Nitric oxide (NO) plays key roles in cell signaling and physiology, with diverse functions mediated by NO concentrations varying over three orders-of-magnitude. In spite of this critical concentration dependence, current approaches to NO delivery in vitro result in biologically irrelevant and poorly controlled levels, with hyperoxic conditions imposed by ambient air. To solve these problems, we developed a system for controlled delivery of NO and O2 over large concentration ranges to mimic biological conditions. Here we describe the fabrication, operation and calibration of the delivery system. We then describe applications for delivery of NO and O2 into cell culture media, with a comparison of experimental results and predictions from mass transfer models that predict the steady-state levels of various NO-derived reactive species. We also determined that components of culture media do not affect the steady-state levels of NO or O2 in the device. This system provides critical control of NO delivery for in vitro models of NO biology and chemistry.
Nitric oxide and secondary oxides of nitrogen react with unsaturated fatty acids such as linoleic acid to yield oxidized and nitrated products. Fatty acid nitroalkene derivatives, (e.g. nitrolinoleate [LNO2]) are produced by oxidative inflammatory reactions, detected clinically, display potent electrophilic reactivity and induce post-translational protein modifications that mediate adaptive inflammatory signaling responses. LNO2 signaling was examined in lung epithelial cells because the alveolar compartment is a rich site for the transduction of redox and inflammatory reactions. LNO2 did not directly induce Ca2+ influx in cultured lung epithelial cells, but inhibited bradykinin-induced Ca2+ influx in a cGMP-independent manner. In contrast, LNO2 activated MAP kinase (Erk1/2) by a mechanism independent of bradykinin. It was hypothesized that these unique responses were transduced by activation of different protein kinase C isotypes, supported by the observation that LNO2-mediated inhibition of Ca2+ influx was blocked by the non-selective PKC inhibitors chelerythine chloride and calphostin C, but not by the calcium dependent “classic” PKC inhibitor Gö6976. Western blot analysis showed that atypical PKCf was activated by LNO2 stimulation, with PKCf and Erk activation also demonstrated in primary culture of human lung type II cells. Addition of pseudotypical PKCζ substrate peptide reversed LNO2-mediated induction of Ca2+ influx and MAP kinase activation. Finally, the electrophilic nature of LNO2 resulted in a novel mode of PKCζ activation, covalent adduction of the enzyme. In summary, LNO2 mediated signaling in lung type II epithelial cells occurs via a unique pathway involving PKCζ.
Signal transduction; Protein kinase C; Pulmonary epithelial cell; Calcium mobilization; Nitrated lipids
Regulation of protein function by S-nitrosation of critical cysteines is known to be an important mechanism for nitric oxide signaling. Evidence for this comes from several different experimental approaches including the ascorbate-based biotin switch method. However technical problems with specificity and sensitivity of ascorbate reduction of S-nitrosothiols limit its usefulness and reliability. In the current study we report the use of triphenylphosphine ester derivatives to selectively reduce SNO bonds in proteins. After triphenylphosphine ester reduction thiols were tagged with biotin or fluorescently labeled maleimide reagents. Importantly we demonstrate that these compounds are specific reductants of SNO in complex biological samples and do not reduce protein disulfides or protein thiols modified by hydrogen peroxide. Reduction proceeds efficiently in cell extracts and in whole fixed cells. Application of this approach allowed us to demonstrate S-nitrosation of specific cellular proteins, label S-nitrosoproteins in whole fixed cells (especially the nuclear compartment) and demonstrate S-nitrosoprotein formation in cells expressing inducible nitric oxide synthase.
S-nitrosoprotein; triphenylphosphine; nitric oxide
Protein S-nitrosation is a reversible post-translation modification critical for redox-sensitive cell signaling that is typically studied using the Biotin Switch method. This method and subsequent modifications usually require avidin binding or Western blot analysis to detect biotin labeled proteins. We describe here a modification of the Biotin Switch assay that eliminates the need for Western blot or avidin enrichment protocols and allows direct comparison of the S-nitrosation state proteins from two different samples in the same gel lane or on the same 2D gel. This S-FLOS method offers detection, identification and quantification of S-nitrosated proteins, with the potential for site-specific identification of nitrosation events.
