David J. Singel

Adverse vascular effects of homocysteine are modulated by endothelium-derived relaxing factor and related oxides of nitrogen.

Elevated levels of homocysteine are associated with an increased risk of atherosclerosis and thrombosis. The reactivity of the sulfhydryl group of homocysteine has been implicated in molecular mechanisms underlying this increased risk. There is also increasingly compelling evidence that thiols react in the presence of nitric oxide (NO) and endothelium-derived relaxing factor (EDRF) to form S-nitrosothiols, compounds with potent vasodilatory and antiplatelet effects. We, therefore, hypothesized that S-nitrosation of homocysteine would confer these beneficial bioactivities to the thiol, and at the same time attenuate its pathogenicity. We found that prolonged (> 3 h) exposure of endothelial cells to homocysteine results in impaired EDRF responses. By contrast, brief (15 min) exposure of endothelial cells, stimulated to secrete EDRF, to homocysteine results in the formation of S-NO-homocysteine, a potent antiplatelet agent and vasodilator. In contrast to homocysteine, S-NO-homocysteine does not support H2O2 generation and does not undergo conversion to homocysteine thiolactone, reaction products believed to contribute to endothelial toxicity. These results suggest that the normal endothelium modulates the potential, adverse effects of homocysteine by releasing EDRF and forming the adduct S-NO-homocysteine. The adverse vascular properties of homocysteine may result from an inability to sustain S-NO formation owing to a progressive imbalance between the production of NO by progressively dysfunctional endothelial cells and the levels of homocysteine.

Nitric oxide in the human respiratory cycle

Interactions of nitric oxide (NO) with hemoglobin (Hb) could regulate the uptake and delivery of oxygen (O2) by subserving the classical physiological responses of hypoxic vasodilation and hyperoxic vasconstriction in the human respiratory cycle. Here we show that in in vitro and ex vivo systems as well as healthy adults alternately exposed to hypoxia or hyperoxia (to dilate or constrict pulmonary and systemic arteries in vivo), binding of NO to hemes (FeNO) and thiols (SNO) of Hb varies as a function of HbO2 saturation (FeO2). Moreover, we show that red blood cell (RBC)/SNO-mediated vasodilator activity is inversely proportional to FeO2 over a wide range, whereas RBC-induced vasoconstriction correlates directly with FeO2. Thus, native RBCs respond to changes in oxygen tension (pO2) with graded vasodilator and vasoconstrictor activity, which emulates the human physiological response subserving O2 uptake and delivery. The ability to monitor and manipulate blood levels of NO, in conjunction with O2 and carbon dioxide, may therefore prove useful in the diagnosis and treatment of many human conditions and in the development of new therapies. Our results also help elucidate the link between RBC dyscrasias and cardiovascular morbidity.

Peroxynitrite-induced rat colitis—A new model of colonic inflammation

BACKGROUND Excessive production of nitric oxide, characteristic of inflamed states, may have deleterious effects through its facile conversion (in the presence of O2) to peroxynitrite, which promotes lipid and sulfhydryl oxidation. This study assessed the effect of peroxynitrite on the rat colon. METHODS Peroxynitrite was administered intrarectally to rats. One, 3, 7, and 21 days after treatment, a distal colonic segment was isolated and tissue was obtained for histological evaluation and determination of myeloperoxidase activity and NOX, and eicosanoids generation. RESULTS Within 24 hours, the exposed segment was edematous and congested with occasional hemorrhagic mucosal ulceration. On day 7, the lumen was narrow; at day 21, there were signs of stenosis. Histological analysis showed transmucosal necrosis, acute inflammation, and exudative edema 24 hours after treatment. Surface re-epithelization and infiltration of granulation tissue were present at 1 week. Resolution of edema, mucin repletion, thickening of muscularis mucosa and propria, and fibrosis were observed at 3 weeks. Significant increase in NOX generation and myeloperoxidase and NO synthase activities were observed at 24 hours, whereas enhanced leukotriene generation was observed only at 21 days. CONCLUSIONS Peroxynitrite-induced colonic inflammation provides a novel model of NO-related tissue injury and offers the opportunity to further explore the potential role of NO in the pathogenesis of inflammatory bowel disease.

