ABSTRACT
The medical use of nitroglycerin (GTN) is limited by patient tolerance. The present study evaluated the role of mitochondrial Complex I in GTN biotransformation and the therapeutic effect of mitochondrial antioxidants. The development of GTN tolerance (in rat and human vessels) produced a decrease in mitochondrial O(2) consumption. Co-incubation with the mitochondria-targeted antioxidant mitoquinone (MQ, 10(-6)mol/L) or with glutathione ester (GEE, 10(-4)mol/L) blocked GTN tolerance and the effects of GTN on mitochondrial respiration and aldehyde dehydrogenase 2 (ALDH-2) activity. Biotransformation of GTN depended on the mitochondria being functionally active, particularly mitochondrial Complex I. Tolerance induced mitochondrial ROS production and oxidative stress, though these effects were not detected in HUVECρ(0) cells or Complex I mutant cells. Experiments performed to evaluate Complex I-dependent respiration demonstrated that its inhibition by GTN was prevented by the antioxidants in control samples. These results point to a key role for mitochondrial Complex I in the adequate functioning of ALDH-2. In addition, we have identified mitochondrial Complex I as one of the targets at which the initial oxidative stress responsible for GTN tolerance takes place. Our data also suggest a role for mitochondrial-antioxidants as therapeutic tools in the control of the tolerance that accompanies chronic nitrate use.
Subject(s)
Aldehyde Dehydrogenase/metabolism , Antioxidants/pharmacology , Drug Tolerance , Electron Transport Complex I/metabolism , Mitochondria/enzymology , Nitroglycerin/pharmacology , Animals , Aorta/drug effects , Aorta/metabolism , Biotransformation/drug effects , Cell Line , Cyclic GMP/biosynthesis , Dose-Response Relationship, Drug , Glutathione/metabolism , Human Umbilical Vein Endothelial Cells/drug effects , Human Umbilical Vein Endothelial Cells/metabolism , Humans , In Vitro Techniques , Male , Mice , Mitochondria/drug effects , Mitochondria/metabolism , Mutation/genetics , Oxygen Consumption/drug effects , Rats , Rats, Sprague-Dawley , Reactive Oxygen Species/metabolism , Rotenone/pharmacology , Vasodilation/drug effectsABSTRACT
PURPOSE: Mitochondrial dysfunction plays a key role in sepsis. METHODS: We used a sepsis model of human endothelial cells (HUVEC) to study mitochondrial function during normoxic (21% O(2)) and hypoxic (1% O(2)) conditions. RESULTS: When stimulated with a LPS cocktail, HUVEC displayed an increase of nitric oxide (NO) in normoxic and hipoxic conditions, being higher at 21% O(2). LPS-activation for 24 h at 1% O(2) increased ROS production, which was reversed with the mitochondrial antioxidant Mitoquinone (MQ) and Glutathione Ethyl Ester (GEE). Activated cells displayed diminished mitochondrial O(2) consumption with specific inhibition of Complex I, accompanied by increase in tyrosine nitration and Type II NOS protein expression, effects which were recovered by antioxidants and/or with L-NAME. These parameters varied with O(2) environment, namely inhibition of respiration observed in both O(2) environments at 24 h was very similar, whereas O(2) consumption rate fell earlier in 1% O(2)-exposed cells. While no significant differences were detected at earlier time points, at 24 h tyrosine nitration was higher in normoxic vs. hypoxic cells. CONCLUSIONS: Mitochondria are heavily implicated in sepsis. Mitochondrial antioxidants provide a mechanistic model for the development of potential therapies.
Subject(s)
Antioxidants/physiology , Nitrosation/physiology , Oxidative Stress/physiology , Reactive Oxygen Species/metabolism , Sepsis/metabolism , Antioxidants/analysis , Antioxidants/metabolism , Endotoxins/administration & dosage , Endotoxins/metabolism , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Escherichia coli , Glutathione/analysis , Human Umbilical Vein Endothelial Cells , Humans , Mitochondria/drug effects , Mitochondria/metabolism , Models, Biological , Molecular Targeted Therapy , NAD/analysis , NG-Nitroarginine Methyl Ester/administration & dosage , NG-Nitroarginine Methyl Ester/metabolism , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide/analysis , Oxygen/analysis , Peroxynitrous Acid/analysisABSTRACT
Cellular response to limiting oxygen levels is managed, in part, by the transcription factor hypoxia-inducible factor 1 (HIF-1), and the prolyl hydroxylase (PHD) family of oxygen-requiring enzymes. In the process of analyzing the expression of PHD3, we observed the presence of two alternatively processed PHD3 transcripts, designated PHD3Delta1 and PHD3Delta4 . The expression of both PHD3 and PHD3Delta1 was observed in all tissues and cell lines tested, although the expression of the novel PHD3Delta4 appeared to be restricted to primary cancer tissues. The function of PHD3Delta4 was assessed in transfection experiments showing a preserved prolyl hydroxylase activity. We would submit that PHD3 variants generated by alternative splicing may be intrinsically involved in the complex system of oxygen sensing.
Subject(s)
Procollagen-Proline Dioxygenase/genetics , Alternative Splicing , Amino Acid Sequence , Base Sequence , Cell Line, Tumor , Dioxygenases , Humans , Hydroxylation , Hypoxia-Inducible Factor 1, alpha Subunit/physiology , Hypoxia-Inducible Factor-Proline Dioxygenases , Molecular Sequence Data , Procollagen-Proline Dioxygenase/chemistry , Procollagen-Proline Dioxygenase/metabolismABSTRACT
Exposure to limiting oxygen in cells and tissues induce the stabilization and transcriptional activation of the hypoxia-inducible factor 1 alpha (HIF-1alpha) protein, a key regulator of the hypoxic response. Reactive oxygen species (ROS) generation has been implicated in the stabilization of HIF-1alpha during this response, but this is still a matter of some debate. In this study we utilize a mitochondria-targeted antioxidant, mitoubiquinone (MitoQ), and examine its effects on the hypoxic stabilization of HIF-1alpha. Our results show that under conditions of reduced oxygen (3% O(2)), MitoQ ablated the hypoxic induction of ROS generation and destabilized HIF-1alpha protein. This in turn led to an abrogation of HIF-1 transcriptional activity. Normoxic stabilization of HIF-1alpha, on the other hand, was unchanged in the presence of MitoQ suggesting that ROS were not involved. This study strongly suggests that mitochondrial ROS contribute to the hypoxic stabilization of HIF-1alpha.
