Ming-Hui Zou

Oxidation of the zinc-thiolate complex and uncoupling of endothelial nitric oxide synthase by peroxynitrite.

Nitric oxide (NO) is produced by NO synthase (NOS) in many cells and plays important roles in the neuronal, muscular, cardiovascular, and immune systems. In various disease conditions, all three types of NOS (neuronal, inducible, and endothelial) are reported to generate oxidants through unknown mechanisms. We present here the first evidence that peroxynitrite (ONOO(-)) releases zinc from the zinc-thiolate cluster of endothelial NOS (eNOS) and presumably forms disulfide bonds between the monomers. As a result, disruption of the otherwise SDS-resistant eNOS dimers occurs under reducing conditions. eNOS catalytic activity is exquisitely sensitive to ONOO(-), which decreases NO synthesis and increases superoxide anion (O(2)(.-)) production by the enzyme. The reducing cofactor tetrahydrobiopterin is not oxidized, nor does it prevent oxidation of eNOS by the same low concentrations of OONO(-). Furthermore, eNOS derived from endothelial cells exposed to elevated glucose produces more O(2)(.-), and, like eNOS purified from diabetic LDL receptor-deficient mice, contains less zinc and fewer SDS-resistant dimers. Hence, eNOS exposure to oxidants including ONOO(-) causes increased enzymatic uncoupling and generation of O(2)(.-) in diabetes, contributing further to endothelial cell oxidant stress. Regulation of the zinc-thiolate center of NOS by ONOO(-) provides a novel mechanism for modulation of the enzyme function in disease.

Tyrosine nitration as a mechanism of selective inactivation of prostacyclin synthase by peroxynitrite.

Vascular tone critically depends on the endothelial release of nitric oxide and prostacyclin. Superoxide anions counteract these relaxations by trapping nitric oxide under formation of peroxynitrite. As we have recently reported, peroxynitrite is able to inhibit prostacyclin formation in aortic microsomes (Zou et al., 1996). Here we show that peroxynitrite also blocks purified prostacyclin synthase with an IC50 value of about 50 nM and with a similar sensitivity also inhibits the enzyme activity in the EaHy 926 endothelial cell line. Thromboxane synthase, having the same heme-thiolate (P450) structure and a closely-related mechanism was unaffected by peroxynitrite. Anti-nitrotyrosine antibodies reacted positive by a Western blot after treatment of the purified enzyme with 1 microM peroxynitrite. Tetranitromethane also inhibited the enzyme activity which, like the inhibition by peroxynitrite, could be partially prevented in the presence of the substrate analog U46619. The simultaneous generation of superoxide and nitric oxide proved to be as efficient as a bolus of peroxynitrite which supports a possible inactivation of prostacyclin synthase under in vivo conditions. This substantiates an often suggested crucial role of superoxide in the pathophysiology of the cardiovascular system.

Molecular insights and therapeutic targets for diabetic endothelial dysfunction.

Diabetes mellitus and its associated complications are major health problems in the developed world. Diabetes mellitus is associated with an increased risk of cardiovascular disease (CVD) even in the presence of intensive glycemic control. Indeed, 75% of diabetic patients will die of CVD. Patients with type 1 and type 2 diabetes mellitus have an increased risk for the 3 major types of macrovascular disease (coronary heart disease, peripheral vascular disease, and stroke).1 A striking feature of diabetic cardiovascular complications is the appearance of accelerated atherosclerosis, which anatomically resembles atherosclerosis in nondiabetic individuals but is more extensive and occurs at an earlier age. Substantial clinical and experimental evidence suggests that endothelial dysfunction is a crucial early step in the development of atherosclerosis. Evidence also suggests that it participates in plaque progression and the clinical emergence of cardiovascular events. Endothelial dysfunction is characterized by impaired endothelium-dependent vasodilation and “endothelial activation,” which is associated with a proinflammatory, proliferative, and procoagulatory milieu that promotes initiation and complications of atherogenesis.2 Endothelial dysfunction associated with insulin resistance appears to precede the development of overt hyperglycemia in patients with type 2 diabetes mellitus.3 Therefore, endothelial dysfunction may be a critical early target for the prevention of atherosclerosis and CVD in patients with diabetes mellitus or insulin resistance.2 A synergistic cross talk exists among the conventional cardiovascular risk factors associated with diabetes mellitus, and such cross talk contributes to disruption of endothelial integrity and acceleration of atherosclerosis. However, the biochemical and cellular links between elevated blood glucose levels and endothelial dysfunction remain incompletely understood. This review will focus on the multifactorial nature of endothelial dysfunction in diabetes mellitus, the relationship between endothelial dysfunction and conventional cardiovascular risk factors, and the translational potential of molecular targets such as the AMP-activated protein kinase (AMPK) for treating …

