Bradford G. Hill

What Part of NO Don't You Understand? Some Answers to the Cardinal Questions in Nitric Oxide Biology

Nitric oxide (NO) regulates biological processes through signaling mechanisms that exploit its unique biochemical properties as a free radical. For the last several decades, the key aspects of the chemical properties of NO relevant to biological systems have been defined, but it has been a challenge to assign these to specific cellular processes. Nevertheless, it is now clear that the high affinity of NO for transition metal centers, particularly iron, and the rapid reaction of NO with oxygen-derived free radicals can explain many of its biological and pathological properties. Emerging studies also highlight a growing importance of the secondary metabolites of NO-dependent reactions in the post-translational modification of key metabolic and signaling proteins. In this minireview, we emphasize the current understanding of the biochemistry of NO and place it in a biological context.

Mitochondrial reserve capacity in endothelial cells: The impact of nitric oxide and reactive oxygen species

The endothelium is not considered to be a major energy-requiring organ, but nevertheless endothelial cells have an extensive mitochondrial network. This suggests that mitochondrial function may be important in response to stress and signaling in these cells. In this study, we used extracellular flux analysis to measure mitochondrial function in adherent bovine aortic endothelial cells (BAEC). Under basal conditions, BAEC use only approximately 35% of their maximal respiratory capacity. We calculate that this represents an intermediate respiratory state between States 3 and 4, which we define as State(apparent) equal to 3.64. Interestingly, the apparent respiratory control ratio (maximal mitochondrial oxygen consumption/non-ADP-linked respiration) in these cells is on the order of 23, which is substantially higher than that which is frequently obtained with isolated mitochondria. These results suggest that mitochondria in endothelial cells are highly coupled and possess a considerable bioenergetic reserve. Because endothelial cells are exposed to both reactive oxygen (ROS) and reactive nitrogen species in the course of vascular disease, we hypothesized that this reserve capacity is important in responding to oxidative stress. To test this, we exposed BAEC to NO or ROS alone or in combination. We found that exposure to nontoxic concentrations of NO or low levels of hydrogen peroxide generated from 2,3-dimethoxy-1,4-napthoquinone (DMNQ) had little impact on basal mitochondrial function but both treatments reversibly decreased mitochondrial reserve capacity. However, combined NO and DMNQ treatment resulted in an irreversible loss of reserve capacity and was associated with cell death. These data are consistent with a critical role for the mitochondrial reserve capacity in endothelial cells in responding to oxidative stress.

Assessing bioenergetic function in response to oxidative stress by metabolic profiling

It is now clear that mitochondria are an important target for oxidative stress in a broad range of pathologies, including cardiovascular disease, diabetes, neurodegeneration, and cancer. Methods for assessing the impact of reactive species on isolated mitochondria are well established but constrained by the need for large amounts of material to prepare intact mitochondria for polarographic measurements. With the availability of high-resolution polarography and fluorescence techniques for the measurement of oxygen concentration in solution, measurements of mitochondrial function in intact cells can be made. Recently, the development of extracellular flux methods to monitor changes in oxygen concentration and pH in cultures of adherent cells in multiple-sample wells simultaneously has greatly enhanced the ability to measure bioenergetic function in response to oxidative stress. Here we describe these methods in detail using representative cell types from renal, cardiovascular, nervous, and tumorigenic model systems while illustrating the application of three protocols to analyze the bioenergetic response of cells to oxidative stress.