S-nitrosylation; Nitric oxide; Biotin switch; SNO; Proteomics
Pulmonary hypertension (PH) is a rare disorder that without treatment is progressive and often fatal within 3 years. The treatment of PH involves the use of a diverse group of drugs and lung transplantation. Although nitrite was once thought to be an inactive metabolite of endothelial-derived nitric oxide (NO), there is increasing evidence that nitrite may be useful in the treatment of PH, but the mechanism by which nitrite exerts its beneficial effect remains uncertain. The purpose of this study was to investigate the effect of chronic sodium nitrite treatment in a PH model in the rat. Following induction of PH with a single injection of monocrotaline, 60 mg; daily ip injections of sodium nitrite (3 mg/kg) starting on day 14 and continuing for 21 days, resulted in a significantly lower pulmonary arterial pressure on day 35 when compared to values in untreated animals with monocrotaline-induced PH. In monocrotaline-treated rats, daily treatment with ip nitrite injections for 21 days decreased right ventricular mass and pathologic changes in small pulmonary arteries. Nitrite therapy did not change systemic arterial pressure or cardiac output when values were measured on day 35. The decreases in pulmonary arterial pressure in response to iv injections of sodium nitroprusside, sodium nitrite, and BAY 41-8543 were not different in rats with monocrotaline-induced pulmonary hypertension and rats with chronic nitrite therapy when compared to responses in animals in which pulmonary arterial pressure was increased with U46619. These findings are consistent with the hypothesis that the mechanisms that convert nitrite to vasoactive NO, activate soluble guanylyl cyclase and mediate the vasodilator response to NO or an NO derivative are not impaired. The present data are consistent with the results of a previous study in monocrotaline-induced PH in which systemic arterial pressure and cardiac output were not evaluated and are consistent with the hypothesis that nitrite is effective in the treatment of monocrotaline-induced PH in the rodent.
pulmonary vascular bed; systemic vascular bed; pulmonary hypertension/therapy; right ventricular hypertrophy; sodium nitrite; nitric oxide; soluble guanylyl cyclase; monocrotaline
Burkholderia pseudomallei infections are fastidious to treat with conventional antibiotic therapy, often involving a combination of drugs and long-term regimes. Bacterial genetic determinants contribute to the resistance of B. pseudomallei to many classes of antibiotics. In addition, anaerobiosis and hypoxia in abscesses typical of melioidosis select for persistent populations of B. pseudomallei refractory to a broad spectrum of antibacterials. We tested the susceptibility of B. pseudomallei to the drugs hydroxyurea, spermine NONOate and DETA NONOate that release nitric oxide (NO). Our investigations indicate that B. pseudomallei are killed by NO in a concentration and time-dependent fashion. The cytoxicity of this diatomic radical against B. pseudomallei depends on both the culture medium and growth phase of the bacteria. Rapidly growing, but not stationary phase, B. pseudomallei are readily killed upon exposure to the NO donor spermine NONOate. NO also has excellent antimicrobial activity against anaerobic B. pseudomallei. In addition, persistent bacteria highly resistant to most conventional antibiotics are remarkably susceptible to NO. Sublethal concentrations of NO inhibited the enzymatic activity of [4Fe-4S]-cofactored aconitase of aerobic and anaerobic B. pseudomallei. The strong anti-B. pseudomallei activity of NO described herein merits further studies on the application of NO-based antibiotics for the treatment of melioidosis.