Modulation by Peroxynitrite of Akt- and AMP-activated Kinase-dependent Ser1179 Phosphorylation of Endothelial Nitric Oxide Synthase

Peroxynitrite (ONOO−), a nitric oxide-derived oxidant, uncouples endothelial nitric oxide synthase (eNOS) and increases enzymatic production of superoxide anions (O 2 ⨪ ) (Zou, M. H., Shi, C., and Cohen, R. A. (2002) J. Clin. Invest. 109, 817–826). Here we studied how ONOO−influences eNOS activity. In cultured bovine aortic endothelial cells (BAEC), ONOO− increased basal and agonist-stimulated Ser1179 phosphorylation of eNOS, whereas it decreased nitric oxide production and bioactivity. However, ONOO−strongly inhibited the phosphorylation and activity of Akt, which is known to phosphorylate eNOS-Ser1179. Moreover, expression of an Akt dominant-negative mutant did not prevent ONOO−-enhanced eNOS-Ser1179 phosphorylation. In contrast to Akt, ONOO− significantly activated 5′-AMP-activated kinase (AMPK), as evidenced by its increased Thr172 phosphorylation as well as increased Ser92 phosphorylation of acetyl-coenzyme A carboxylase, a downstream target of AMPK. Associated with the increased release of O 2 ⨪ , ONOO− significantly increased the co-immunoprecipitation of eNOS with AMPK. Further, overexpression of the AMPK-constitutive active adenovirus significantly enhanced ONOO− up-regulated eNOS-Ser(P)1179. In contrast, overexpression of a dominant-negative AMPK mutant attenuated the ONOO−-enhanced eNOS-Ser1179phosphorylation as well as O 2 ⨪ release. We conclude that ONOO− inhibits Akt and increases AMPK-dependent Ser1179 phosphorylation of eNOS resulting in enhanced O 2 ⨪ release.

Selective Nitration of Prostacyclin Synthase and Defective Vasorelaxation in Atherosclerotic Bovine Coronary Arteries

Prostacyclin synthase (PCS) is an enzyme with antithrombotic, antiproliferative, and dilatory functions in the normal vasculature, and inactivation of PCS by tyrosine nitration may favor atherosclerotic processes. Here, we show that PCS is nitrated and inactivated in early stage atherosclerotic lesions (focal intimal thickenings). Immunoprecipitation with antibodies raised against nitrotyrosine yielded PCS as the main nitrated protein in blood vessels. Moreover, we identified two nitrated degradation products of PCS with molecular mass of 30 and 46 kd, which were selective for atherosclerotic tissue. Agonist (acetylcholine, angiotensin II)-induced prostacyclin formation was decreased in atherosclerotic vessels compared with normal tissue, whereas PGE2 formation was increased and cyclooxygenase activity remained unchanged. A selective loss of PCS activity was confirmed by direct measurement of enzymatic activity. In line with this, we observed defective relaxation of early atherosclerotic vessels following vasoconstrictive stimulation. This functional impairment was completely reversed by coincubation with an antagonist of the thromboxane/PGH2 receptor but not by a thromboxane synthase inhibitor. These data suggest that reduced PCS activity in atherosclerotic arteries prevents the rapid use of PGH2, which accumulates and acts as an agonist on the vasoconstrictive thromboxane receptor.

Prostaglandin endoperoxide-dependent vasospasm in bovine coronary arteries after nitration of prostacyclin synthase

In the present study we used a bioassay to study the effects of peroxynitrite (ONOO−) on angiotensin II (A‐II)‐triggered tension in isolated bovine coronary arteries in order to show the consequences of the previously reported PGI2‐synthase inhibition by ONOO− in this model. The following results were obtained:

Ebselen as a peroxynitrite scavenger in vitro and ex vivo.

