Role of Cl(-) channels in alpha-adrenoceptor-mediated vasoconstriction in the anesthetized rat.

In vitro studies have provided evidence that Cl(-) ion currents are important for activation of vascular smooth muscle contraction. The stilbene, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), disrupts Cl(-) metabolism by blocking Cl(-) channels and by inhibiting Cl(-) bicarbonate exchange. The aims of this study were to: (i) characterize the hemodynamic responses produced by DIDS in pentobarbital anesthetized rats, and (ii) examine vasoconstrictor responses to norepinephrine before and after administration of DIDS. DIDS (2.5-50 micromol/kg, 92.5 micromol/kg total dose, i.v.) produced dose-dependent but transient reductions in mean arterial blood pressure and in hindquarter, renal and mesenteric vascular resistances. Prior to the administration of DIDS, norepinephrine (1. 0-5.0 microgram/kg, i.v.) produced dose-dependent increases in mean arterial pressure, renal resistance and mesenteric resistance, but decreases in hindquarter resistance that were inversely related to dose. After administration of DIDS, the peak pressor responses produced by norepinephrine were either slightly diminished (1.0, 2.5 microgram/kg) or unchanged (5.0 microgram/kg). Peak norepinephrine-induced changes in hindquarter and renal vascular resistance were unaffected by DIDS, while increases in mesenteric resistance were augmented. The total norepinephrine-induced increases in mean arterial pressure (mm Hgxs) were markedly reduced by DIDS. These effects of DIDS on norepinephrine-induced responses were similar, but not identical to those of the voltage-sensitive Ca(2+) channel blocker, nifedipine (500 nmol/kg, i.v.). These findings suggest that DIDS may interfere with norepinephrine-induced depolarization of resistance arteries, thereby preventing activation of voltage-sensitive Ca(2+) channels.

Peroxynitrite-mediated attenuation of alpha- and beta-adrenoceptor agonist-induced vascular responses in vivo.

Peroxynitrite is produced by vascular endothelial and smooth muscle cells in response to inflammation, induces vascular relaxation, and alters vascular responses to endothelial-derived relaxing factors. The present study examined the changes in mean arterial pressure and hindquarter, renal, and mesenteric vascular resistances produced by the systemic administration of (i) the catecholamines epinephrine or norepinephrine, (ii) the alpha1-adrenoceptor agonist phenylephrine, (iii) the beta-adrenoceptor agonist isoproterenol or (iv) [Arg delta] vasopressin in pentobarbital-anesthetized rats prior to and following the systemic administration of peroxynitrite. The systemic administration of peroxynitrite significantly inhibited (i) epinephrine-induced pressor and renal and mesenteric vasoconstrictor responses, (ii) norepinephrine-induced pressor and hindquarter, renal, and mesenteric vasoconstrictor responses, (iii) phenylephrine-induced hindquarter and mesenteric vasoconstrictor responses, and (iv) isoproterenol-induced depressor and hindquarter and renal vasodilator responses. In comparison, the systemic administration of peroxynitrite had no effect on arginine vasopressin-induced pressor or vasoconstrictor responses. These results demonstrate selective and consequential attenuation of the hemodynamic effects produced by alpha- and beta-adrenoceptor agonists, suggesting that selective impairment of adrenoceptors by peroxynitrite may play a critical role in the hemodynamic dysfunction associated with inflammatory conditions.

Attenuation of vascular relaxation after development of tachyphylaxis to peroxynitrite in vivo.

Peroxynitrite, formed endogenously by the near diffusion-limited reaction of nitric oxide with superoxide anion, induces vascular relaxation. This effect is subject to rapid tachyphylaxis, suggesting that peroxynitrite may alter subsequent vasorelaxant responses. The present study examined the effects of peroxynitrite on mean arterial pressure and hindquarter, renal, and mesenteric vascular resistances in pentobarbital-anesthetized rats. Peroxynitrite induced dose-dependent decreases in mean arterial pressure and hindquarter and mesenteric vascular resistances. The repetitive administration of peroxynitrite resulted in the rapid development of tachyphylaxis, with subsequent doses producing progressively smaller effects. After the development of tachyphylaxis to peroxynitrite, the hemodynamic effects produced by the systemic administration of acetylcholine and prostacyclin were significantly attenuated, whereas the hemodynamic responses to bradykinin and the nitric oxide donor (Z)-1-¿N-methyl-N-[6(N-methylammoniohexyl)amino]¿diazen-1-++ +ium-1, 2-diolate (MAHMA NONOate) remained unchanged. These results demonstrate that 1) peroxynitrite is a potent vasorelaxant in vivo, 2) peroxynitrite-mediated vasodilatation is subject to the development of rapid tachyphylaxis, and 3) peroxynitrite alters the vascular smooth muscle response to prostacyclin, perhaps via inactivation of vascular smooth muscle ATP-sensitive potassium channel function.