antibiotics; antimicrobials; melioidosis; reactive nitrogen species; therapy; [4Fe-4S] clusters
The presence of acellular hemoglobin (Hb) within the circulation is generally viewed as a pathological state that can result in toxic consequences. Haptoglobin (Hp), a globular protein found in the plasma, binds with high avidity the αβ dimers derived from the dissociation of Hb tetramer and thus helps clear free Hb. More recently there have been compelling indications that the redox properties of the Hp bound dimer (Hb–Hp) may play a more active role in controlling toxicity by limiting the potential tissue damage caused by propagation of the free-radicals generated within the heme containing globin chains. The present study further examines the potential protective effect of Hp through its impact on the production of nitric oxide (NO) from nitrite through nitrite reductase activity of the Hp bound αβ Hb dimer. The presented results show that the Hb dimer in the Hb–Hp complex has oxygen binding, CO recombination and spectroscopic properties consistent with an Hb species having properties similar to but not exactly the same as the R quaternary state of the Hb tetramer. Consistent with these observations is the finding that the initial nitrite reductase rate for Hb–Hp is approximately ten times that of HbA under the same conditions. These results in conjunction with the earlier redox properties of the Hb–Hp are discussed in terms of limiting the pathophysiological consequences of acellular Hb in the circulation.
Nitrite reductase; Haptoglobin; Hemoglobin
Doxorubicin (DOX) is one of the most powerful and widely prescribed chemotherapeutic agents to treat divergent human cancers. However, the clinical use of DOX is restricted due to its severe cardiotoxic side-effects. There has been ongoing search for cardioprotectants against DOX toxicity. Inorganic nitrate has emerged as a bioactive compound that can be reduced into nitrite and nitric oxide in vivo and in turn plays a therapeutic role in diseases associated with nitric oxide insufficiency or dysregulation. In this review, we describe a novel concept of using dietary supplementation of inorganic nitrate to reduce DOX-induced cardiac cellular damage and dysfunction, based on our recent promising studies in a mouse model of DOX cardiotoxicity. Our data show that chronic oral ingestion of sodium nitrate, at a dose equivalent to ~400% of the Acceptable Daily Intake of the World Health Organization, alleviated DOX-induced left ventricular dysfunction and mitochondrial respiratory chain damage. Such cardioprotective effects were associated with reduction of cardiomyocyte necrosis/apoptosis, tissue lipid peroxidation, and mitochondrial H2O2 generation following DOX treatment. Furthermore, proteomic studies revealed enhanced cardiac expression of mitochondrial antioxidant enzyme – peroxiredoxin 5 in the nitrate-treated animals. These studies suggest that inorganic nitrate could be an inexpensive therapeutic agent for long-term oral administration in preventing DOX-induced cardiac toxicity and myopathy during the prolonged pathological process. Future clinical trials in the cancer patients undergoing DOX chemotherapy are warranted to translate these experimental findings into an effective new therapy in preventing the DOX-induced cardiomyopathy.
anthracycline; cardioprotection; cardiotoxicity; mitochondria; nitrate; ventricular function
Peripheral Artery Disease (PAD) represents a burgeoning form of cardiovascular disease associated with significant clinical morbidity and increased 5 year cardiovascular disease mortality. It is characterized by impaired blood flow to the lower extremities, claudication pain and severe exercise intolerance. Pathophysiological factors contributing to PAD include atherosclerosis, endothelial cell dysfunction, and defective nitric oxide metabolite physiology and biochemistry that collectively lead to intermittent or chronic tissue ischemia. Recent work from our laboratories is revealing that nitrite/nitrate anion and nitric oxide metabolism plays an important role in modulating functional and pathophysiological responses during this disease. In this review, we discuss experimental and clinical findings demonstrating that nitrite anion acts to ameliorate numerous pathophysiological events associated with PAD and chronic tissue ischemia. We also highlight future directions for this promising line of therapy.
ischemia; angiogenesis; arteriogenesis; vasodilation; exercise; blood flow; claudication
Since their initial discovery over a century ago, our knowledge of the functions of myoglobin and the mitochondrion has gradually evolved. The mitochondrion, once thought to be solely responsible for energy production, is now known to be an integral redox and apoptotic signal tranducer within the cell. Likewise, myoglobin, traditionally thought of only as an oxygen store, has emerged as a physiological catalyst that can modulate reactive oxygen species levels, facilitate oxygen diffusion and scavenge or generate nitric oxide (NO) depending on oxygen tensions within the cell. By virtue of its unique ability to regulate O2 and NO levels within the cell, myoglobin can modulate mitochondrial function in energy-demanding tissues such as the beating heart and exercising muscle. In this review, we present the conventional functions of myoglobin and mitochondria, and describe how these roles have been reassessed and advanced, particularly in the context of NO and nitrite signaling. We present the mechanisms by which mitochondria and myoglobin regulate one another within the cell through their interactions with NO and oxygen and discuss the implications of these interactions in terms of health and disease.
