Miriam M. Cortese-Krott

Key bioactive reaction products of the NO/H2S interaction are S/N-hybrid species, polysulfides, and nitroxyl

Significance Reactions of sulfur-centered nucleophiles with nitrogenous species have been studied independently for more than a century for synthetic/industrial purposes; to understand geochemical, atmospheric, and biological processes; and to explain the origins of life. Various products and reaction mechanisms were proposed. We here identify a singular process comprising a network of cascading chemical reactions that form three main bioactive products at physiological pH: nitrosopersulfide, polysulfides, and dinitrososulfite. These anionic products scavenge, transport, and release NO/HNO or sulfide/sulfane sulfur, each displaying distinct chemistries and bioactivities. Our observations provide a chemical foundation for the cross-talk between the NO and H2S signaling pathways in biology and suggest that the biological actions of these entities can be neither considered nor studied in isolation. Experimental evidence suggests that nitric oxide (NO) and hydrogen sulfide (H2S) signaling pathways are intimately intertwined, with mutual attenuation or potentiation of biological responses in the cardiovascular system and elsewhere. The chemical basis of this interaction is elusive. Moreover, polysulfides recently emerged as potential mediators of H2S/sulfide signaling, but their biosynthesis and relationship to NO remain enigmatic. We sought to characterize the nature, chemical biology, and bioactivity of key reaction products formed in the NO/sulfide system. At physiological pH, we find that NO and sulfide form a network of cascading chemical reactions that generate radical intermediates as well as anionic and uncharged solutes, with accumulation of three major products: nitrosopersulfide (SSNO−), polysulfides, and dinitrososulfite [N-nitrosohydroxylamine-N-sulfonate (SULFI/NO)], each with a distinct chemical biology and in vitro and in vivo bioactivity. SSNO− is resistant to thiols and cyanolysis, efficiently donates both sulfane sulfur and NO, and potently lowers blood pressure. Polysulfides are both intermediates and products of SSNO− synthesis/decomposition, and they also decrease blood pressure and enhance arterial compliance. SULFI/NO is a weak combined NO/nitroxyl donor that releases mainly N2O on decomposition; although it affects blood pressure only mildly, it markedly increases cardiac contractility, and formation of its precursor sulfite likely contributes to NO scavenging. Our results unveil an unexpectedly rich network of coupled chemical reactions between NO and H2S/sulfide, suggesting that the bioactivity of either transmitter is governed by concomitant formation of polysulfides and anionic S/N-hybrid species. This conceptual framework would seem to offer ample opportunities for the modulation of fundamental biological processes governed by redox switching and sulfur trafficking.

Nitrosopersulfide (SSNO−) accounts for sustained NO bioactivity of S-nitrosothiols following reaction with sulfide

Sulfide salts are known to promote the release of nitric oxide (NO) from S-nitrosothiols and potentiate their vasorelaxant activity, but much of the cross-talk between hydrogen sulfide and NO is believed to occur via functional interactions of cell regulatory elements such as phosphodiesterases. Using RFL-6 cells as an NO reporter system we sought to investigate whether sulfide can also modulate nitrosothiol-mediated soluble guanylyl cyclase (sGC) activation following direct chemical interaction. We find a U-shaped dose response relationship where low sulfide concentrations attenuate sGC stimulation by S-nitrosopenicillamine (SNAP) and cyclic GMP levels are restored at equimolar ratios. Similar results are observed when intracellular sulfide levels are raised by pre-incubation with the sulfide donor, GYY4137. The outcome of direct sulfide/nitrosothiol interactions also critically depends on molar reactant ratios and is accompanied by oxygen consumption. With sulfide in excess, a ‘yellow compound’ accumulates that is indistinguishable from the product of solid-phase transnitrosation of either hydrosulfide or hydrodisulfide and assigned to be nitrosopersulfide (perthionitrite, SSNO−; λmax 412 nm in aqueous buffers, pH 7.4; 448 nm in DMF). Time-resolved chemiluminescence and UV–visible spectroscopy analyses suggest that its generation is preceded by formation of the short-lived NO-donor, thionitrite (SNO−). In contrast to the latter, SSNO− is rather stable at physiological pH and generates both NO and polysulfides on decomposition, resulting in sustained potentiation of SNAP-induced sGC stimulation. Thus, sulfide reacts with nitrosothiols to form multiple bioactive products; SSNO− rather than SNO− may account for some of the longer-lived effects of nitrosothiols and contribute to sulfide and NO signaling.