Peroxynitrite-mediated vasorelaxation: evidence against the formation of circulating S-nitrosothiols.

Peroxynitrite-induced relaxation of isolated vessels may involve the formation of S-nitrosothiols. This study characterized the hemodynamic effects of systemically injected peroxynitrite in penotobarbital sodium-anesthetized rats and determined whether these effects were due to the formation of S-nitrosothiols. We utilized L-penicillamine, which attenuates the hemodynamic effects of systemically injected S-nitrosothiols. The hemodynamic effects of peroxynitrite and the S-nitrosothiols L-S-nitrosocysteine, L-S-nitrosoglutathione, and S-nitrosoalbumin were determined before and 25 min after the administration of L-penicillamine or saline. Peroxynitrite and the S-nitrosothiols produced dose-dependent reductions in mean arterial pressure and mesenteric and hindquarter vascular resistances. The hypotensive and vasodilator effects of the S-nitrosothiols were significantly reduced by L-penicillamine. In contrast, the hemodynamic actions of peroxynitrite were unaffected by L-penicillamine. Therefore, peroxynitrite produces hypotensive and vasodilator responses in anesthetized rats that are unlikely to be due to the formation of circulating S-nitrosothiols. The mechanisms by which peroxynitrite produces vasodilatation in vivo remain to be determined.

Complete heart block in association with graft-versus-host disease.

An infant who received haploidentical BM for severe combined immunodeficiency (SCID) developed acute, reversible complete heart block in association with an exacerbation of GVHD. Respiratory distress and myocardial dysfunction were also seen with this and previous GVHD exacerbations. The patient had not received chemotherapy or radiation prior to BMT. The complete heart block resolved after 1 week of intensive immunosuppression. The association of complete heart block with GVHD is important because the heart block is potentially reversible with prompt, aggressive control of the GVHD.

Oxidation of 2',7'-dichlorofluorescin by peroxynitrite.

The simultaneous production of nitric oxide and superoxide anion leads to the formation of peroxynitrite, a potent oxidant which may be an important mediator of cellular injury. Oxidation of dichlorofluorescin to the fluorescent dichlorofluorescein has been used as a marker for cellular oxidant production. The mechanisms of peroxynitrite-mediated oxidation of dichlorofluorescin to dichlorofluorescein were investigated. Chemically synthesized peroxynitrite (50-500 nM) induced the oxidation of dichlorofluorescin to dichlorofluorescein in a linear fashion. In addition, the simultaneous generation of nitric oxide and superoxide anion induced the oxidation of dichlorofluorescin to dichlorofluorescein, while nitric oxide (1-10 microM) alone under aerobic conditions did not. Peroxynitrite-mediated oxidation of dichlorofluorescin was not inhibited by the hydroxyl radical scavengers mannitol (100 mM) or dimethylsulfoxide (100 mM). Moreover, peroxynitrite-mediated oxidation of dichlorofluorescin was not dependent upon metal ion-catalyzed reactions. Furthermore, dichlorofluorescein formation was diminished at alkaline pH. These findings suggest that peroxynitrite-mediated dichlorofluorescein formation results directly from the protonation of peroxynitrite to form the conjugate peroxynitrous acid. L-cysteine was an efficient inhibitor (KI approximately 25 microM) of dichlorofluorescin oxidation through competitive oxidation of free sulfhydryls. Urate was a less efficient with a maximum inhibition of only 49%. These results demonstrate that dichlorofluorescin is efficiently oxidized by peroxynitrite. Therefore, under conditions where nitric oxide and superoxide are produced simultaneously, oxidation of dichlorofluorescin may be mediated by the formation of peroxynitrite.

Extensive tyrosine nitration in human myocardial inflammation: evidence for the presence of peroxynitrite.