cytochrome c oxidase; nitrite; nitrite reductase; facilitated diffusion; hypoxia
Responses to glyceryl trinitrate/nitroglycerin (GTN), S-nitrosoglutathione (GSNO), and sodium nitrite were compared in the intact chest rat. The iv injections of GTN, sodium nitrite, and GSNO produced dose-dependent decreases in pulmonary and systemic arterial pressures. In as much as cardiac output was not reduced, the decreases in pulmonary and systemic arterial pressures indicate that GTN, sodium nitrite, and GSNO have significant vasodilator activity in the pulmonary and systemic vascular beds in the rat. Responses to GTN were attenuated by cyanamide, but not allopurinol, whereas responses to nitrite formed by the metabolism of GTN were attenuated by allopurinol and cyanamide. The results with allopurinol and cyanamide suggest that only mitochondrial aldehyde dehydrogenase is involved in the bioactivation of GTN, sodium nitrite, and GSNO, whereas both pathways are involved in the bioactivation of nitrite anion in the intact rat. The comparison of vasodilator activity indicates that GSNO and GTN are more than 1000 fold more potent than sodium nitrite in decreasing pulmonary and systemic arterial pressures in the rat. Following administration of 1H-[1,2,4]-oxadizaolo[4,3-]quinoxaline-1-one (ODQ), responses to GTN were significantly attenuated, indicating that responses are mediated by the activation of soluble guanylyl cyclase. These data suggest that the reduction of nitrite to nitric oxide formed from the metabolism of GTN, cannot account for the vasodilator activity of GTN in the intact rat and that another mechanism; perhaps the formation of an S-NO, may mediate the vasodilator response to GTN in this species.
Mitochondrial aldehyde dehydrogenase; Xanthine oxidoreductase; Nitric oxide; S-nitrosothiols; Glyceryl trinitrate/nitroglycerin; Sodium nitrite; Sodium nitroprusside; Allopurinol; Cyanamide; L-NAME
While much research has been directed to harnessing the antimicrobial properties of exogenous NO, the possibility of bacteria developing resistance to such therapy has not been thoroughly studied. Herein, we evaluate potential NO resistance using spontaneous and serial passage mutagenesis assays. Specifically, Staphylococcus aureus, Methicillin-resistant S. aureus (MRSA), Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa were systematically exposed to NO-releasing 75mol% MPTMS-TEOS nitrosothiol particles at or below minimum inhibitory concentration (MIC) levels. In the spontaneous mutagenesis assay, bacteria that survived exposure to lethal concentrations of NO showed no increase in MIC. Similarly, no increase in MIC was observed in the serial passage mutagenesis assay after exposure of these species to sub-inhibitory concentrations of NO through 20 d.
antimicrobial resistance; nitric oxide; resistance; spontaneous mutagenesis; serial passage mutagenesis
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 utilised 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 sensitise 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 hypothesised, 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.
sepsis; lipopolysaccharide; nitrosative; S-nitrosylation; nitration; blood pressure
Nitric oxide synthases (NOSs) have been shown to modulate thermal hyperalgesia and mechanical hypersensitivity in inflammatory and neuropathic pain. However, little is known about the effect of NOSs on baseline function of sensory nerve fibers. Using genetic deficiency and pharmacologic inhibition of NOSs, we examined the impact of the three isoforms NOS1, NOS2, and NOS3 on baseline nocifensive behavior by measuring current vocalization threshold in response to electrical stimulation at 5, 250, 2000 Hz that preferentially stimulate C, Aδ, and Aβ fibers. In response to 5, 250 and 2000 Hz, NOS1-deficient animals had significantly higher current vocalization thresholds compared with wild-type. Genetic deficiency of NOS2 was associated with higher current vocalization thresholds in response to 5 Hz (C-fiber) stimulation. In contrast, NOS3-deficient animals had an overall weak trend toward lower current vocalization thresholds at 5 Hz and significantly lower current vocalization threshold compared with wild-type animals at 250 and 2000 Hz. Therefore, NOSs distinctively affect baseline mouse current vocalization threshold and appear to play a role on nocifensive response to electrical stimulation of sensory nerve fibers.