Human red blood cells at work: identification and visualization of erythrocytic eNOS activity in health and disease

A nitric oxide synthase (NOS)-like activity has been demonstrated in human red blood cells (RBCs), but doubts about its functional significance, isoform identity and disease relevance remain. Using flow cytometry in combination with the nitric oxide (NO)-imaging probe DAF-FM we find that all blood cells form NO intracellularly, with a rank order of monocytes > neutrophils > lymphocytes > RBCs > platelets. The observation of a NO-related fluorescence within RBCs was unexpected given the abundance of the NO-scavenger oxyhemoglobin. Constitutive normoxic NO formation was abolished by NOS inhibition and intracellular NO scavenging, confirmed by laser-scanning microscopy and unequivocally validated by detection of the DAF-FM reaction product with NO using HPLC and LC-MS/MS. Using immunoprecipitation, ESI-MS/MS-based peptide sequencing and enzymatic assay we further demonstrate that human RBCs contain an endothelial NOS (eNOS) that converts L-(3)H-arginine to L-(3)H-citrulline in a Ca(2+)/calmodulin-dependent fashion. Moreover, in patients with coronary artery disease, red cell eNOS expression and activity are both lower than in age-matched healthy individuals and correlate with the degree of endothelial dysfunction. Thus, human RBCs constitutively produce NO under normoxic conditions via an active eNOS isoform, the activity of which is compromised in patients with coronary artery disease.

Silver ions induce oxidative stress and intracellular zinc release in human skin fibroblasts

Silver compounds used as topical antimicrobial agents are known to exert toxic effects on skin cells. The aim of this study was to investigate whether the toxicity of silver ions, in analogy to other transition metal ions, depends on pro-oxidant effects. We treated human skin fibroblasts with concentrations of AgNO(3) not affecting cell proliferation, mitochondrial activity, or cell viability and found that Ag(+) strongly increases the production of reactive oxygen species, including superoxide anion radicals. These effects correspond to a strong decrease in intracellular reduced glutathione and to an increased susceptibility to H(2)O(2)-induced cell death. In addition, AgNO(3) down-regulates the expression of antioxidant genes such as the transcription factor Nrf2 and its target gene glutamate-cysteine ligase catalytic subunit. Furthermore Ag(+) induces a transient intracellular zinc release and increases the mRNA and protein expression of the zinc-binding protein metallothionein by activating the metal-responsive transcription factor 1, as verified by RNA interference. In conclusion, we show for the first time that Ag(+) induces oxidative stress and affects intracellular zinc homeostasis in human skin fibroblasts. The understanding of the mechanism involved in silver toxicity might contribute to new strategies for managing the therapy of skin infections.

Circulating Blood Endothelial Nitric Oxide Synthase Contributes to the Regulation of Systemic Blood Pressure and Nitrite Homeostasis

