peroxynitrite on hemodynamic changes produced by (Z)-1-{N-methyl-N-[6(N-methylammoniohexyl)amino]}diazen-1-ium-1,2-diolate (MAHMA NONOate). MAHMA NONOate (2.5-50 nmol/kg) was systemically administered to pentobarbital-anesthetized rats pre- and post-ONOO (100 µmol/kg total). Changes in MAP and hindquarter, renal, and mesenteric vascular resistances produced by MAHMA NONOate are expressed as means ± SE (n = 6) of %changes from baseline. * P < 0.05, post-ONOO vs. pre-ONOO.

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Effects of peroxynitrite on hemodynamic changes produced by prostacyclin. Prostacyclin (1-5 nmol/kg) was systemically administered to pentobarbital-anesthetized rats pre- and post-ONOO (100 µmol/kg total). Changes in MAP and hindquarter, renal, and mesenteric vascular resistances produced by prostacyclin are expressed as means ± SE (n = 6) of %changes from baseline. * P < 0.05, post-ONOO vs. pre-ONOO.

	DISCUSSION

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The present study demonstrates that peroxynitrite is a potent vasorelaxant in the hindquarter and mesenteric vascular beds, but not in the renal vascular bed, of pentobarbital-anesthetized rats. Autoregulatory mechanisms may account for the lack of vasorelaxant effect within the renal vascular bed, suggesting that the in vivo effects produced by peroxynitrite are principally mediated at the resistance vessel level. The calculated biological half-life for peroxynitrite is ~0.6 s (29). On the basis of a cardiac output of 40 ml · min¹ · 100 g¹ and a circulating volume of 20 ml, the circulatory time for a 300-g rat would be 10 s, or 15 half-lives. Therefore, the concentration of peroxynitrite in the blood reaching resistance arteries after injection of 10 µmol/kg peroxynitrite would be ~5 nM. In isolated blood vessels, this concentration of peroxynitrite produces minimal relaxation (31), raising the possibility that peroxynitrite reacts with serum components to form biological vasorelaxants.

Peroxynitrite may be involved in the formation of a number of potential endogenous vasodilators including low-molecular-mass S-nitrosothiols (23), S-nitrosoalbumin (13), or nitrosoglucose adducts (24). In vitro peroxynitrite-mediated relaxation of coronary (19, 31) and pulmonary arteries (33), inhibition of platelet aggregation (23), and activation of soluble guanylate cyclase (20) are dependent on the presence of reduced thiols, suggesting peroxynitrite-mediated nitrosylation of thiols as the mechanism for peroxynitrite activity. Although the addition of peroxynitrite to buffer containing glutathione results in the formation of S-nitrosoglutathione, the yield is only 1-2%, and the addition of peroxynitrite to plasma does not result in the formation of detectable amounts of S-nitrosothiols (23). Moreover, in pentobarbital-anesthetized rats, L-penicillamine significantly attenuates the vasodilatory responses to S-nitrosocysteine, S-nitrosoglutathione, and S-nitrosoalbumin but has no effect on the hemodynamic responses to peroxynitrite (9), suggesting that the formation of S-nitrosothiols in the circulation is an unlikely mechanism for peroxynitrite-mediated vasorelaxation. When added to physiological buffer, peroxynitrite forms a nitrosoglucose adduct that has vasodilatory capabilities (24). With a yield of only 0.1%, however, the maximum serum concentration of the nitrosoglucose adduct formed from peroxynitrite administration in the present study would be ~150 nM. A comparable degree of vasodilatation was achieved at an approximate serum concentration of 2,500 nM for MAHMA NONOate. Whereas the presumed mechanism of nitrosoglucose adduct-mediated vasodilatation is through the release of nitric oxide, the nitrosoglucose adduct would not be expected to be a more potent vasodilator than MAHMA NONOate. Therefore, the low concentrations formed in the present study make the formation of a nitrosoglucose adduct unlikely to be of importance in peroxynitrite-mediated vasorelaxation. Furthermore, the repeated administration of peroxynitrite results in the development of tachyphylaxis but does not alter the hemodynamic effects produced by the nitric oxide donor MAHMA NONOate. Taken together, these findings raise the possibility that peroxynitrite does not mediate in vivo hemodynamic effects through the formation of chemical compounds that mediate vasorelaxation through the release of nitric oxide.