Nociception; Pain; Vocalization; Nitric oxide; Mouse
Soluble guanylyl cyclase (sGC) is a key protein in the nitric oxide (NO)/-cGMP signaling pathway. sGC activity is involved in a number of important physiological processes including smooth muscle relaxation, neurotransmission and platelet aggregation and adhesion. Regulation of sGC expression and activity emerges as a crucial factor in control of sGC function in normal and pathological conditions. Recently accumulated evidence strongly indicates that the regulation of sGC expression is a complex process modulated on several levels including transcription, post-transcriptional regulation, translation and protein stability. Presently our understanding of mechanisms governing regulation of sGC expression remains very limited and awaits systematic investigation. Among other ways, the expression of sGC subunits is modulated at the levels of mRNA abundance and transcript diversity. In this review we summarize available information on different mechanisms (including transcriptional activation, mRNA stability and alternative splicing) involved in the modulation of mRNA levels of sGC subunits in response to various environmental clues. We also summarize and cross-reference the information on human sGC splice forms available in the literature and in genomic databases. This review highlights the fact that the study of the biological role and regulation of sGC splicing will bring new insights to our understanding of NO/cGMP biology.
nitric oxide; soluble guanylyl cyclase; splicing; regulation
The biosynthesis of nitric oxide (NO) and prostaglandin share many similarities. Two major forms of nitric oxide synthase (NOS) and cyclooxygenase (COX) have been identified: constitutive vs inducible. In general, the constitutive form functions in housekeeping and physiologic roles whereas the inducible form is up-regulated by mitogenic or inflammatory stimuli and is responsible for pathophysiological responses. The cross talk between the COX and NOS pathways was initially reported 1993 and since then, numerous studies have been undertaken to delineate the functional consequences of this interaction as well as the potential mechanism by which each pathway interacts. This review will focus in particular on recent advances in this field that extend our understanding of these two pathways under various systems.
nitric oxide; nitric oxide synthase; cyclooxygenase; prostaglandin; s-nitrosylation; peroxynitrite
Nitric oxide (NO) is present in exhaled breath and is generally considered to be a noninvasive marker of airway inflammation, and is thus of particular relevance to monitoring asthma. NO is produced when l-arginine is converted to l-citrulline by NO synthase (NOS); however, l-arginine is also the substrate for arginase and both enzymes are upregulated in asthma. Recent reports have speculated that enhanced expression of one or both enzymes could lead to a limitation in substrate availability, and hence impact downstream targets or markers such as exhaled NO. The non-linear nature and vastly different kinetics of the enzymes make predictions difficult, particularly over the wide range of enzyme activity between baseline and inflammation. In this study, we developed a steady state model of l-arginine transmembrane transport, NO production, diffusion, and gas phase NO release from lung epithelial cells. We validated our model with experimental results of gas phase NO release and intracellular l-arginine concentration in A549 cells, and then performed a sensitivity analysis to determine relative impact of each enzyme on NO production. Our model predicts intracellular l-arginine and gas phase NO release over a wide range of initial extracellular l-arginine concentrations following stimulation with cytomix (10 ng/ml TNF-α, IL-1β, and INF-γ). Relative sensitivity analysis demonstrates that enhanced arginase activity has little impact on l-arginine bioavailability for NOS. In addition, NOS activity is the dominant parameter which impacts gas phase NO release.
