3-nitrotyrosine and Hypotension

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Arterial Pressure; Cyclooxygenase 2; Epoprostenol; Gene Expression Regulation, Enzymologic; Heart Rate; Hypotension; I-kappa B Proteins; Inflammation; Lipopolysaccharides; Male; Nitric Oxide; Nitric Oxide Synthase Type II; Peroxidase; Peroxynitrous Acid; Rats; Rats, Wistar; Ribosomal Protein S6; Signal Transduction; TOR Serine-Threonine Kinases; Transcription Factor RelA; Tumor Necrosis Factor-alpha; Tyrosine

Bioconversion of glyceryl trinitrate (GTN) into nitric oxide (NO) by aldehyde dehydrogenase-2 (ALDH-2) is a crucial mechanism which drives vasodilatory and antiplatelet effect of organic nitrates in vitro and in vivo. Oxidative stress generated by overproduction of free radical species, mostly superoxide anions and NO-derived peroxynitrite, has been suggested to play a pivotal role in the development of nitrate tolerance, though the mechanism still remains unclear. Here we studied the free radical-dependent impairment of ALDH-2 in platelets as well as vascular tissues undergoing organic nitrate ester tolerance and potential benefit when using the selective peroxynitrite decomposition catalyst Mn(III) tetrakis (4-Benzoic acid) porphyrin (MnTBAP). Washed human platelets were made tolerant to nitrates via incubation with GTN for 4h. This was expressed by attenuation of platelet aggregation induced by thrombin (40U/mL), an effect accompanied by GTN-related induction of cGMP levels in platelets undergoing thrombin-induced aggregation. Both effects were associated to attenuated GTN-induced nitrite formation in platelets supernatants and to prominent nitration of ALDH-2, the GTN to NO metabolizing enzyme, suggesting that GTN tolerance was associated to reduced NO formation via impairment of ALDH-2. These effects were all antagonized by co-incubation of platelets with MnTBAP, which restored GTN-induced responses in tolerant platelets. Comparable effect was found under in in vivo settings. Indeed, MnTBAP (10mg/kg, i.p.) significantly restored the hypotensive effect of bolus injection of GTN in rats made tolerants to organic nitrates via chronic administration of isosorbide-5-mononitrate (IS-5-MN), thus confirming the role of peroxynitrite overproduction in the development of tolerance to vascular responses induced by organic nitrates. In conclusion, oxidative stress subsequent to prolonged use of organic nitrates, which occurs via nitration of ALDH-2, represents a key event in GTN tolerance, an effect counteracted both in vitro and in vivo by novel peroxynitrite decomposition catalyst.

Topics: Aldehyde Dehydrogenase; Animals; Aorta; Blood Platelets; Cyclic GMP; Drug Tolerance; Endothelium, Vascular; Humans; Hypotension; Isosorbide Dinitrate; Male; Metalloporphyrins; Muscle, Smooth, Vascular; Nitrates; Nitric Oxide; Nitrites; Nitroglycerin; Platelet Aggregation; Rats; Thrombin; Tyrosine; Vasodilator Agents

Formation of nitric oxide and its derivative reactive nitrogen species during endotoxemia has been implicated in the pathogenesis of the associated cardiovascular dysfunction. This stress can promote nitrosative post-translational modifications of proteins that may alter their activity and contribute to dysregulation. We utilized the ascorbate-dependent biotin-switch method to assay protein S-nitrosylation and immunoblotted for tyrosine nitration to monitor changes in nitrosative protein oxidation during endotoxemia. Hearts from lipopolysaccharide (LPS)-treated rats showed no apparent variation in global protein S-nitrosylation, but this may be due to the poor sensitivity of the biotin-switch method. To sensitize our monitoring of protein S-nitrosylation we exposed isolated hearts to the efficient trans-nitrosylating agent nitrosocysteine (which generated a robust biotin-switch signal) and then identified a number of target proteins using mass spectrometry. We were then able to probe for these target proteins in affinity-capture preparations of S-nitrosylated proteins prepared from vehicle- or LPS-treated animals. Unexpectedly this showed a time-dependent loss in S-nitrosylation during sepsis, which we hypothesized, may be due to concomitant superoxide formation that may lower nitric oxide but simultaneously generate the tyrosine-nitrating agent peroxynitrite. Indeed, this was confirmed by immunoblotting for global tyrosine nitration, which increased time-dependently and temporally correlated with a decrease in mean arterial pressure. We assessed if tyrosine nitration was causative in lowering blood pressure using the putative peroxynitrite scavenger FeTPPS. However, FeTPPS was ineffective in reducing global protein nitration and actually exacerbated LPS-induced hypotension.

