Renal dysfunction in adults following cardiopulmonary bypass is linked to declines in S-nitroso hemoglobin: a case series.

Background: Impaired kidney function is frequently observed in patients following cardiopulmonary bypass (CPB). Our group has previously linked blood transfusion to acute declines in S-nitroso haemoglobin (SNO-Hb; the main regulator of tissue oxygen delivery), reductions in intraoperative renal blood flow, and postoperative kidney dysfunction. While not all CPB patients receive blood, kidney injury is still common.

Protocol for preparing Thiopropyl Sepharose resin used for capturing S-nitrosylated proteins.

S-nitrosothiol (SNO)-Resin Assisted Capture (SNO-RAC) relies on a Thiopropyl Sepharose resin to identify S-nitrosylated proteins (SNO-proteins) and sites of S-nitrosylation. Here, we present a protocol for preparing Thiopropyl Sepharose resin with efficiency of SNO-protein capture comparable to the discontinued commercial version. We describe steps for amine coupling, disulfide reduction, and generation of thiol reactive resin.

Identification of a Selective SCoR2 Inhibitor That Protects Against Acute Kidney Injury.

Acute kidney injury (AKI) is associated with high morbidity and mortality, and no drugs are available clinically. Metabolic reprogramming resulting from the deletion of S-nitroso-coenzyme A reductase 2 (SCoR2; AKR1A1) protects mice against AKI, identifying SCoR2 as a potential drug target. Of the few known inhibitors of SCoR2, none are selective versus the related oxidoreductase AKR1B1, limiting therapeutic utility.

Control of tissue oxygenation by S-nitrosohemoglobin in human subjects.

S-Nitrosohemoglobin (SNO-Hb) is unique among vasodilators in coupling blood flow to tissue oxygen requirements, thus fulfilling an essential function of the microcirculation. However, this essential physiology has not been tested clinically. Reactive hyperemia following limb ischemia/occlusion is a standard clinical test of microcirculatory function, which has been ascribed to endothelial nitric oxide (NO).

S-nitrosylation is required for βAR desensitization and experimental asthma.

The β-adrenergic receptor (βAR), a prototypic G-protein-coupled receptor (GPCR), is a powerful driver of bronchorelaxation, but the effectiveness of β-agonist drugs in asthma is limited by desensitization and tachyphylaxis. We find that during activation, the βAR is modified by S-nitrosylation, which is essential for both classic desensitization by PKA as well as desensitization of NO-based signaling that mediates bronchorelaxation. Strikingly, S-nitrosylation alone can drive βAR internalization in the absence of traditional agonist.

Optimized S-nitrosohemoglobin Synthesis in Red Blood Cells to Preserve Hypoxic Vasodilation Via Cys93.

Classic physiology links tissue hypoxia to oxygen delivery through control of microvascular blood flow (autoregulation of blood flow). Hemoglobin (Hb) serves both as the source of oxygen and the mediator of microvascular blood flow through its ability to release vasodilatory S-nitrosothiol (SNO) in proportion to degree of hypoxia. -globin Cys93Ala (Cys93Ala) mutant mice deficient in S-nitrosohemoglobin (SNO-Hb) show profound deficits in microvascular blood flow and tissue oxygenation that recapitulate microcirculatory dysfunction in multiple clinical conditions.

S-Nitrosylated hemoglobin predicts organ yield in neurologically-deceased human donors.

Current human donor care protocols following death by neurologic criteria (DNC) can stabilize macro-hemodynamic parameters but have minimal ability to preserve systemic blood flow and microvascular oxygen delivery. S-nitrosylated hemoglobin (SNO-Hb) within red blood cells (RBCs) is the main regulator of tissue oxygenation (StO). Based on various pre-clinical studies, we hypothesized that brain death (BD) would decrease post-mortem SNO-Hb levels to negatively-impact StO and reduce organ yields.

Hypoxic vasodilatory defect and pulmonary hypertension in mice lacking hemoglobin β-cysteine93 S-nitrosylation.

Systemic hypoxia is characterized by peripheral vasodilation and pulmonary vasoconstriction. However, the system-wide mechanism for signaling hypoxia remains unknown. Accumulating evidence suggests that hemoglobin (Hb) in RBCs may serve as an O2 sensor and O2-responsive NO signal transducer to regulate systemic and pulmonary vascular tone, but this remains unexamined at the integrated system level.