Although the calculated concentration of peroxynitrite present at the resistance vessel level is not sufficient to cause vasorelaxation in vitro, in experiments utilizing isolated vessels, peroxynitrite is added to a bath containing physiological buffer and the final concentration is calculated by assuming no degradation of peroxynitrite. Because of the short half-life of peroxynitrite at physiological pH, this assumption is incorrect, and the actual concentration of peroxynitrite experienced by the vascular smooth muscle may be much less. Moreover, the vasorelaxant effects of peroxynitrite are endothelium independent (19), suggesting that the vasodilator effects of peroxynitrite may be mediated by a direct interaction with the vascular smooth muscle. Indeed, recent evidence suggests that peroxynitrite may directly hyperpolarize vascular smooth muscle via the activation of ATP-sensitive potassium channels (32).

The present study confirms the development of rapid tachyphylaxis to peroxynitrite in pentobarbital-anesthetized rats (14, 17) with subsequent increases in vascular resistance over time (14). These vasoconstrictor effects are unlikely to be due to augmented sympathetic neurogenic vasomotor tone because the catecholamines norepinephrine and epinephrine produce substantially smaller vasoconstrictor responses in peroxynitrite-tolerant rats (17). Therefore, the peroxynitrite-mediated increase in vascular resistance may be due to an altered release of vasodilatory substances from the endothelium or, alternatively, an altered response of the vascular smooth muscle to endogenous vasodilatory substances.

The hemodynamic responses produced by the systemic administration of the endothelium-dependent vasodilator acetylcholine were significantly attenuated after the development of tachyphylaxis to peroxynitrite. In contrast, the hemodynamic responses produced by the systemic administration of bradykinin were not altered by the prior administration of peroxynitrite, demonstrating that peroxynitrite does not inhibit the endothelial release of vasorelaxing substances. Therefore, attenuation of acetylcholine-induced vasorelaxation may occur via peroxynitrite-mediated selective modification of the endothelial acetylcholine receptor or, alternatively, via peroxynitrite-mediated attenuation of the vascular smooth muscle response to acetylcholine-induced, endothelium-derived relaxing factors. Whereas the vasorelaxant responses produced by peroxynitrite are endothelium independent (19), the development of tachyphylaxis to peroxynitrite suggests that peroxynitrite directly alters vascular smooth muscle function.

The vascular smooth muscle cellular responses to prostacyclin and nitric oxide are similar; nevertheless, the systemic administration of peroxynitrite attenuates the vasodilatory actions of prostacyclin but not those of nitric oxide (MAHMA NONOate). In contrast to nitric oxide (27), however, prostacyclin mediates vascular smooth muscle relaxation, in part, via the activation of ATP-sensitive potassium channels (26). Moreover, recent evidence also suggests that peroxynitrite mediates vasorelaxation through the activation of vascular smooth muscle ATP-sensitive potassium channels (32). Therefore, tachyphylaxis to peroxynitrite and the peroxynitrite-mediated inhibition of prostacyclin-induced vasorelaxation may occur through inhibition of vascular smooth muscle ATP-sensitive potassium channel activation.

In conclusion, peroxynitrite is a potent in vivo vasodilator, the effects of which are subject to the development of rapid tachyphylaxis. The development of tachyphylaxis may be due to a direct peroxynitrite-mediated modification of ATP-sensitive potassium channels in the vascular smooth muscle, because the vasodilatory actions of prostacyclin, but not nitric oxide, were also attenuated. The peroxynitrite-mediated attenuation of prostacyclin-induced vasorelaxation may account for the development of increased vascular resistance after the development of tachyphylaxis to peroxynitrite. Therefore, the endogenous production of peroxynitrite within the vasculature may be important in the vascular pathophysiology of sepsis and other inflammatory conditions.