Integration of cellular bioenergetics with mitochondrial quality control and autophagy

Abstract Bioenergetic dysfunction is emerging as a cornerstone for establishing a framework for understanding the pathophysiology of cardiovascular disease, diabetes, cancer and neurodegeneration. Recent advances in cellular bioenergetics have shown that many cells maintain a substantial bioenergetic reserve capacity, which is a prospective index of ‘healthy’ mitochondrial populations. The bioenergetics of the cell are likely regulated by energy requirements and substrate availability. Additionally, the overall quality of the mitochondrial population and the relative abundance of mitochondria in cells and tissues also impinge on overall bioenergetic capacity and resistance to stress. Because mitochondria are susceptible to damage mediated by reactive oxygen/nitrogen and lipid species, maintaining a ‘healthy’ population of mitochondria through quality control mechanisms appears to be essential for cell survival under conditions of pathological stress. Accumulating evidence suggest that mitophagy is particularly important for preventing amplification of initial oxidative insults, which otherwise would further impair the respiratory chain or promote mutations in mitochondrial DNA (mtDNA). The processes underlying the regulation of mitophagy depend on several factors, including the integrity of mtDNA, electron transport chain activity, and the interaction and regulation of the autophagic machinery. The integration and interpretation of cellular bioenergetics in the context of mitochondrial quality control and genetics is the theme of this review.

Cardioprotection by N-Acetylglucosamine Linkage to Cellular Proteins

Background— The modification of proteins with O-linked &bgr;-N-acetylglucosamine (O-GlcNAc) represents a key posttranslational modification that modulates cellular function. Previous data suggest that O-GlcNAc may act as an intracellular metabolic or stress sensor, linking glucose metabolism to cellular function. Considering this, we hypothesized that augmentation of O-GlcNAc levels represents an endogenously recruitable mechanism of cardioprotection. Methods and Results— In mouse hearts subjected to in vivo ischemic preconditioning, O-GlcNAc levels were significantly elevated. Pharmacological augmentation of O-GlcNAc levels in vivo was sufficient to reduce myocardial infarct size. We investigated the influence of O-GlcNAc levels on cardiac injury at the cellular level. Lethal oxidant stress of cardiac myocytes produced a time-dependent loss of cellular O-GlcNAc levels. This pathological response was largely reversible by pharmacological augmentation of O-GlcNAc levels and was associated with improved cardiac myocyte survival. The diminution of O-GlcNAc levels occurred synchronously with the loss of mitochondrial membrane potential in isolated cardiac myocytes. Pharmacological enhancement of O-GlcNAc levels attenuated the loss of mitochondrial membrane potential. Proteomic analysis identified voltage-dependent anion channel as a potential target of O-GlcNAc modification. Mitochondria isolated from adult mouse hearts with elevated O-GlcNAc levels had more O-GlcNAc–modified voltage-dependent anion channel and were more resistant to calcium-induced swelling than cardiac mitochondria from vehicle mice. Conclusions— O-GlcNAc signaling represents a unique endogenously recruitable mechanism of cardioprotection that may involve direct modification of mitochondrial proteins critical for survival such as voltage-dependent anion channel.

Protein O-GlcNAcylation: a new signaling paradigm for the cardiovascular system

The posttranslational modification of serine and threonine residues of nuclear and cytoplasmic proteins by the O-linked attachment of the monosaccharide beta-N-acetylglucosamine (O-GlcNAc) is a highly dynamic and ubiquitous protein modification. Protein O-GlcNAcylation is rapidly emerging as a key regulator of critical biological processes including nuclear transport, translation and transcription, signal transduction, cytoskeletal reorganization, proteasomal degradation, and apoptosis. Increased levels of O-GlcNAc have been implicated as a pathogenic contributor to glucose toxicity and insulin resistance, which are both major hallmarks of diabetes mellitus and diabetes-related cardiovascular complications. Conversely, there is a growing body of data demonstrating that the acute activation of O-GlcNAc levels is an endogenous stress response designed to enhance cell survival. Reports on the effect of altered O-GlcNAc levels on the heart and cardiovascular system have been growing rapidly over the past few years and have implicated a role for O-GlcNAc in contributing to the adverse effects of diabetes on cardiovascular function as well as mediating the response to ischemic injury. Here, we summarize our present understanding of protein O-GlcNAcylation and its effect on the regulation of cardiovascular function. We examine the pathways regulating protein O-GlcNAcylation and discuss, in more detail, our understanding of the role of O-GlcNAc in both mediating the adverse effects of diabetes as well as its role in mediating cellular protective mechanisms in the cardiovascular system. In addition, we also explore the parallels between O-GlcNAc signaling and redox signaling, as an alternative paradigm for understanding the role of O-GlcNAcylation in regulating cell function.

