thromboxane-a2 and 3-nitrotyrosine

Prostaglandins generated by cyclooxygenase (COX) have been implicated in hyperglycemia-induced endothelial dysfunction. However, the role of individual COX isoenzymes as well as the molecular mechanisms linking oxidative stress and endothelial dysfunction in diabetes remains to be clarified.. Human aortic endothelial cells were exposed to normal (5.5 mmol/L) and high (22.2 mmol/L) glucose. Glucose selectively increased mRNA and protein expression of COX-2. Its upregulation was associated with an increase of thromboxane A2 and a reduction of prostacyclin (PGI2) release. Glucose-induced activation of PKC resulted in the formation of peroxynitrite and tyrosine nitration of PGI2 synthase. NO release was reduced despite 2-fold increase of endothelial NO synthase expression. Phorbol ester caused an increase of COX-2 and endothelial NO synthase expression similar to that elicited by glucose. These effects were prevented by the PKC inhibitor calphostin C. N-acetylcysteine, vitamin C, and calphostin C prevented ROS formation, restored NO release, and reduced colocalization of nitrotyrosine and PGI2 synthase. Expression of p22(phox), a subunit of NAD(P)H oxidase, was increased, and diphenyleneiodonium inhibited ROS formation. By contrast, indomethacin did not affect glucose-induced ROS generation.. Thus, high glucose, via PKC signaling, induces oxidative stress and upregulation of COX-2, resulting in reduced NO availability and altered prostanoid profile.

Topics: Cells, Cultured; Cyclooxygenase 2; Cytochrome P-450 Enzyme System; Endothelium, Vascular; Epoprostenol; Gene Expression Regulation; Glucose; Humans; Intramolecular Oxidoreductases; Isoenzymes; Membrane Proteins; Models, Biological; NADPH Oxidases; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Oxidative Stress; Prostaglandin-Endoperoxide Synthases; Prostaglandins; Protein Kinase C; Reactive Oxygen Species; Signal Transduction; Tetradecanoylphorbol Acetate; Thromboxane A2; Tyrosine; Up-Regulation

Peroxynitrite (ONOO(-)), a reactive oxidant produced by the reaction between nitric oxide and superoxide, was found to diffuse into the platelet cytosol and inhibit arachidonic acid-induced platelet aggregations with IC(50) value of 5.8 +/- 1.2 microM. A fluorescence assay established that ONOO(-) diffused into the platelet cytosol in a manner that was inhibited (50-70%) by 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid, an inhibitor of HCO(3)(-)/Cl(-) anion exchanger. Treatment of platelets with (-)-epigallocatechin gallate (2 microM), a tea polyphenol and inhibitor of tyrosine nitration, abolished the inhibitory effect of ONOO(-) on arachidonate-induced aggregations by 88%. ONOO(-) (50-300 microM), added to platelets 1 min before arachidonic acid, inhibited (20-100%) formation of platelet cyclooxygenase (COX) products thromboxane A(2) and 12-hydroxyheptadecatrienoic acid. Interestingly, simultaneous addition of ONOO(-) and arachidonic acid stimulated eicosanoid production by 20 to 60%. The inhibition of thromboxane A(2) generation correlated with the 5- to 10-fold increase in the 3-nitrotyrosine levels of the platelet COX. Experiments with purified COX-1 and COX-2 also showed 9-fold increase of 3-nitrotyrosine levels, which correlated with decreased (93-98%) production of prostaglandin H(2) when ONOO(-) (50 microM) was added 1 min before arachidonic acid. However, the addition of ONOO(-) (50-100 microM) simultaneously with arachidonic acid increased prostaglandin H(2) formation by 30 to 60%. Thus, the inhibitory effect of ONOO(-) involved nitration of COX tyrosine residues, whereas the stimulatory effect was likely to be a result of ONOO(-) functioning as a peroxide activator of eicosanoid signaling. Increasing doses of ONOO(-) not only inhibited platelet COX but also induced formation of unique eicosanoids: iso-prostaglandin F(2alpha), epoxyhydroxyeicosatrienoic acid, and trans-arachidonic acids, suggesting that OH and NO(2) radicals were generated from ONOO(-) in platelets. Formation of ONOO(-) from NO and superoxide may function as a platelet hormone-like COX regulatory mechanism in inflammatory processes in which large amounts of these molecules are produced.

Topics: Blood Platelets; Cell Membrane; Cell Membrane Permeability; Chromatography, High Pressure Liquid; Cyclooxygenase 1; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Diffusion; Eicosanoids; Gas Chromatography-Mass Spectrometry; Humans; Immunoblotting; In Vitro Techniques; Indicators and Reagents; Isoenzymes; Membrane Proteins; Nitrates; Oxidants; Platelet Activation; Platelet Aggregation; Prostaglandin-Endoperoxide Synthases; Thromboxane A2; Tyrosine; Vasoconstriction