Arginase; Nitric oxide synthase; l-Arginine
Nitric oxide (NO) regulates vascular smooth muscle cell (VSMC) structure and function, in part by activating soluble guanylate cyclase (sGC) to synthesize cGMP. The objective of this study was to further characterize the signaling mechanisms by which NO regulates VSMC gene expression using transcription profiling. DNA microarrays were hybridized with RNA extracted from rat pulmonary artery smooth muscle cells (RPaSMC) exposed to the NO donor compound, S-nitroso-glutathione (GSNO). Many of the genes, whose expression was induced by GSNO, contain a cAMP-response element (CRE), of which one encoded the inducible cAMP early repressor (ICER). sGC and cAMP-dependent protein kinase, but not cGMP-dependent protein kinase, were required for NO-mediated phosphorylation of CRE-binding protein (CREB) and induction of ICER gene expression. Expression of a dominant-negative CREB in RPaSMC prevented the NO-mediated induction of CRE-dependent gene transcription and ICER gene expression. Pre-treatment of RPaSMC with the intracellular calcium (Ca2+) chelator, BAPTA-AM, blocked the induction of ICER gene expression by GSNO. The store-operated Ca2+ channel inhibitors, 2-ABP and SKF-96365, reduced the GSNO-mediated increase in ICER mRNA levels, while 2-ABP did not inhibit GSNO-induced CREB phosphorylation. Our results suggest that induction of ICER gene expression by NO requires both CREB phosphorylation and Ca2+ signaling. Transcription profiling of RPaSMC exposed to GSNO revealed important roles for sGC, PKA, CREB, and Ca2+ in the regulation of gene expression by NO. The induction of ICER in GSNO-treated RPaSMC highlights a novel cross-talk mechanism between cGMP and cAMP signaling pathways.
nitric oxide; vascular smooth muscle; cAMP-response element; cyclic GMP; protein kinase A
Induction and activation of nitric oxide (NO) synthases (NOS) and excessive production of NO are common features of almost all diseases associated with infection and acute or chronic inflammation, although the contribution of NO to the pathophysiology of these diseases is highly multifactorial and often still a matter of controversy. Because of its direct impact on tissue oxygenation and cellular oxygen (O2) consumption and redistribution, the ability of NO to regulate various aspects of hypoxia-induced signaling has received widespread attention. Conditions of tissue hypoxia and the activation of hypoxia-inducible factors (HIF) have been implicated in hypoxia or in cancer biology, but are also being increasingly recognized as important features of acute and chronic inflammation. Thus, the activation of HIF transcription factors has been increasingly implicated in inflammatory diseases, and recent studies have indicated its critical importance in regulating phagocyte function, inflammatory mediator production, and regulation of epithelial integrity and repair processes. Finally, HIF also appears to contribute to important features of tissue fibrosis and epithelial-to-mesenchymal transition, processes that are associated with tissue remodeling in various non-malignant chronic inflammatory disorders. In this review, we briefly summarize the current state of knowledge with respect to the general mechanisms involved in HIF regulation and the impact of NO on HIF activation. Secondly, we will summarize the major recent findings demonstrating a role for HIF signaling in infection, inflammation, and tissue repair and remodeling, and will address the involvement of NO. The growing interest in hypoxia-induced signaling and its relation with NO biology is expected to lead to further insights into the complex roles of NO in acute or chronic inflammatory diseases and may point to the importance of HIF signaling as key feature of NO-mediated events during these disorders.
Recent data suggest that transitions between the relaxed (R) and tense (T) state of hemoglobin control the reduction of nitrite to nitric oxide (NO) by deoxyhemoglobin. This reaction may play a role in physiologic NO homeostasis and be a novel consideration for the development of the next generation of hemoglobin-based blood oxygen carriers (HBOCs, i.e. artificial blood substitutes). Herein we tested the effects of chemical stabilization of bovine hemoglobin in either the T- (THb) or R-state (RHb) on nitrite reduction kinetics, NO-gas formation and ability to stimulate NO-dependent signaling. These studies were performed over a range of fractional saturations that is expected to mimic biological conditions. The initial rate for nitrite-reduction decreased in the following order RHb > bHb > THb, consistent with the hypothesis that the rate constant for nitrite reduction is faster with R-state Hb and slower with T-state Hb. Moreover, RHb produced more NO-gas and inhibited mitochondrial respiration more potently than both bHb and THb. Interestingly, at low oxygen fractional saturations, THb produced more NO and stimulated nitrite-dependent vasodilation more potently than bHb despite both derivatives having similar initial rates for nitrite reduction and a more negative reduction potential in THb versus bHb. These data suggest that cross-linking of bovine hemoglobin in the T-state conformation leads to a more effective coupling of nitrite reduction to NO-formation. Our results support the model of allosteric regulation of nitrite reduction by deoxyhemoglobin and show that cross-linking hemoglobins in distinct quaternary states can generate products with increased NO yields from nitrite reduction that could be harnessed to promote NO-signaling in vivo.
