NO 2 Tyr (3-Nitrotyrosine) is a modified amino acid that is formed by nitric oxide-derived species and has been implicated in the pathology of diverse human diseases. Nitration of active-site tyrosine residues is known to compromise protein structure and function. Although free NO 2 Tyr is produced in abundant concentrations under pathological conditions, its capacity to alter protein structure and function at the translational or posttranslational level is unknown. Here, we report that free NO 2 Tyr is transported into mammalian cells and selectively incorporated into the extreme carboxyl terminus of ␣-tubulin via a posttranslational mechanism catalyzed by the enzyme tubulin-tyrosine ligase. In contrast to the enzymatically regulated carboxylterminal tyrosination͞detyrosination cycle of ␣-tubulin, incorporation of NO 2 Tyr shows apparent irreversibility. Nitrotyrosination of ␣-tubulin induces alterations in cell morphology, changes in microtubule organization, loss of epithelialbarrier function, and intracellular redistribution of the motor protein cytoplasmic dynein. These observations imply that posttranslational nitrotyrosination of ␣-tubulin invokes conformational changes, either directly or via allosteric interactions, in the surface-exposed carboxyl terminus of ␣-tubulin that compromises the function of this critical domain in regulating microtubule organization and binding of motorand microtubule-associated proteins. Collectively, these observations illustrate a mechanism whereby free NO 2 Tyr can impact deleteriously on cell function under pathological conditions encompassing reactive nitrogen species production. The data also yield further insight into the role that the ␣-tubulin tyrosination͞detyrosination cycle plays in microtubule function. Nitric oxide (• NO) is a pervasive signaling molecule generated from L-arginine via the catalytic action of both constitutive and inducible forms of • NO synthases (1). A large body of evidence has amassed in the last decade, establishing the operative role of inducible • NO synthase in the pathogenesis of inflammatory, infectious, and degenerative human diseases (2). The detrimental effects ascribed to • NO often arise from its conversion to more reactive species through reactions with partially reduced oxygen species (3).The pathophysiological actions of• NO congeners are primarily rooted in their capacity to alter the function of biological macromolecules through covalent modifications. A metabolite generally reflecting in vivo production of reactive nitrogen intermediates is the amino acid derivative 3-nitrotyrosine (NO 2 Tyr). Evidence for NO 2 Tyr formation in vivo was found when the free amino acid and its deaminated͞ decarboxylated metabolite 3-nitro-4-hydroxyphenylacetic acid were detected as excretory products in human urine (4). The significance of NO 2 Tyr in vivo is highlighted further by observations that protein-linked NO 2 Tyr is markedly elevated in a broad range of human diseases and clinical disorders (5). In vitro studies have identifi...

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Ischemia/reperfusion (IR) injury in transplanted livers contributes to organ dysfunction and failure and is characterized in part by loss of NO bioavailability. Inhalation of NO is nontoxic and at high concentrations (80 ppm) inhibits IR injury in extrapulmonary tissues. In this prospective, blinded, placebo-controlled study, we evaluated the hypothesis that administration of inhaled NO (iNO; 80 ppm) to patients undergoing orthotopic liver transplantation inhibits hepatic IR injury, resulting in improved liver function. Patients were randomized to receive either placebo or iNO (n = 10 per group) during the operative period only. When results were adjusted for cold ischemia time and sex, iNO significantly decreased hospital length of stay, and evaluation of serum transaminases (alanine transaminase, aspartate aminotransferase) and coagulation times (prothrombin time, partial thromboplastin time) indicated that iNO improved the rate at which liver function was restored after transplantation. iNO did not significantly affect changes in inflammatory markers in liver tissue 1 hour after reperfusion but significantly lowered hepatocyte apoptosis. Evaluation of circulating NO metabolites indicated that the most likely candidate transducer of extrapulmonary effects of iNO was nitrite. In summary, this study supports the clinical use of iNO as an extrapulmonary therapeutic to improve organ function following transplantation.

The reaction between nitric oxide (*NO) and lipid peroxyl radicals (LOO*) has been proposed to account for the potent inhibitory properties of *NO toward lipid peroxidation processes; however, the mechanisms of this reaction, including kinetic parameters and nature of termination products, have not been defined. Here, the reaction between linoleate peroxyl radicals and *NO was examined using 2, 2'-azobis(2-amidinopropane) hydrochloride-dependent oxidation of linoleate. Addition of *NO (0.5-20 microM) to peroxidizing lipid led to cessation of oxygen uptake, which resumed at original rates when all *NO had been consumed. At high *NO concentrations (>3 microM), the time of inhibition (Tinh) of chain propagation became increasingly dependent on oxygen concentration, due to the competing reaction of oxygen with *NO. Kinetic analysis revealed that a simple radical-radical termination reaction (*NO:ROO* = 1:1) does not account for the inhibition of lipid oxidation by *NO, and at least two molecules of *NO are consumed per termination reaction. A mechanism is proposed whereby *NO first reacts with LOO* (k = 2 x 10(9) M-1 s-1) to form LOONO. Following decomposition of LOONO to LO* and *NO2, a second *NO is consumed via reaction with LO*, with the composite rate constant for this reaction being k = 7 x 10(4) M-1 s-1. At equal concentrations, greater inhibition of oxidation was observed with *NO than with alpha-tocopherol. Since *NO reacts with LOO* at an almost diffusion-limited rate, steady state concentrations of 30 nM *NO would effectively compete with endogenous alpha-tocopherol concentrations (about 20 microM) as a scavenger of LOO* in the lipid phase. This indicates that biological *NO concentrations (up to 2 microM) will significantly influence peroxidation reactions in vivo.