A Novel Method to Improve Perfusion of Ex Vivo Pumped Human Kidneys.

Objective: To determine if addition of the S-nitrosylating agent ethyl nitrite (ENO) to the preservation solution can improve perfusion parameters in pumped human kidneys.

Background: A significant percentage of actively stored kidneys experience elevations in resistance and decreases in flow rate during the ex vivo storage period. Preclinical work indicates that renal status after brain death is negatively impacted by inflammation and reduced perfusion-processes regulated by protein S-nitrosylation.

Anaerobic Transcription by OxyR: A Novel Paradigm for Nitrosative Stress.

S-nitrosylation, the post-translational modification by nitric oxide (NO) to form S-nitrosothiols (SNOs), regulates diverse aspects of cellular function, and aberrant S-nitrosylation (nitrosative stress) is implicated in disease, from neurodegeneration to cancer. Essential roles for S-nitrosylation have been demonstrated in microbes, plants, and animals; notably, bacteria have often served as model systems for elucidation of general principles. Recent conceptual advances include the idea of a molecular code through which proteins sense and differentiate S-nitrosothiol (SNO) from alternative oxidative modifications, providing the basis for specificity in SNO signaling.

AKR1A1 is a novel mammalian -nitroso-glutathione reductase.

Oxidative modification of Cys residues by NO results in -nitrosylation, a ubiquitous post-translational modification and a primary mediator of redox-based cellular signaling. Steady-state levels of -nitrosylated proteins are largely determined by denitrosylase enzymes that couple NAD(P)H oxidation with reduction of -nitrosothiols, including protein and low-molecular-weight (LMW) -nitrosothiols (-nitroso-GSH (GSNO) and -nitroso-CoA (SNO-CoA)). SNO-CoA reductases require NADPH, whereas enzymatic reduction of GSNO can involve either NADH or NADPH.

A pilot study on the kinetics of metabolites and microvascular cutaneous effects of nitric oxide inhalation in healthy volunteers.

Rationale: Inhaled nitric oxide (NO) exerts a variety of effects through metabolites and these play an important role in regulation of hemodynamics in the body. A detailed investigation into the generation of these metabolites has been overlooked.

Objectives: We investigated the kinetics of nitrite and S-nitrosothiol-hemoglobin (SNO-Hb) in plasma derived from inhaled NO subjects and how this modifies the cutaneous microvascular response.

Author Correction: Metabolic reprogramming by the S-nitroso-CoA reductase system protects against kidney injury.

Change history: In Fig. 1j of this Letter, one data point was inadvertently omitted from the graph for the acute kidney injury (AKI), double knockout (-/-), S-nitrosothiol (SNO) condition at a nitrosylation level of 25.9 pmol mg and the statistical significance given of P = 0.

Metabolic reprogramming by the S-nitroso-CoA reductase system protects against kidney injury.

Endothelial nitric oxide synthase (eNOS) is protective against kidney injury, but the molecular mechanisms of this protection are poorly understood. Nitric oxide-based cellular signalling is generally mediated by protein S-nitrosylation, the oxidative modification of Cys residues to form S-nitrosothiols (SNOs). S-nitrosylation regulates proteins in all functional classes, and is controlled by enzymatic machinery that includes S-nitrosylases and denitrosylases, which add and remove SNO from proteins, respectively.

A Multiplex Enzymatic Machinery for Cellular Protein S-nitrosylation.

S-nitrosylation, the oxidative modification of Cys residues by nitric oxide (NO) to form S-nitrosothiols (SNOs), modifies all main classes of proteins and provides a fundamental redox-based cellular signaling mechanism. However, in contrast to other post-translational protein modifications, S-nitrosylation is generally considered to be non-enzymatic, involving multiple chemical routes. We report here that endogenous protein S-nitrosylation in the model organism E.

Endothelial cell-surface tissue transglutaminase inhibits neutrophil adhesion by binding and releasing nitric oxide.