Hypoxia; blood flow; oxygen sensing; blood substitute; nitrite reduction
Ischemia/reperfusion (IR) injury is a central component in the pathogenesis of several diseases and is a leading cause of morbidity and mortality in the western world. Subcellularly, mitochondrial dysfunction, characterized by depletion of ATP, calcium-induced opening of the mitochondrial permeability transition pore, and exacerbated reactive oxygen species (ROS) formation, plays an integral role in the progression of IR injury. Nitric oxide (NO) and more recently nitrite (NO2-) are known to modulate mitochondrial function, mediate cytoprotection after IR and have been implicated in the signaling of the highly protective ischemic preconditioning (IPC) program. Here, we review what is known about the role of NO and nitrite in cytoprotection after IR and consider the putative role of nitrite in IPC. Focus is placed on the potential cytoprotective mechanisms involving NO and nitrite-dependent modulation of mitochondrial function.
The distinctive function of nitric oxide (NO) in biology is to transmit cellular signals through membranes and regulate cellular functions in adjacent cells. NO conveys signals as a second messenger from a cell where NO is generated to contiguous cells in two ways; one is as gaseous molecule by free diffusion resulting in an activation of soluble guanylate cyclase (NO/cGMP pathway), and another form is by binding with a molecule such as cysteine or protein thiol through S-nitrosylation (SNO pathway). Both pathways transmit much of the biological influence of NO from cell where other messenger molecules but NO are confined, through the plasma membrane to the adjacent cells. Since SNO pathway cannot utilize free-diffusion mechanism to get through the membrane as the molecular size is significantly larger than NO molecule, it utilizes amino acid transporter to convey signals as a form of S-nitrosylated cysteine (CysNO). Although S-nitrosylated glutathione (GSNO) is the molecule which act as a determinant of the total S-nitrosothiol level in cell, transnitrosylation reaction from GSNO to CysNO is an initial requirement to pass through signal through the membrane. Thus, multiplexed combination of these steps and the regulatory factors involved in this system conform and modify the outcome from stimulus-response coupling via the SNO pathway.
nitrosylation; SNO; transnitrosylation; signal transduction; transmembrane
The gaseous mediator hydrogen sulfide (H2S) is synthesized mainly by cystathionine gamma-lyase in the heart and plays a role in the regulation of cardiovascular homeostasis. Here we first overview the state of the art in the literature on the cardioprotective effects of H2S in various models of cardiac injury. Subsequently, we present original data showing the beneficial effects of parenteral administration of a donor of H2S on myocardial and endothelial function during reperfusion in a canine experimental model of cardiopulmonary bypass. Overview of the literature demonstrates that various formulations of H2S exert cardioprotective effects in cultured cells, isolated hearts and various rodent and large animal models of regional or global myocardial ischemia and heart failure. In addition, the production of H2S plays a role in myocardial pre- and post-conditioning responses. The pathways implicated in the cardioprotective action of H2S are multiple and involve KATP channels, regulation of mitochondrial respiration, and regulation of cytoprotective genes such as Nrf-2. In the experimental part of the current article, we demonstrate the cardioprotective effects of H2S in a canine model of cardiopulmonary bypass surgery. Anesthetized dogs were subjected hypothermic cardiopulmonary bypass with 60 minutes of hypothermic cardiac arrest in the presence of either saline (control, n=8), or H2S infusion (1 mg/kg/h for 2 h). Left ventricular hemodynamic variables (via combined pressure-volume-conductance catheter) as well as coronary blood flow, endothelium-dependent vasodilatation to acetylcholine and endothelium-independent vasodilatation to sodium nitroprusside were measured at baseline and after 60 minutes of reperfusion. Ex vivo vascular function and high-energy phosphate contents were also measured. H2S led to a significantly better recovery of preload recruitable stroke work (p<0.05) after 60 minutes of reperfusion. Coronary blood flow was also significantly higher in the H2S group (p<0.05). While the vasodilatory response to sodium nitroprusside was similar in both groups, acetylcholine resulted in a significantly higher increase in coronary blood flow in the H2S-treated group (p<0.05) both in vivo and ex vivo. Furthermore, high-energy phosphate contents were better preserved in the H2S group. Additionally, the cytoprotective effects of H2S were confirmed also using in vitro cell culture experiments in H9c2 cardiac myocytes exposed to hypoxia and reoxygenation or to the cytotoxic oxidant hydrogen peroxide. Thus, therapeutic administration of H2S exerts cardioprotective effects in a variety of experimental models, including a significant improvement of the recovery of myocardial and endothelial function in a canine model of cardiopulmonary bypass with hypothermic cardiac arrest.