We have previously shown that peroxynitrite (PN) selectively impaired prostacyclin (PGI2)-dependent vasorelaxation by tyrosine nitration of PGI2 synthase in an in situ model (Zou MH, Jendral M and Ullrich V, Br J Pharmacol 126: 1283-1292, 1999). By using this established model, we tested whether or not ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), which reacts rapidly with the anionic form of PN, affected PN inhibition of PGI2 synthase. Administration of ebselen (1 to 50 microM) to bovine coronary strips 5 min prior to PN (1 microM) treatment neither prevented PN-triggered vasoconstriction nor the inhibition of PGI2 release. In line with these results, ebselen affected neither PN inhibition of the conversion of [14C]-PGH2 into 6-keto-PGF1 alpha nor the nitration of PGI2 synthase in bovine aortic microsomes. Following the hypothesis that a reaction of ebselen with cellular thiols could have caused the inefficiency of ebselen, we observed that free ebselen quickly reacted with thiols in both coronary strips and in aortic microsomes to form two metabolites, one of which was identified as the ebselen-glutathione adduct, whereas the other had a similar retention time to that of the ebselen-cysteine adduct. The nitration of phenol by PN in a metal-free solution could be blocked more efficiently in the presence of ebselen or glutathione alone than in the presence of both, indicating that like selenomethionine and other selenocompounds, ebselen-thiol adducts were less reactive towards PN than ebselen itself. Further evidence came from the results that ebselen became effective in preventing the inhibition and nitration of PGI2 synthase after thiol groups of microsomal proteins were previously oxidized with Ellmans reagent. We conclude that in cellular systems ebselen is present as thiol adducts and thus loses its high reactivity towards PN, which is required to compete with the nitration of PGI2 synthase.

Endothelial cell activation by endotoxin involves superoxide/NO-mediated nitration of prostacyclin synthase and thromboxane receptor stimulation

In bovine coronary artery segments, peroxynitrite inhibits prostacyclin (PGI2) synthase by tyrosine nitration. Using this pharmacological model, we show that a 1 h exposure of bovine coronary artery segments to endotoxin (lipopolysaccharide [LPS]) inhibits the relaxation phase following angiotensin II (Ang II) stimulation and causes a vasospasm that can be suppressed by a thromboxane A2 (TxA2) receptor blocker. In parallel, PGI2 synthesis decreases in favor of prostaglandin E2 formation. Immunoprecipitation and costaining with an anti‐nitrotyrosine antibody identified PGI2 synthase as the main nitrated protein in the endothelium. All effects of LPS could be prevented in the presence of the nitric oxide (NO) synthase inhibitor Nω‐monomethyl‐larginine and polyethylene‐glycolated Cu/Zn‐ superoxide dismutase. Thus, the early phase of endothelial cell activation in bovine coronary arteries by inflammatory agents proceeds by a protein synthesis‐independent priming process for a source of superoxide that we tentatively attribute to xanthine oxidase. Upon receptor activation, Ang II stimulates NO and superoxide production, resulting in a peroxynitrite‐mediated nitration and inhibition of PGI2 synthase. The remaining 15‐hydroxy‐prostaglandin 9,11‐endoperoxide (PGH2) first activates the TxA2/PGH2 receptor and then is converted to prostaglandin E2 (PGE2) by smooth muscle cells. PGE2 together with a lack of NO and PGI2 is known to promote the adhesion of white blood cells and their immigration to the inflammatory locus.

PEROXYNITRITE INACTIVATES PROSTACYCLIN SYNTHASE BY HEME–THIOLATE-CATALYZED TYROSINE NITRATION

Previous work has shown a sensitive inhibition of prostacyclin synthase activity by peroxynitrite as well as by superoxide in the presence of NO donors. Neither superoxide nor NO alone nor decomposed peroxynitrite is effective. The inhibition of activity was paralleled by a nitration of a tyrosine residue and both could be prevented by a stable substrate analog. The same IC50 value for peroxynitrite was also found for the cellular prostacyclin activity in endothelial and kidney mesangial cells, indicating that the antioxidant potential of the cell cannot prevent the inactivation. Aortic tissue shows a co-localization of prostacyclin synthase and nitrotyrosine staining after treatment of the tissue with 1 microM peroxynitrite. It can be speculated that this pathway of enzyme nitration is of pathophysiological significance.

PEROXYNITRITE INHIBITION OF NITRIC OXIDE SYNTHASES

Peroxynitrite (PN) can be formed under mainly pathophysiological conditions from nitric oxide (NO) and superoxide anion and may be responsible for oxidative modifications of biomolecules. Preparations of nitric oxide synthases from porcine cerebellum (nNOS), bovine aortic endothelium (eNOS) and cytokine-treated murine macrophages (iNOS) were inhibited by PN in their ability to transform arginine to citrulline and nitric oxide with IC50 values of 15, 28, and 10 microM, respectively. Glutathione, bovine serum albumin and tyrosine provided varying degrees of protection in the three preparations. Intact endothelial cells, upon exposure to PN, rapidly lost their glutathione content but protein-SH groups and eNOS activity remained largely unaffected. Destruction of the heme-thiolate catalytic site was observed when nNOS was exposed to PN suggesting that the irreversible oxidation of this bond may be the common mechanism of NOS inhibition.