Double electron-electron resonance spin-echo modulation : spectroscopic measurement of electron spin pair separations in orientationally disordered solids

A DEER (double electron–electron resonance) spin–echo technique was applied to measure the electron–electron dipolar spectrum of a frozen toluene solution of the biradical, 2,6‐bis[(((2,2,5, 5‐tetramethyl‐1‐oxypyrrolin‐3‐yl)carbonyl)oxy)]‐anthracene. Modulation of the DEER spin–echo envelope was observed and identified as originating from the dipolar coupling between the two nitroxide spins of the biradical. Fourier transformation of the modulated components of the echo envelope yielded a dipolar spectrum from which a spin–pair separation of 19.73±0.14 A was calculated. Constraints on the relative orientation of the two nitroxide spin moieties were obtained by analysis of the effect of the microwave pulse orientational selectivity on the DEER modulation amplitudes. Molecular models of the studied compound exhibit structures that correspond well with the structural information deduced by DEER spectroscopy.

Routes to S-nitroso-hemoglobin formation with heme redox and preferential reactivity in the β subunits

Previous studies of the interactions of NO with human hemoglobin have implied the predominance of reaction channels that alternatively eliminate NO by converting it to nitrate, or tightly complex it on the α subunit ferrous hemes. Both channels could effectively quench NO bioactivity. More recent work has raised the idea that NO groups can efficiently transfer from the hemes to cysteine thiols within the β subunit (cysβ-93) to form bioactive nitrosothiols. The regulation of NO function, through its chemical position in the hemoglobin, is supported by response to oxygen and to redox agents that modulate the molecular and electronic structure of the protein. In this article, we focus on reactions in which Fe(III) hemes could provide the oxidative requirements of this NO-group transfer chemistry. We report a detailed investigation of the reductive nitrosylation of human met-Hb, in which we demonstrate the production of S-nitroso (SNO)-Hb through a heme-Fe(III)NO intermediate. The production of SNO-Hb is strongly favored (over nitrite) when NO is gradually introduced in limited total quantities; in this situation, moreover, heme nitrosylation occurs primarily within the β subunits of the hemoglobin tetramer. SNO-Hb can similarly be produced when Fe(II)NO hemes are subjected to mild oxidation. The reaction of deoxygenated hemoglobin with limited quantities of nitrite leads to the production of β subunit Fe(II)NO hemes, with SNO-Hb produced on subsequent oxygenation. The common theme of these reactions is the effective coupling of heme–iron and NO redox chemistries. Collectively, they establish a connectivity between hemes and thiols in Hb, through which NO is readily dislodged from storage on the heme to form bioactive SNO-Hb.

Analysis of 14N ESEEM patterns of randomly oriented solids

Simulations of 14N ESEEM patterns for an S=1/2, I=1 spin system with an isotropic hyperfine coupling, in a nonordered solid are reported. The dependence of the simulated patterns on the quadrupole and hyperfine coupling constants, and on the external field strength is examined and discussed. Three distinct types of powder patterns are identified, all of which exhibit discrete peaks. The dependence of the frequency, width, and amplitude of these peaks on the coupling constants and field strength is elucidated in simple terms. The results provide a basis for the interpretation of ESEEM patterns and for the development of experimental tactics: the variation of field strength (electron spin‐excitation frequency) can be used to manipulate modulation depths, select specific nuclei, and ascertain accurate quadrupole coupling constants. The anisotropy of the amplitude of modulation components, its effect on ESEEM powder patterns, and its implications as regards orientation selection experiments are also considered.

Antiplatelet properties of protein S-nitrosothiols derived from nitric oxide and endothelium-derived relaxing factor.