OBJECTIVES: Production of nitric oxide via the cytokine-mediated activation of myocardial inducible nitric oxide synthase decreases myocardial contractility. Whether myocardial dysfunction is mediated directly by nitric oxide or indirectly through the formation of secondary reaction products, such as peroxynitrite, has not been established. Peroxynitrite, but not nitric oxide, reacts with the phenolic ring of tyrosine to form the stable product 3-nitro-L-tyrosine. Demonstration of tissue nitrotyrosine residues, therefore, infers the presence of peroxynitrite or related nitrogen-centered oxidants. DESIGN: Retrospective analysis of human autopsy specimens. SETTING: University pathology and basic science laboratories. PATIENTS: Formalin-fixed, paraffin-embedded myocardial tissue samples were obtained from 11 patients with a diagnosis of sepsis, seven patients with a diagnosis of viral myocarditis, and five control patients without clinical or pathologic cardiac disease. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Specific antibodies to nitrotyrosine were utilized to detect nitrotyrosine residues in human autopsy specimens. Cardiac tissue obtained from patients with myocarditis or sepsis demonstrated intense nitrotyrosine immunoreactivity in the endocardium, myocardium, and coronary vascular endothelium and smooth muscle. In contrast, connective tissue elements were without appreciable immunohistochemical staining. Nitrotyrosine antibody binding was blocked by coincubation with nitrotyrosine or nitrated bovine serum albumin, but not by aminotyrosine, phosphotyrosine, or bovine serum albumin. In situ reduction of tissue nitrotyrosine to aminotyrosine by sodium hydrosulfite also blocked antibody binding. Densitometric analysis of nitrotyrosine immunoreactivity demonstrated significantly higher values for specimens from myocarditis and sepsis patients when compared with control tissue specimens. CONCLUSION: These results demonstrate the formation of peroxynitrite within the myocardium during inflammatory disease states, suggesting a role for peroxynitrite in inflammation-associated myocardial dysfunction.

The peroxynitrite product 3-nitro-L-tyrosine attenuates the hemodynamic responses to angiotensin II in vivo.

Peroxynitrite is a potent oxidant formed endogenously by the near diffusion-limited reaction of nitric oxide with superoxide anion. Peroxynitrite specifically adds a nitro group to the ortho position of the phenolic ring of free and protein-associated tyrosines to form the stable product 3-nitro-L-tyrosine. Systemic administration of 3-nitro-L-tyrosine markedly inhibits the subsequent hemodynamic responses to alpha 1- and beta-adrenoceptor agonists in anesthetized rats. Angiotensin II is an important modulator of vascular tone. The vasoconstrictor effects of this hormone are known to involve the release of catecholamines from sympathetic tissues. In the present study, we examined whether 3-nitro-L-tyrosine (2.5 mumol/kg i.v.) would attenuate the hemodynamic responses produced by angiotensin II (0.1-1.0 microgram/kg i.v.). Angiotensin II produced increases in mean arterial pressure, and renal and mesenteric vascular resistances, but no changes in hindquarter vascular resistance. The pressor and renal and mesenteric vasoconstrictor responses produced by angiotensin II were significantly attenuated 30-60 min following the administration of 3-nitro-L-tyrosine. Further attenuation of these responses was evident 120-180 min following the administration of 3-nitro-L-tyrosine. The alpha 1-adrenoceptor antagonist prazosin also diminished the pressor and renal and mesenteric vasoconstrictor responses produced by angiotensin II. These results demonstrate that 3-nitro-L-tyrosine inhibits the hemodynamic responses to angiotensin II, possibly through the inhibition of alpha 1-adrenoceptor-mediated events. The effect of 3-nitro-L-tyrosine on the hemodynamic action of angiotensin II raises the possibility that 3-nitro-L-tyrosine may be involved in the pathogenesis of the hemodynamic disturbances associated with inflammatory conditions, such as atherosclerosis, ischemia-reperfusion, and sepsis, where formation of peroxynitrite is favored.

Nitrotyrosine attenuates the hemodynamic effects of adrenoceptor agonists in vivo: relevance to the pathophysiology of peroxynitrite.

Peroxynitrite, which attenuates catecholamine-mediated hemodynamic responses in vivo, nitrates free tyrosine residues to form the specific product, 3-nitro-L-tyrosine. The chemical structure of 3-nitro-L-tyrosine is similar to that of the endogenous catecholamines. Therefore, 3-nitro-L-tyrosine may interfere with catecholamine hemodynamic function in vivo. The hemodynamic responses produced by norepinephrine (1-4 micrograms/kg, i.v., n = 6), epinephrine (0.5-4 micrograms/kg, i.v., n = 7), phenylephrine (1-8 micrograms/kg, i.v., n = 5), and isoproterenol (100-400 ng/kg, i.v., n = 5) were attenuated, while the hemodynamic responses produced by arginine vasopressin (50-250 ng/kg; i.v., n = 5) were unaffected following the administration of 3-nitro-L-tyrosine (2.5 mumol/kg, i.v.) in pentobarbital-anesthetized rats. These results demonstrate substantial and selective attenuation of the hemodynamic effects produced by alpha- and beta-adrenoceptor agonists, raising the possibility that 3-nitro-L-tyrosine may play a role in the hemodynamic dysfunction associated with inflammatory conditions in which the formation of peroxynitrite is favored.

