Attenuated vasodilatation in lambs with endogenous and exogenous activation of cGMP signaling: role of protein kinase G nitration.
ABSTRACT Pulmonary vasodilation is mediated through the activation of protein kinase G (PKG) via a signaling pathway involving nitric oxide (NO), natriuretic peptides (NP), and cyclic guanosine monophosphate (cGMP). In pulmonary hypertension secondary to congenital heart disease, this pathway is endogenously activated by an early vascular upregulation of NO and increased myocardial B-type NP expression and release. In the treatment of pulmonary hypertension, this pathway is exogenously activated using inhaled NO or other pharmacological agents. Despite this activation of cGMP, vascular dysfunction is present, suggesting that NO-cGMP independent mechanisms are involved and were the focus of this study. Exposure of pulmonary artery endothelial or smooth muscle cells to the NO donor, Spermine NONOate (SpNONOate), increased peroxynitrite (ONOO(-) ) generation and PKG-1α nitration, while PKG-1α activity was decreased. These changes were prevented by superoxide dismutase (SOD) or manganese(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) and mimicked by the ONOO(-) donor, 3-morpholinosydnonimine N-ethylcarbamide (SIN-1). Peripheral lung extracts from 4-week old lambs with increased pulmonary blood flow and pulmonary hypertension (Shunt lambs with endogenous activation of cGMP) or juvenile lambs treated with inhaled NO for 24 h (with exogenous activation of cGMP) revealed increased ONOO(-) levels, elevated PKG-1α nitration, and decreased kinase activity without changes in PKG-1α protein levels. However, in Shunt lambs treated with L-arginine or lambs administered polyethylene glycol conjugated-SOD (PEG-SOD) during inhaled NO exposure, ONOO(-) and PKG-1α nitration were diminished and kinase activity was preserved. Together our data reveal that vascular dysfunction can occur, despite elevated levels of cGMP, due to PKG-1α nitration and subsequent attenuation of activity.
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ABSTRACT: The nitration of tyrosine to 3-nitrotyrosine is an oxidative modification of tyrosine by nitric oxide and is associated with many diseases, and targeting of protein kinase G (PKG)-I represents a potential therapeutic strategy for pulmonary hypertension and chronic pain. The direct assignment of tyrosine residues of PKG-I has remained to be made due to the low sensitivity of the current proteomic approach. In order to assign modified tyrosine residues of PKG-I, we nitrated purified PKG-Iα expressed in insect Sf9 cells by use of peroxynitrite in vitro and analyzed the trypsin-digested fragments by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Among the 21 tyrosine residues of PKG-Iα, 16 tyrosine residues were assigned in 13 fragments; and six tyrosine residues were nitrated, those at Y71, Y141, Y212, Y336, Y345, and Y567, in the peroxynitrite-treated sample. Single mutation of tyrosine residues at Y71, Y212, and Y336 to phenylalanine significantly reduced the nitration of PKG-Iα; and four mutations at Y71, Y141, Y212, and Y336 (Y4F mutant) reduced it additively. PKG-Iα activity was inhibited by peroxynitrite in a concentration-dependent manner from 30 μM to 1 mM, and this inhibition was attenuated in the Y4F mutant. These results demonstrated that PKG-Iα was nitrated at multiple tyrosine residues and that its activity was reduced by nitration of these residues.Analytical and Bioanalytical Chemistry 01/2014; · 3.66 Impact Factor
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ABSTRACT: Acute lung injury (ALI) is a severe hypoxemic respiratory insufficiency associated with lung leak, diffuse alveolar damage, inflammation, and loss of lung function. Decreased dimethylaminohydrolase (DDAH) activity and increases in asymmetric dimethylarginine (ADMA) along with exaggerated oxidative/nitrative stress contributes to the development of ALI in mice exposed to lipopolysaccharide (LPS). Whether restoring DDAH function and suppressing ADMA levels can effectively ameliorate vascular hyperpermeability and lung injury in ALI is unknown and was the focus of this study. In human lung microvascular endothelial cells, DDAH II over-expression prevented the LPS dependent increase in ADMA, superoxide, peroxynitrite, and protein nitration. DDAH II also preserved endothelial barrier function by inhibiting the LPS induced loss of trans-endothelial resistance and the formation of F-actin stress fibers. Similarly, in vivo, we demonstrated that the targeted over-expression of DDAH II in the pulmonary vasculature significantly inhibited the accumulation of ADMA, and the subsequent increase in oxidative/nitrative stress in the lungs of mice exposed to LPS. Increasing pulmonary DDAH II activity was also able to attenuate the LPS mediated increase in lung vascular leak and prevented neutrophil infiltration into the lung tissue, as indicated by reduced myeloperoxidase activity. Finally, DDAH II gene delivery significantly reduced lung injury in mice exposed to LPS. Together, these data suggest that enhancing DDAH II activity may prove a useful adjuvant therapy to treat patients with ALI.American Journal of Respiratory Cell and Molecular Biology 10/2013; · 4.15 Impact Factor
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ABSTRACT: Recent studies have indicated that during the development of pulmonary hypertension (PH) there is a switch from oxidative phosphorylation to glycolysis in the pulmonary endothelium. However, the mechanisms underlying this phenomenon have not been elucidated. Endothelin-1 (ET-1), an endothelial-derived vasoconstrictor peptide, is increased in PH and has been shown to play an important role in the oxidative stress associated with PH. Thus, in this study we investigated whether there was a potential link between increases in ET-1 and mitochondrial remodeling in. Our data indicate that ET-1 induces the redistribution of endothelial nitric oxide synthase (eNOS) from the plasma membrane to the mitochondria in pulmonary arterial endothelial cells (PAEC) and that this was dependent on eNOS uncoupling. We also found that ET-1 disturbed carnitine metabolism resulting in the attenuation of mitochondrial bioenergetics. However, ATP levels were unchanged due to a compensatory increase in glycolysis. Further mechanistic investigations demonstrated that ET-1 mediated the redistribution of eNOS via the phosphorylation of eNOS at Thr495 by protein kinase C delta (PKCδ). In addition, the glycolytic switch appeared to be dependent on mitochondrial-derived ROS that led to the activation of hypoxia inducible factor (HIF) signaling. Finally, the cell culture data were confirmed in vivo, using the monocrotaline (MCT) rat model of PH. Thus, we conclude that ET-1 induces a glycolytic switch in PAEC via the redistribution of uncoupled eNOS to the mitochondria and that preventing this event may be an approach for the treatment of PH.American Journal of Respiratory Cell and Molecular Biology 01/2014; · 4.15 Impact Factor