S-nitrosothiols may serve as carriers in the mechanism of action of endothelium-derived relaxing factor (EDRF) by stabilizing the labile nitric oxide (NO) radical from inactivation by reactive species in the physiological milieu and by delivering NO to the heme activator site of guanylyl cyclase. Low-molecular-weight thiols, such as cysteine and glutathione, form S-nitrosothiol adducts with vasodilatory and antiplatelet properties, and protein thiols can interact in the presence of NO and/or EDRF to form uniquely stable S-nitroso-proteins. We now show that the S-nitroso-proteins, S-nitroso-albumin, S-nitroso-tissue type plasminogen activator, and S-nitroso-cathepsin B, have potent antiplatelet effects with an IC50 of approximately 1.5 microM. In the dog, S-nitroso-albumin inhibits ex vivo platelet aggregation and significantly prolongs the template bleeding time from 2.15 +/- 0.13 (mean +/- SEM) to 9.70 +/- 1.24 minutes. The antiplatelet action of S-nitroso-proteins is associated with the stimulation of guanylyl cyclase and a significant decrease in fibrinogen binding to platelets. S-Nitroso-proteins undergo thiol-nitrosothiol exchange with low-molecular-weight thiols to form low-molecular-weight S-nitroso-thiols, and they also interact directly with the platelet surface, both of which processes facilitate generation of NO. These data suggest that S-nitroso-proteins are potent antiplatelet agents and may be intermediates in the antiplatelet mechanism of EDRF action.

High frequency (140 GHz) dynamic nuclear polarization: Polarization transfer to a solute in frozen aqueous solution

Dynamic nuclear polarization (DNP) transfers the large polarization of unpaired electrons to nuclei and thus significantly enhances the signal strength in nuclear magnetic resonance (NMR) spectroscopy. High frequency/field (140 GHz/5 T) DNP has been implemented in solid state NMR experiments using a nitroxide radical as the paramagnetic polarizing agent in a water:glycerol frozen solution. The 1H and 13C NMR signal strengths of both the solvent and an amino acid solute have been enhanced by a factor of 185, which represents a reduction of ≳102 in sample size requirements or ≳104 in signal acquisition time.

Nitric oxide in the central nervous system.

1. The reactions of nitric oxide with superoxide can lead to neurotoxicity through formation of peroxynitrite, and not by NO. alone, at least under our conditions. 2. Transfer of NO+ groups to thiol(s) on the NMDA receptor can lead to neuroprotection by inhibiting Ca2+ influx. These findings suggest that cell function can be controlled by, or through, protein S-nitrosylation, and raise the possibility that the NO group may initiate signal transduction in or at the plasma membrane. 3. The local redox milieu of a biological system is of critical importance in understanding NO actions as disparate chemical pathways involving distinct redox related congeners of NO may trigger neurotoxic or neuroprotective pathways. These claims are highlighted in the CNS by the recent finding that tissue concentrations of cysteine approach 700 microM in settings of cerebral ischemia (Slivka and Cohen, 1993); these levels of thiol would be expected to influence the redox state of the NO group. 4. Finally, our findings suggest novel therapeutic strategies. For example, downregulation of NMDA receptor activity via S-nitrosylation with NO+ donors could be implemented in the treatment of focal ischemia, AIDS dementia, and other neurological disorders associated, at least in part, with excessive activation of NMDA receptors.

Assessment of nitric oxide signals by triiodide chemiluminescence

Nitric oxide (NO) bioactivity is mainly conveyed through reactions with iron and thiols, furnishing iron nitrosyls and S-nitrosothiols with wide-ranging stabilities and reactivities. Triiodide chemiluminescence methodology has been popularized as uniquely capable of quantifying these species together with NO byproducts, such as nitrite and nitrosamines. Studies with triiodide, however, have challenged basic ideas of NO biochemistry. The assay, which involves addition of multiple reagents whose chemistry is not fully understood, thus requires extensive validation: Few protein standards have in fact been characterized; NO mass balance in biological mixtures has not been verified; and recovery of species that span the range of NO-group reactivities has not been assessed. Here we report on the performance of the triiodide assay vs. photolysis chemiluminescence in side-by-side assays of multiple nitrosylated standards of varied reactivities and in assays of endogenous Fe- and S-nitrosylated hemoglobin. Although the photolysis method consistently gives quantitative recoveries, the yields by triiodide are variable and generally low (approaching zero with some standards and endogenous samples). Moreover, in triiodide, added chemical reagents, changes in sample pH, and altered ionic composition result in decreased recoveries and misidentification of NO species. We further show that triiodide, rather than directly and exclusively producing NO, also produces the highly potent nitrosating agent, nitrosyliodide. Overall, we find that the triiodide assay is strongly influenced by sample composition and reactivity and does not reliably identify, quantify, or differentiate NO species in complex biological mixtures.