Unsaturated lipid peroxidation-derived aldehydes activate autophagy in vascular smooth-muscle cells

Proteins modified by aldehydes generated from oxidized lipids accumulate in cells during oxidative stress and are commonly detected in diseased or aged tissue. The mechanisms by which cells remove aldehyde-adducted proteins, however, remain unclear. Here, we report that products of lipid peroxidation such as 4-HNE (4-hydroxynonenal) and acrolein activate autophagy in rat aortic smooth-muscle cells in culture. Exposure to 4-HNE led to the modification of several proteins, as detected by anti-protein-4-HNE antibodies or protein-bound radioactivity in [3H]4-HNE-treated cells. The 4-HNE-modified proteins were gradually removed from cells. The removal of 4-HNE-modified proteins was not affected by the oxidized protein hydrolase inhibitor, acetyl leucine chloromethyl ketone, or lactacystin, although it was significantly decreased by PSI (proteasome inhibitor I), the lysosome/proteasome inhibitor MG-132 (carbobenzoxy-L-leucyl-L-leucyl-leucinal), insulin or the autophagy inhibitor 3-MA (3-methyladenine). Pre-incubation of cells with rapamycin accelerated the removal of 4-HNE-modified proteins. Treatment with 4-HNE, nonenal and acrolein, but not nonanal or POVPC (1-palmitoyl-2-oxovaleroyl phosphatidyl choline), caused a robust increase in LC3-II (microtubule-associated protein 1 light chain 3-II) formation, which was increased also by rapamycin, but prevented by insulin. Electron micrographs of 4-HNE-treated cells showed extensive vacuolization, pinocytic body formation, crescent-shaped phagophores, and multilamellar vesicles. Treatment with 3-MA and MG-132, but not proteasome-specific inhibitors, induced cell death in 4-HNE-treated cells. Collectively, these results show that lipid peroxidation-derived aldehydes stimulate autophagy, which removes aldehyde-modified proteins, and that inhibition of autophagy precipitates cell death in aldehyde-treated cells. Autophagy may be an important mechanism for the survival of arterial smooth-muscle cells under conditions associated with excessive lipid peroxidation.

Redox regulation of antioxidants, autophagy, and the response to stress: Implications for electrophile therapeutics

Redox networks in the cell integrate signaling pathways that control metabolism, energetics, cell survival, and death. The physiological second messengers that modulate these pathways include nitric oxide, hydrogen peroxide, and electrophiles. Electrophiles are produced in the cell via both enzymatic and nonenzymatic lipid peroxidation and are also relatively abundant constituents of the diet. These compounds bind covalently to families of cysteine-containing, redox-sensing proteins that constitute the electrophile-responsive proteome, the subproteomes of which are found in localized intracellular domains. These include those proteins controlling responses to oxidative stress in the cytosol—notably the Keap1-Nrf2 pathway, the autophagy-lysosomal pathway, and proteins in other compartments including mitochondria and endoplasmic reticulum. The signaling pathways through which electro-philes function have unique characteristics that could be exploited for novel therapeutic interventions; however, development of such therapeutic strategies has been challenging due to a lack of basic understanding of the mechanisms controlling this form of redox signaling. In this review, we discuss current knowledge of the basic mechanisms of thiol-electrophile signaling and its potential impact on the translation of this important field of redox biology to the clinic. Emerging understanding of thiolelectrophile interactions and redox signaling suggests replacement of the oxidative stress hypothesis with a new redox biology paradigm, which provides an exciting and influential framework for guiding translational research.

