Effects Of ONOO Research Articles

Effect of Korean Red Ginseng Extract on Cell Death Responses in Peroxynitrite-Treated Keratinocytes

Korean red ginseng (KRG) has been used worldwide as a traditional medicine for the treatment of various diseases, includ- including cancer. In this study, we determined the effect of KRG on the responses of HaCaT cells to peroxynitrite (ONOO?). Cells treated with ONOO? (2 mM) prior to incubation with control medium for 12 hours displayed reduced viability, as determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (viability about 48% of that of non-treated control cells). When KRG was added to the post-incubation medium, the negative effects of ONOO? on cell viability were significantly reduced. Reverse transcription-polymerase chain reaction analysis indicated that KRG alone did not significantly alter p53 or “growth arrest and DNA damage” (GADD)45 mRNA levels. However, the addition of KRG to the post-incubation medium significantly and dose-dependently reduced levels of p53 and GADD45 mRNA in ONOO?-treated cells. Western blot analyses revealed that incubation with KRG decreased p53 and GADD45 protein levels in ONOO?-treated cells, relative to those in cells incubated with control medium. Collectively, these results suggest that Korean red ginseng extract protects cells against ONOO?-induced genotoxicity by increasing cell viability through modulating the expression of p53 signaling intermediates.

Heme catalyzes tyrosine 385 nitration and inactivation of prostaglandin H2 synthase-1 by peroxynitrite

The mechanism by which the inflammatory enzyme prostaglandin H(2) synthase-1 (PGHS-1) deactivates remains undefined. This study aimed to determine the stabilizing parameters of PGHS-1 and identify factors leading to deactivation by nitric oxide species (NO(x)). Purified PGHS-1 was stabilized when solubilized in beta-octylglucoside (rather than Tween-20 or CHAPS) and when reconstituted with hemin chloride (rather than hematin). Peroxynitrite (ONOO(-)) activated the peroxidase site of PGHS-1 independently of the cyclooxygenase site. After ONOO(-) exposure, holoPGHS-1 could not metabolize arachidonic acid and was structurally compromised, whereas apoPGHS-1 retained full activity once reconstituted with heme. After incubation of holoPGHS-1 with ONOO(-), heme absorbance was diminished but to a lesser extent than the loss in enzymatic function, suggesting the contribution of more than one process to enzyme inactivation. Hydroperoxide scavengers improved enzyme activity, whereas hydroxyl radical scavengers provided no protection from the effects of ONOO(-). Mass spectral analyses revealed that tyrosine 385 (Tyr 385) is a target for nitration by ONOO(-) only when heme is present. Multimer formation was also observed and required heme but could be attenuated by arachidonic acid substrate. We conclude that the heme plays a role in catalyzing Tyr 385 nitration by ONOO(-) and the demise of PGHS-1.

Peroxynitrite is a contractile agonist of cerebral artery smooth muscle cells.

On reperfusion of ischemic tissue, a prolonged phase of vasoconstriction occurs, the mechanism of which is poorly understood. However, it is known that peroxynitrite (ONOO-) is formed during reperfusion. In this study the contractile properties of ONOO- were investigated in Wistar rat middle cerebral arteries. The effects of ONOO- on vessel diameter were dose dependent. Low-dose ONOO- (10 microM) caused vessels to constrict by 15%. At an intermediate concentration of 25 microM, the effect of ONOO- was variable, whereas at the highest concentration (100 microM), vessels underwent persistent dilation and became insensitive to the endogenous vasoconstrictor 5-hydroxytryptamine. At the single cell level, ONOO- caused cerebral artery smooth muscle cells to contract. Reduced, but not oxidized, glutathione completely inhibited the contractile action of ONOO- on single cells. Vehicle and decomposed ONOO- each had minimal effect on cell length. These data show that ONOO- is a contractile agonist of middle cerebral arteries, at the single cell and whole vessel levels, suggesting that formation of ONOO- may contribute mechanistically to ischemic brain injury during stroke. Moreover, relatively high concentrations of ONOO- result in vascular paralysis.

Peroxynitrite inhibits leukocyte-endothelial cell interactions and protects against ischemia-reperfusion injury in rats.