Elevation in arterial blood pressure following the development of tachyphylaxis to peroxynitrite.

The systemic administration of peroxynitrite produces transient reductions in mean arterial pressure and vascular resistances in anesthetized rats. The repeated administration of peroxynitrite results in tachyphylaxis. We now report that anesthetized rats (n = 8) treated with peroxynitrite (10 injections of 10 mumol/kg i.v.) subsequently develop increases in mean arterial pressure (20 +/- 4%) and hindquarter (153 +/- 28%), renal (93 +/- 21%), and mesenteric (133 +/- 25%) vascular resistances. These findings suggest that the in vivo production of peroxynitrite may contribute to the pathogenesis of hypertension.

Evidence for in vivo peroxynitrite production in human acute lung injury.

Oxidant-mediated toxicity resulting from acute pulmonary inflammation has been demonstrated in acute lung injury. A potent biological oxidant, peroxynitrite, is formed by the near diffusion-limited reaction of nitric oxide with superoxide. In addition to having hydroxyl radical-like oxidative reactivity, peroxynitrite is capable of nitrating phenolic rings, including protein-associated tyrosine residues. Nitric oxide does not directly nitrate tyrosine residues, therefore, demonstration of tissue nitrotyrosine residues infers the action of peroxynitrite or related nitrogen-centered oxidants. Lung tissue was obtained from formalin-fixed, paraffin-embedded autopsy specimens, and specific polyclonal and monoclonal antibodies to nitrotyrosine were visualized by diaminobenzidene-peroxidase staining. Acute lung injury resulted in intense staining throughout the lung, including lung interstitium, alveolar epithelium, proteinaceous alveolar exudate, and inflammatory cells. In addition, staining of the vascular endothelium and subendothelial tissues was present in those patients with sepsis-induced acute lung injury. Antibody binding was blocked by coincubation with nitrotyrosine or nitrated bovine serum albumin but not by aminotyrosine, phosphotyrosine, or bovine serum albumin. Reduction of tissue nitrotyrosine to aminotyrosine by sodium hydrosulfite also blocked antibody binding. In control specimens with no overt pulmonary disease, there was only slight staining of the alveolar septum. These results demonstrate that nitrogen-derived oxidants are formed in human acute lung injury and suggest that peroxynitrite may be an important oxidant in inflammatory lung disease.

Nitric oxide-related oxidants in acute lung injury.

Nitric oxide (NO.) is a free radical and will react efficiently with other radicals. The reaction between NO. and superoxide anion (O2.-) is a pivotal reaction by which NO. affects oxidant metabolism. This reaction may scavenge O2.- before further reactions can occur that lead to the biosynthesis of more potent oxidants such as hydroxyl radical. The product of the reaction between NO. and O2.-, however, is peroxynitrite anion, which is also a potent oxidant capable of participating in several oxidative reactions. Among these reactions are oxidation of sulfhydryl groups, oxidation of lipids, and nitration of tyrosine by noncatalyzed and catalyzed mechanisms. The conformation, and therefore specific reactivity, of peroxynitrite are dependent on pH. Based on an understanding of this concept, sulfhydryl oxidation should be the predominant oxidative reaction of peroxynitrite in biological systems. Some experimental data support this conclusion. There is increasing evidence from isolated cell systems that peroxynitrite is produced under the influence of inflammatory mediators. Most data from animal models suggest that increased NO. production in acute lung injury is detrimental. We have performed immunohistochemical evaluation of lung tissue from pediatric patients with acute lung injury using an antinitrotyrosine antibody and have found evidence of extensive nitrotyrosine formation. This observation suggests a significant effect of peroxynitrite on lung tissue in this disorder. NO. has a variety of nonoxidant effects that also may also have a role in acute lung injury. With the information currently available, one cannot conclude with certainty whether the net effect of increased NO. production in inflammatory disorders of the lung is beneficial or injurious. However, simultaneous increases in NO. and O2.- occurring during inflammation may lead to peroxynitrite formation and subsequent oxidative tissue injury.

Agonist-induced peroxynitrite production from endothelial cells.