Topics: Animals; Biotin; Blood Pressure; Cysteine; Endotoxemia; Escherichia coli; Heart; HEK293 Cells; Humans; Hypotension; Immunoblotting; In Vitro Techniques; Lipopolysaccharides; Mass Spectrometry; Metalloporphyrins; Mice; Mice, Inbred C57BL; Models, Animal; Nitric Oxide; Nitric Oxide Synthase; Oxidation-Reduction; Peroxynitrous Acid; Protein Processing, Post-Translational; Proteins; Rats; Rats, Wistar; S-Nitroso-N-Acetylpenicillamine; Sensitivity and Specificity; Sepsis; Signal Transduction; Telemetry; Tyrosine

Increased production of inducible nitric oxide (NO) synthase (iNOS)-derived NO contributes to fall in blood pressure and vascular reactivity during endotoxemia. We investigated whether an increase in protein expression and activity of the enzymes involved in mitogen-activated protein kinase kinase 1 (MEK1)/extracellular signal-regulated kinase 1/2 (ERK1/2)/iNOS/soluble guanylyl cyclase (sGC)/protein kinase G (PKG) pathway associated with peroxynitrite production would contribute to endotoxin-induced decrease in mean blood pressure (MAP) and vascular reactivity in rats. A selective iNOS inhibitor, 1,3-PBIT (10 mg/kg, i.p.), or a selective inhibitor ERK1/2 phosphorylation by MEK1, U0126 (5mg/kg, i.p.), prevented endotoxin (10mg/kg, i.p.)-induced decrease in MAP and vascular reactivity to norepinephrine (0.001-100 μM) in endothelium-intact and -denuded arteries associated with increased levels of nitrite (an index for NO production), cyclic GMP (an index for sGC activity), phosphorylated vasodilator stimulated phosphoprotein (an index for PKG activity), and nitrotyrosine (an index for peroxynitrite production). Endotoxin-induced increase in the phosphorylated MEK1 protein levels were not changed by 1,3-PBIT or U0126. U0126 prevented the endotoxin-induced increase in phosphorylated ERK1/2 and iNOS expressions. A selective sGC inhibitor, ODQ (3μM), prevented the endotoxin-induced decrease in the E(max) values and increase in the EC(50) values of norepinephrine in endothelium-intact aortic rings isolated from endotoxemic rats in vitro. ODQ also reversed the effect of endotoxin on the increase in the EC(50) values of norepinephrine in endothelium-denuded rings. A selective PKG inhibitor, KT5823 (1 μM), only prevented the endotoxin-induced decrease in the E(max) values of norepinephrine in arteries with endothelium. These results suggest that activation of MEK1/ERK1/2 pathway leading to an increase in iNOS protein expression and NO production associated with an increase in sGC and PKG activity and peroxynitrite formation results in hypotension and vascular hyporeactivity in endotoxemic rats. However, further study is needed to confirm the involvement of PKG to the fall in vascular reactivity in the rat model of endotoxemia.

Topics: Animals; Aorta, Thoracic; Blood Pressure; Cyclic GMP-Dependent Protein Kinases; Endotoxemia; Endotoxins; Enzyme Activation; Enzyme Inhibitors; Guanylate Cyclase; Heart Rate; Hypotension; Male; MAP Kinase Kinase 1; Mitogen-Activated Protein Kinase 3; Nitric Oxide Synthase Type II; Nitrites; Norepinephrine; Oxidative Stress; Peroxynitrous Acid; Rats; Rats, Wistar; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Soluble Guanylyl Cyclase; Tyrosine

Nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) mediates hypotension and metabolic derangements in sepsis. We hypothesized that selective iNOS-inhibition would prevent hypotension in septic rats without inhibiting endothelium-dependent vasodilation caused by the physiologically important endothelial NOS. Rats were exposed to lipopolysaccharide (LPS) for 6 h and the selective iNOS-inhibitor L-N6-(1-iminoethyl)-lysine (L-NIL), the nonselective NOS-inhibitor N:(G)-nitro-L-arginine methyl ester (L-NAME), or control. Mean arterial pressure (MAP) and vasodilation to acetylcholine (ACh, endothelium-dependent), sodium nitroprusside (SNP, endothelium-independent), and isoproterenol (ISO, endothelium-independent beta agonist) were determined. Exhaled NO, nitrate/nitrite-(NOx) levels, metabolic data, and immunohistochemical staining for nitrotyrosine, a tracer of peroxynitrite-formation were also determined. In control rats, L-NAME increased MAP, decreased the response to ACh, and increased the response to SNP, whereas L-NIL did not alter these variables. LPS decreased MAP by 18% +/- 1%, decreased vasodilation (ACh, SNP, and ISO), increased exhaled NO, NOx, nitrotyrosine staining, and caused acidosis and hypoglycemia. L-NIL restored MAP and vasodilation (ACh, SNP, and ISO) to baseline and prevented the changes in exhaled NO, NOx, pH, and glucose levels. In contrast, L-NAME restored MAP and SNP vasodilation, but did not alter the decreased response to ACh and ISO or prevent the changes in exhaled NO and glucose levels. Finally, L-NIL but not L-NAME decreased nitrotyrosine staining in LPS rats. In conclusion, L-NIL prevents hypotension and metabolic derangements in septic rats without affecting endothelium-dependent vasodilation whereas L-NAME does not.. Sepsis causes hypotension and metabolic derangements partly because of increased nitric oxide. Selective inhibition of nitric oxide produced by the inducible nitric oxide synthase enzyme prevents hypotension and attenuates metabolic derangements while preserving the important vascular function associated with endothelium-dependent vasodilation in septic rats.

Topics: Acetylcholine; Animals; Blood Pressure; Endothelium, Vascular; Enzyme Inhibitors; Hypotension; Lipopolysaccharides; Lysine; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Rats, Sprague-Dawley; Sepsis; Survival Rate; Tyrosine; Vasodilation