Peroxynitrite (ONOO-) anion, formed by the interaction of superoxide with nitric oxide (NO), has previously been implicated as a cytotoxic agent. However, the effects of this free radical species on neutrophil (PMN)-endothelial cell interactions is largely unknown. We investigated the direct actions of ONOO- on PMN adhesion to endothelial cells in vitro and in vivo, as well as the effects of ONOO- on PMN-mediated myocardial ischemia-reperfusion injury. In vitro, peroxynitrite (100-1,000 nM) inhibited the adhesion of rat PMNs to the endothelium of isolated thrombin- or H2O2-stimulated rat mesenteric artery (P < 0.01 vs. thrombin or H2O2 alone). In vivo, in the rat mesentery, thrombin (0.5 U/ml) or N(G)-nitro-L-arginine-methyl ester (50 microM) significantly increased venular leukocyte rolling and adherence, which were also significantly (P < 0.01) attenuated by ONOO (800 nM) accompanied by reduced P-selectin expression on the endothelial cell surface. Isolated perfused rat hearts were subjected to global ischemia and reperfusion with rat PMNs (10(8) cells), which resulted in profound cardiac depression (i.e., a marked reduction in left ventricular developed pressure and maximal rate of development of left ventricular pressure). Infusion of ONOO- reversed the myocardial contractile dysfunction of ischemic-reperfused rat hearts to near baseline levels, and markedly attenuated the accumulation of PMNs in the postischemic heart. The present study provides strong evidence that nanomolar concentrations of ONOO- both inhibit leukocyte-endothelial cell interactions and exert cytoprotective effects in myocardial ischemia-reperfusion injury. Furthermore, our results suggest that the inhibition of P-selectin expression by peroxynitrite is a key mechanism of the modulatory actions of ONOO- on leukocyte-endothelial cell interactions.

75 EFFECTS OF PEROXYNITRITE ON ESOPHAGEAL MOTOR FUNCTION.

Introduction: Nitric oxide (NO)·) is the primary mediator of nerve-induced esophageal peristalsis, and the lower esophageal sphincter (LES) relaxation. Disordered esophageal motor function accompanies esophageal inflammation. Superoxide radicals are formed during all inflammatory states and are known to scavange NO·. The reaction of NO· with superoxide generates peroxynitrite (ONOO-). Peroxynitrite may alter cellular functions in several ways: it is a potent oxidant and it nitrates tyrosine residues of cellular proteins. It is generated in or near myenteric ganglion neurons in the inflamed small bowel and is known to produce neuronal injury. It causes relaxation of vascular smooth muscle. Hypothesis: Peroxynitrite alters esophageal motor function in vitro. Methods: Circular esophageal muscle strips from opossum were placed in tissue baths containing oxygenated Krebs solution maintained at 37°C. The tissues were stimulated by an electrical field (4-s trains of 50V, 0.5ms square-wave pulses at 5 Hz) previously shown to activate intrinsic esophageal nerves. Responses to nerve stimulation were observed in the presence of a) peroxynitrite (1×10-6M to 5×10-4M) with or without superoxide dismutase (SOD), Nw-nitro-L-arginine (L-NNA) or oxyhemoglobin (Hb); b) decomposed peroxynitrite (1×10-6M to 5×10-3M) with or without Hb; or c) sodium nitrite (NaNO2)(1×10-6M to 5×10-3M) with or without Hb. Head space gas in tissue baths was analyzed for NO· by chemiluminescense assay. Results: ONOO-, its decomposed form, or its major breakdown product NaNO2 relax the LES, and attenuate esophageal contraction in a concentration-dependent manner. ONOO- is the most potent. The effects of ONOO- are not inhibited by a superoxide scavanger, SOD, or a nitric oxide synthase inhibitor, L-NNA. The effects of ONOO-, decomposed ONOO-, or NaNO2 are inhibited by a NO· scavanger, Hb. The inhibition is most pronounced with ONOO-. NO· is produced from ONOO-, but not from its decomposed form or NaNO2. Conclusions: Disordered esophageal motility may be related to the formation of ONOO- and its breakdown products during inflammation. NO· may be generated directly from ONOO- under physiologic conditions.