Nitric oxide reacts with superoxide to form peroxynitrite, a potential mediator of oxidant-induced cellular injury. The endothelium is a primary target of injury in many pathological states, including acute lung injury, sepsis, multiple organ failure syndrome, and atherosclerosis, where enhanced production of nitric oxide and superoxide occurs simultaneously. It was hypothesized that stimulation of endothelial cell nitric oxide production would result in formation of peroxynitrite. Immediate oxidant production was detected by luminol- and lucigenin-enhanced chemiluminescence from cultured bovine aortic endothelial cells exposed to bradykinin or to the calcium ionophore A23187. Luminol-enhanced chemiluminescence was efficiently inhibited by the nitric oxide synthase inhibitor nitro-L-arginine methyl ester and by superoxide dismutase, implying dependence on the presence of both nitric oxide and superoxide for oxidant production. Inhibition of luminol-enhanced chemiluminescence by nitro-L-arginine methyl ester was partially reversed by L-arginine, but not by D-arginine. Cysteine, methionine, and urate, known inhibitors of peroxynitrite-mediated oxidation, inhibited luminol-enhanced chemiluminescence, while the hydroxyl radical scavengers, mannitol and dimethylsulfoxide, and catalase did not. Bicarbonate increased luminol-enhanced chemiluminescence in a concentration-dependent manner. Superoxide production, detected by lucigenin-enhanced chemiluminescence, was slightly increased in the presence of nitro-L-arginine methyl ester, suggesting that endothelial cell-produced superoxide was partially metabolized by reaction with nitric oxide. These results are consistent with agonist-induced peroxynitrite production by endothelial cells and suggests that peroxynitrite may have an important role in oxidant-induced endothelial injury.

Peroxynitrite-mediated oxidation of dihydrorhodamine 123.

Nitric oxide reacts with superoxide to form peroxynitrite, which may be an important mediator of free radical-induced cellular injury. Oxidation of dihydrorhodamine to fluorescent rhodamine is a marker of cellular oxidant production. We investigated the mechanisms of peroxynitrite-mediated formation of rhodamine from dihydrorhodamine. Peroxynitrite at low levels (0-1000 nM) induced a linear, concentration-dependent, oxidation of dihydrorhodamine. Hydroxyl radical scavengers mannitol and dimethylsulfoxide had minimal effect (< 10%) on rhodamine production. Peroxynitrite-mediated formation of rhodamine was not dependent on metal ion catalyzed reactions because studies were performed in metal ion-free buffer and rhodamine formation was not enhanced in the presence of Fe3+ ethylenediaminetetraacetic acid (EDTA). Thus, rhodamine formation appears to be mediated directly by peroxynitrite. Superoxide dismutase slightly enhanced rhodamine production. L-cysteine was an efficient inhibitor (KI approximately 25 microM) of dihydrorhodamine oxidation through competetive oxidation of free sulfhydryls. Urate was also an efficient inhibitor (KI approximately 2.5 microM), possibly by reduction of an intermediate dihydrorhodamine radical and recycling of dihydrorhodamine. Under anaerobic conditions, nitric oxide did not oxidize dihydrorhodamine and inhibited spontaneous oxidation of dihydrorhodamine. In the presence of oxygen, nitric oxide induces a relatively slow oxidation of dihydrorhodamine due to the formation of nitrogen dioxide. We conclude that dihydrorhodamine is a sensitive and efficient trap for peroxynitrite and may serve as a probe for peroxynitrite production.

Effect of vitamin E deficiency and selenium deficiency on insulin secretory reserve and free radical scavenging systems in islets: decrease of islet manganosuperoxide dismutase.

There is increasing evidence that islet beta cells may be susceptible to redox insult, and that this susceptibility may contribute to the pathogenesis of experimental models of diabetes mellitus. We investigated the effect of vitamin E deficiency, selenium deficiency, and combined deficiency on islet function and free radical scavenging systems. The tissue levels of glutathione peroxidase, catalase, and immunoreactive superoxide dismutases were measured in four groups of rats (i.e., controls and those with vitamin E, selenium, and combined deficiency). Glucose tolerance tests were performed for each animal before sacrifice. Superoxide dismutase concentrations in liver, heart, and skeletal muscle were within 20% of the control levels in all groups. However, the manganosuperoxide dismutase concentrations in islets were significantly lower than control levels in response to vitamin E, selenium, and combined deficiency. Combined deficiency appeared to have an additive effect. In contrast, cuprozinc superoxide dismutase concentration in islets was higher in the deficient groups than in controls. Insulin secretory reserve was decreased in each of the three deficient groups. This decrease was reflected as glucose intolerance only in the group with combined deficiency. Glutathione peroxidase activity was markedly decreased in selenium-deficient animals in all tissues studied. Catalase activity did not change significantly among groups in any tissue studied. Islets had the lowest glutathione peroxidase and cuprozinc and total superoxide dismutase levels among tissues studied.