Objective—Mice genetically deficient in endothelial nitric oxide synthase (eNOS−/−) are hypertensive with lower circulating nitrite levels, indicating the importance of constitutively produced nitric oxide (NO•) to blood pressure regulation and vascular homeostasis. Although the current paradigm holds that this bioactivity derives specifically from the expression of eNOS in endothelium, circulating blood cells also express eNOS protein. A functional red cell eNOS that modulates vascular NO• signaling has been proposed. Approach and Results—To test the hypothesis that blood cells contribute to mammalian blood pressure regulation via eNOS-dependent NO• generation, we cross-transplanted wild-type and eNOS−/− mice, producing chimeras competent or deficient for eNOS expression in circulating blood cells. Surprisingly, we observed a significant contribution of both endothelial and circulating blood cell eNOS to blood pressure and systemic nitrite levels, the latter being a major component of the circulating NO• reservoir. These effects were abolished by the NOS inhibitor L-NG-nitroarginine methyl ester and repristinated by the NOS substrate L-arginine and were independent of platelet or leukocyte depletion. Mouse erythrocytes were also found to carry an eNOS protein and convert 14C-arginine into 14C-citrulline in NOS-dependent fashion. Conclusions—These are the first studies to definitively establish a role for a blood-borne eNOS, using cross-transplant chimera models, that contributes to the regulation of blood pressure and nitrite homeostasis. This work provides evidence suggesting that erythrocyte eNOS may mediate this effect.

Endothelial nitric oxide synthase in red blood cells: key to a new erythrocrine function?

Red blood cells (RBC) have been considered almost exclusively as a transporter of metabolic gases and nutrients for the tissues. It is an accepted dogma that RBCs take up and inactivate endothelium-derived NO via rapid reaction with oxyhemoglobin to form methemoglobin and nitrate, thereby limiting NO available for vasodilatation. Yet it has also been shown that RBCs not only act as “NO sinks”, but exert an erythrocrine function – i.e an endocrine function of RBC – by synthesizing, transporting and releasing NO metabolic products and ATP, thereby potentially controlling systemic NO bioavailability and vascular tone. Recent work from our and others laboratory demonstrated that human RBCs carry an active type 3, endothelial NO synthase (eNOS), constitutively producing NO under normoxic conditions, the activity of which is compromised in patients with coronary artery disease. In this review we aim to discuss the potential role of red cell eNOS in RBC signaling and function, and to critically revise evidence to this date showing a role of non-endothelial circulating eNOS in cardiovascular pathophysiology.

On the chemical biology of the nitrite/sulfide interaction

Sulfide (H2S/HS(-)) has been demonstrated to exert an astounding breadth of biological effects, some of which resemble those of nitric oxide (NO). While the chemistry, biochemistry and potential pathophysiology of the cross-talk between sulfide and NO have received considerable attention lately, a comparable assessment of the potential biological implications of an interaction between nitrite and sulfide is lacking. This is surprising inasmuch as nitrite is not only a known bioactive oxidation product of NO, but also efficiently converted to S-nitrosothiols in vivo; the latter have been shown to rapidly react with sulfide in vitro, leading to formation of S/N-hybrid species including thionitrite (SNO(-)) and nitrosopersulfide (SSNO(-)). Moreover, nitrite is used as a potent remedy against sulfide poisoning in the clinic. The chemistry of interaction between nitrite and sulfide or related bioactive metabolites including polysulfides and elemental sulfur has been extensively studied in the past, yet much of this information appears to have been forgotten. In this review, we focus on the potential chemical biology of the interaction between nitrite and sulfide or sulfane sulfur molecules, calling attention to the fundamental chemical properties and reactivities of either species and discuss their possible contribution to the biology, pharmacology and toxicology of both nitrite and sulfide.

Nitric Oxide Synthase Expression and Functional Response to Nitric Oxide Are Both Important Modulators of Circulating Angiogenic Cell Response to Angiogenic Stimuli