Cardiac Myocyte–Specific Expression of Inducible Nitric Oxide Synthase Protects Against Ischemia/Reperfusion Injury by Preventing Mitochondrial Permeability Transition

Background— Inducible nitric oxide synthase (iNOS) is an obligatory mediator of the late phase of ischemic preconditioning, but the mechanisms of its cardioprotective actions are unknown. In addition, it remains unclear whether sustained elevation of iNOS in myocytes provides chronic protection against ischemia/reperfusion injury. Methods and Results— Constitutive overexpression of iNOS in transgenic mice (α-myosin heavy chain promoter) did not induce contractile dysfunction and did not affect mitochondrial respiration or biogenesis, but it profoundly decreased infarct size in mice subjected to 30 minutes of coronary occlusion and 24 hours of reperfusion. In comparison with wild-type hearts, isolated iNOS-transgenic hearts subjected to ischemia for 30 minutes followed by 40 minutes of reperfusion displayed better contractile recovery, smaller infarct size, and less mitochondrial entrapment of 2-deoxy-[3H]-glucose. Reperfusion-induced loss of NAD+ and mitochondrial release of cytochrome c were attenuated in iNOS-transgenic hearts, indicating reduced mitochondrial permeability transition. The NO donor NOC-22 prevented permeability transition in isolated mitochondria, and mitochondrial permeability transition–induced NAD+ loss was decreased in wild-type but not iNOS-null mice treated with the NO donor diethylene triamine/NO 24 hours before ischemia and reperfusion ex vivo. iNOS-mediated cardioprotection was not abolished by atractyloside. Reperfusion-induced production of oxygen-derived free radicals (measured by electron paramagnetic resonance spectroscopy) was attenuated in iNOS-transgenic hearts and was increased in wild-type hearts treated with the mitochondrial permeability transition inhibitor cyclosporin A. Conclusions— Cardiomyocyte-restricted expression of iNOS provides sustained cardioprotection. This cardioprotection is associated with a decrease in reperfusion-induced oxygen radicals and inhibition of mitochondrial swelling and permeability transition.

Protein glutathiolation by nitric oxide: an intracellular mechanism regulating redox protein modification

This study was designed to examine whether NO regulates protein glutathiolation. Exposure to NO donors increased protein glutathiolation in COS‐7 or rat aortic smooth muscle cells as detected by anti‐protein glutathione (GSH) antibodies. This process was reversible and saturable. Stimulation with acetylcholine (ACh) increased protein glutathiolation in isolated rat aortic rings. This was prevented by inhibiting endothelial NO synthase (eNOS). In ACh‐treated rings, proteins showing positive immunoreactivity with the anti‐PSSG antibody (Ab) were identified by matrix assisted laser desorption‐time‐of‐flight mass spectrometry to be actin, vimentin, and heat shock protein 70. Purified actin was more readily glutathiolated by Snitrosoglutathione than by oxidized GSH as determined by electrospray‐ionization mass spectrometry, and nitrosylated actin was glutathiolated by reduced GSH. Relative to wild‐type (WT) mice, increased protein glutathiolation was observed in hearts of mice with cardiac‐specific expression of inducible NO synthase (iNOS). Proteins immunoprecipitated from transgenic hearts revealed GSH‐adducted peptides corresponding to adenine nucleotide translocator and the α‐subunit of F1F0ATPase. These data suggest that exogenous NO or NO generated by eNOS or iNOS regulates protein adduction with GSH. This could be due to a direct reaction of proteins with S‐nitrosoglutathione or denitrosylation of S‐nitrosylated proteins by reduced GSH. Glutathiolation of cytoskeletal and mitochondrial proteins may be a significant feature of NO bioreactivity.—West, M. B., Hill, B. G., Xuan, Y.‐T., Bhatnagar, A. Protein glutathiolation by nitric oxide: An intracellular mechanism regulating redox protein modification FASEB J. 20, E1049–E1060 (2006)