cardiopulmonary bypass; ischemia/reperfusion; cardiac function; vascular reactivity; myocardial protection; hydrogen sulfide
Nitric oxide (NO) is a messenger molecule that is highly diffusible and short-lived. Despite these two characteristics, seemingly unsuitable for intracellular reactions, NO modulates a variety of cellular processes via the mechanism of S-nitrosylation. An important factor that determines the specificity of S-nitrosylation as a signaling mechanism is the compartmentalization of nitric oxide synthase (NOS) with its target proteins. Endothelial NOS (eNOS) is unique among the NOS family members by being localized mainly near specific intracellular membrane domains including the cytoplasmic face of the Golgi apparatus and plasma membrane caveolae. Nitric oxide produced by eNOS localized on the Golgi apparatus can react with thiol groups on nearby Golgi proteins via a redox mechanism resulting in S-nitrosylation of these proteins. This modification influences their function as regulators of cellular processes such as protein trafficking (e.g., exocytosis and endocytosis), redox state, and cell cycle. Thus, eNOS-derived NO regulates a wide range of endothelial cell functions, such as inflammation, apoptosis, permeability, migration and cell growth.
NOS; endothelium; Golgi apparatus; trafficking; vesicles; cardiovascular
The mechanism(s) regulating nitric oxide synthase-1 (NOS1) localization within the cardiac myocytes in health and disease remains unknown. Here we tested the hypothesis that the PDZ binding-domain interaction between CAPON (carboxy-terminal PDZ ligand of NOS1), a NOS1 adaptor protein, contributes to NOS1 localization in specific organelles within cardiomyocytes. Ventricular cardiomyocytes and whole heart homogenates were isolated from sham and post-myocardial infarction (MI) wild-type (C57BL/6) and NOS1-/- female mice for quantification of CAPON protein expression levels. NOS1, CAPON, xanthine oxidoreductase and Dexras1, a CAPON binding partner, were all present and enriched in isolated cardiac sarcoplasmic reticulum (SR) fractions. CAPON co-immunoprecipitated with the mu and alpha isoforms of NOS1 in whole heart lysates, and co-localization of CAPON and NOS1 was demonstrated in the SR and mitochondria with dual immuno-gold electron microscopy. Following MI, CAPON and NOS1 both redistributed to caveolae and co-localized with caveolin-3. In addition, following MI, expression level of CAPON remained unchanged and Dexras1 was reduced, CAPON binding to xanthine oxidoreductase was augmented and the plasma membrane calcium ATPase (PMCA) increased. In NOS1 deficient myocytes, CAPON abundance in the SR was reduced, and redistribution to caveolae and PMCA binding was absent. Together these findings support the hypothesis that NOS1 redistribution in injured myocardium requires the formation of a complex with the PDZ adaptor protein CAPON.
Nitric oxide synthase (NOS1); Heart failure; CAPON; PDZ- interaction; PMCA; Dexras1