Objective—Circulating angiogenic cells (CACs), also termed endothelial progenitor cells, play an integral role in vascular repair and are functionally impaired in coronary artery disease (CAD). The role of nitric oxide (NO) in CAC function is poorly understood. We hypothesized that CAC migration toward angiogenic signals is modulated by both NO synthase (NOS) expression and functional response to NO. Methods and Results—Similar to endothelial cells, CAC chemotaxis to vascular endothelial growth factor (VEGF) was blocked by inhibition of NOS, phosphatidylinositol 3-kinase, or guanylyl cyclase or by treatment with an NO scavenger. Addition of an NO donor (S-nitroso-N-acetylpenicillamine) and the NOS substrate l-arginine increased random cell migration (chemokinesis) and enhanced VEGF-dependent chemotaxis. Healthy CACs expressed endothelial NOS, but endothelial NOS was not detected in CAD patient CACs. Both chemokinesis and chemotaxis to VEGF of patient CACs were decreased compared with healthy CACs but were restored to healthy values by S-nitroso-N-acetylpenicillamine. In parallel, CAD patients exhibited lower flow-mediated vasodilation and plasma NO source nitrite than young, healthy subjects, indicating endothelial dysfunction with reduced NO bioavailability. Conclusion—NOS activity is required for CAC chemotaxis. In CAD patients, impairment of NOS expression and NO bioavailability, rather than response to NO, may contribute to dysfunction of CACs and limit their regenerative capacity.

Circulating Microparticles Carry a Functional Endothelial Nitric Oxide Synthase That Is Decreased in Patients With Endothelial Dysfunction

Background Microparticles (MPs) are circulating membrane particles of less than a micrometer in diameter shed from endothelial and blood cells. Recent literature suggests that MPs are not just functionally inert cell debris but may possess biological functions and mediate the communication between vascular cells. As a significant proportion of MPs originate from platelets and endothelial cells, we hypothesized that MPs may harbor functional enzymes including an endothelial NO synthase (eNOS). Methods and Results Using immunoprecipitation and Western blot analysis, we found that human circulating MPs carry an eNOS. Ca2+ and l‐arginine‐dependent NOS activity of crude enzyme extract from MPs was determined by measuring the conversion of [3H]‐L‐arginine to [3H]‐citrulline and NOS‐dependent nitrite production. NOS‐dependent NO production in intact MPs was assessed by the NO‐specific fluorescent probe MNIP‐Cu. In patients with cardiovascular disease, endothelial dysfunction was associated with an increase in the total number of circulating MPs as well as a significant decrease in the expression and activity of eNOS in MPs. No difference in reactive oxygen species was noted in MPs isolated from either group. Conclusions Our data further support the concept that circulating MPs may not only retain phenotypic markers but also preserve the functionality of enzymes of the cells they originate from, including eNOS.

The Reactive Species Interactome: Evolutionary Emergence, Biological Significance, and Opportunities for Redox Metabolomics and Personalized Medicine

Abstract Significance: Oxidative stress is thought to account for aberrant redox homeostasis and contribute to aging and disease. However, more often than not, administration of antioxidants is ineffective, suggesting that our current understanding of the underlying regulatory processes is incomplete. Recent Advances: Similar to reactive oxygen species and reactive nitrogen species, reactive sulfur species are now emerging as important signaling molecules, targeting regulatory cysteine redox switches in proteins, affecting gene regulation, ion transport, intermediary metabolism, and mitochondrial function. To rationalize the complexity of chemical interactions of reactive species with themselves and their targets and help define their role in systemic metabolic control, we here introduce a novel integrative concept defined as the reactive species interactome (RSI). The RSI is a primeval multilevel redox regulatory system whose architecture, together with the physicochemical characteristics of its constituents, allows efficient sensing and rapid adaptation to environmental changes and various other stressors to enhance fitness and resilience at the local and whole-organism level. Critical Issues: To better characterize the RSI-related processes that determine fluxes through specific pathways and enable integration, it is necessary to disentangle the chemical biology and activity of reactive species (including precursors and reaction products), their targets, communication systems, and effects on cellular, organ, and whole-organism bioenergetics using system-level/network analyses. Future Directions: Understanding the mechanisms through which the RSI operates will enable a better appreciation of the possibilities to modulate the entire biological system; moreover, unveiling molecular signatures that characterize specific environmental challenges or other forms of stress will provide new prevention/intervention opportunities for personalized medicine. Antioxid. Redox Signal. 27, 684–712.