Nitric oxide (NO) produced by endothelial cells in response to cytokines displays anti-inflammatory activity by preventing the adherence, migration and activation of neutrophils. The molecular mechanism by which NO operates at the blood-endothelium interface to exert anti-inflammatory properties is largely unknown. Here we show that on endothelial surfaces, NO is associated with the sulfhydryl-rich protein tissue transglutaminase (TG2), thereby endowing the membrane surfaces with anti-inflammatory properties.

-Nitrosoglutathione Reductase Deficiency Confers Improved Survival and Neurological Outcome in Experimental Cerebral Malaria.

Artesunate remains the mainstay of treatment for cerebral malaria, but it is less effective in later stages of disease when the host inflammatory response and blood-brain barrier integrity dictate clinical outcomes. Nitric oxide (NO) is an important regulator of inflammation and microvascular integrity, and impaired NO bioactivity is associated with fatal outcomes in malaria. Endogenous NO bioactivity in mammals is largely mediated by -nitrosothiols (SNOs).

Endothelial Function Is Preserved in Veins Harvested by Either Endoscopic or Surgical Techniques.

Background: Endoscopic vein harvest for lower extremity arterial bypass grafting has been questioned due to concern for endothelial damage during procurement. We sought to compare nitric oxide (NO)-mediated endothelial-dependent relaxation (EDR) in vein segments harvested using open surgical techniques (OH) versus endoscopic vein harvest (EH) techniques.

Methods: Saphenous vein segments were harvested for lower extremity bypass, and a single, minimally handled section of saphenous vein, free of branches, was taken from the end of the graft.

The LargPAD Trial: Phase IIA evaluation of l-arginine infusion in patients with peripheral arterial disease.

Objective: Endothelial function is improved by l-arginine (l-arg) supplementation in preclinical and clinical studies of mildly diseased vasculature; however, endothelial function and responsiveness to l-arg in severely diseased arteries is not known. Our objective was to evaluate the acute effects of catheter-directed l-arg delivery in patients with chronic lower extremity ischemia secondary to peripheral arterial disease.

Methods: The study enrolled 22 patients (45% male) with peripheral arterial disease (mean age, 62 years) requiring lower extremity angiography.

Hemoglobin βCys93 is essential for cardiovascular function and integrated response to hypoxia.

Oxygen delivery by Hb is essential for vertebrate life. Three amino acids in Hb are strictly conserved in all mammals and birds, but only two of those, a His and a Phe that stabilize the heme moiety, are needed to carry O2. The third conserved residue is a Cys within the β-chain (βCys93) that has been assigned a role in S-nitrosothiol (SNO)-based hypoxic vasodilation by RBCs.

Identification of S-nitroso-CoA reductases that regulate protein S-nitrosylation.

Coenzyme A (CoA) mediates thiol-based acyl-group transfer (acetylation and palmitoylation). However, a role for CoA in the thiol-based transfer of NO groups (S-nitrosylation) has not been considered. Here we describe protein S-nitrosylation in yeast (heretofore unknown) that is mediated by S-nitroso-CoA (SNO-CoA).

Endogenous protein S-Nitrosylation in E. coli: regulation by OxyR.

Endogenous S-nitrosylation of proteins, a principal mechanism of cellular signaling in eukaryotes, has not been observed in microbes. We report that protein S-nitrosylation is an obligate concomitant of anaerobic respiration on nitrate in Escherichia coli. Endogenous S-nitrosylation during anaerobic respiration is controlled by the transcription factor OxyR, previously thought to operate only under aerobic conditions.

Host S-nitrosylation inhibits clostridial small molecule-activated glucosylating toxins.

The global prevalence of severe Clostridium difficile infection highlights the profound clinical significance of clostridial glucosylating toxins. Virulence is dependent on the autoactivation of a toxin cysteine protease, which is promoted by the allosteric cofactor inositol hexakisphosphate (InsP(6)). Host mechanisms that protect against such exotoxins are poorly understood.

Thioredoxin-interacting protein (Txnip) is a feedback regulator of S-nitrosylation.

Nitric oxide exerts a plethora of biological effects via protein S-nitrosylation, a redox-based reaction that converts a protein Cys thiol to a S-nitrosothiol. However, although the regulation of protein S-nitrosylation has been the subject of extensive study, much less is known about the systems governing protein denitrosylation. Most recently, thioredoxin/thioredoxin reductases were shown to mediate both basal and stimulus-coupled protein denitrosylation.