How do red blood cells dilate blood vessels?--Reply.
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Animals↗
Biomedical subjects
Neil Hogg
Publications and source records attributed to Neil Hogg.
Biological activity of nitric oxide in the plasmatic compartment.
There exist reaction products of nitric oxide (NO) with blood that conserve its bioactivity and transduce an endocrine vasomotor function under certain conditions. Although S-nitrosated albumin has been considered the major species subserving this activity, recent data suggest that additional NO species, such as nitrite, nitrated lipids, N-nitrosamine, and iron-nitrosyl complexes, may contribute. We therefore examined the end products of NO reactions in plasma and blood in vitro and in vivo by using reductive chemiluminescent assays and electron paramagnetic resonance spectroscopy. We found that NO complexes in plasma previously considered to be S-nitrosated albumin were <10 nM after elimination of nitrite and were mercury-stable, consistent with iron-nitrosyl or N-nitrosamine complex. During clinical NO gas inhalation protocols or in vitro NO donor treatment of human plasma, S-nitroso-albumin did not form with NO exposure <2 microM, but plasma methemoglobin was detectable by paramagnetic resonance spectroscopy. Consistent with this formation of methemoglobin, human plasma was found to consume approximately 2 microM NO at a rate equivalent to that of hemoglobin. This NO consumption was mediated by the reaction of NO with plasma haptoglobin-hemoglobin complexes and limited slower reaction pathways required for S-nitrosation. These data suggest that high-affinity, metal-based reactions in plasma with the haptoglobin-hemoglobin complex modulate plasmatic NO reaction products and limit S-nitrosation at low NO flux. The studies further suggest that alternative NO reaction end products in plasma, such as nitrite, N-nitrosamines, iron-nitrosyls, and nitrated lipids, should be evaluated in blood NO transport along the vasculature.
Administration, Inhalation↗
Impact of pulmonary arterial endothelial cells on duroquinone redox status.
The study objective was to use pulmonary arterial endothelial cells to examine kinetics and mechanisms contributing to the disposition of the quinone 2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone, DQ) observed during passage through the pulmonary circulation. The approach was to add DQ, durohydroquinone (DQH2), or DQ with the cell membrane-impermeant oxidizing agent, ferricyanide (Fe(CN)6(3)-), to the cell medium, and to measure the medium concentrations of substrates and products over time. Studies were carried out under control conditions and with dicumarol, to inhibit NAD(P)H:quinone oxidoreductase 1 (NQO1), or cyanide, to inhibit mitochondrial electron transport. In control cells, DQH2 appears in the extracellular medium of cells incubated with DQ, and DQ appears when the cells are incubated with DQH2. Dicumarol blocked the appearance of DQH2 when DQ was added to the cell medium, and cyanide blocked the appearance of DQ when DQH2 was added to the cell medium, suggesting that the two electron reductase NQO1 dominates DQ reduction and mitochondrial electron transport complex III is the predominant route of DQH2 oxidation. In the presence of cyanide, the addition of DQ also resulted in an increased rate of appearance of DQH2 and stimulation of cyanide-insensitive oxygen consumption. As DQH2 does not autoxidize-comproportionate over the study time course, these observations suggest a cyanide-stimulated one-electron DQ reduction and durosemiquinone (DQ*-) autoxidation. The latter processes are apparently confined to the cell interior, as the cell membrane impermeant oxidant, ferricyanide, did not inhibit the DQ-stimulated cyanide-insensitive oxygen consumption. Thus, regardless of whether DQ is reduced via a one- or two-electron reduction pathway, the net effect in the extracellular medium is the appearance of DQH2. These endothelial redox functions and their apposition to the vessel lumen are consistent with the pulmonary endothelium being an important site of DQ reduction to DQH2 observed in the lungs.
Animals↗
Transduction of NO-bioactivity by the red blood cell in sepsis: novel mechanisms of vasodilation during acute inflammatory disease.
Sepsis is an acute inflammatory disease characterized by dysfunctional blood flow and hypotension. Nitric oxide (NO) is elevated during sepsis and plays an integral role in the associated vascular pathology. However, precise mechanisms and functions of NO in sepsis remain unclear. In this study, we show that red blood cells (RBCs) are foci for nitrosative reactions during acute inflammation, resulting in the formation of cells that can promote systemic vascular relaxation in an uncontrolled manner. Specifically, using experimental models of endotoxemia and surgical sepsis, NO adducts were found in the RBCs, including S-nitrosohemoglobin (SNOHb). These RBCs, referred to as septic RBCs, spontaneously stimulated vasodilation in a manner consistent with elevated SNOHb concentrations. Moreover, relaxation was cyclic guanosine monophosphate (cGMP) dependent and was inhibited by RBC lysis and glutathione but not by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5 tetramethylimidazoline 1-oxyl 3-oxide (C-PTIO). The potential mechanism of septic RBC-mediated vasorelaxation is discussed and may involve the intermediate, nitroxyl (HNO). Coupled with data showing that NO adducts in septic RBCs were dependent on the inducible nitric oxide synthase and correlated with plasma nitrite, these findings provide a novel framework to understand mechanisms underlying dysfunctional blood flow responses during sepsis. Specifically, the concept that RBCs directly mediate systemic hypotension through NO-dependent mechanisms is discussed.
Acute Disease↗
The mechanism of transmembrane S-nitrosothiol transport.
S-nitrosothiols have been suggested to play an important role in nitric oxide (NO)-mediated biological events. However, the mechanisms by which an S-nitrosothiol (or the S-nitroso functional group) is transferred across cell membrane are still poorly understood. We have demonstrated previously that the degradation of S-nitrosoglutathione (GSNO) by cells absolutely required the presence of cystine in the extracellular medium and proposed a mechanism that involved the reduction of cystine to cysteine, followed by the reaction of cysteine with GSNO to form S-nitrosocysteine (CysNO), mixed disulfides, and nitrosyl anion. In the present study we have assessed the effect of cystine on the transfer of the S-nitroso functional group from the extracellular to the intracellular space. Using RAW 264.7 cells, we found that the presence of L-cystine enhanced GSNO-dependent S-nitrosothiol uptake, increasing the intracellular S-nitrosothiol level from approximately 60 pmol/mg of protein to approximately 3 nmol/mg of protein. The uptake seems to depend on the reduction of L-cystine to L-cysteine, which involves the xc- amino acid transport system, the transnitrosation between GSNO and L-cysteine to form L-CysNO, and uptake of L-CysNO via amino acid transport system L. Compared with GSNO, (Z)-1-[N-(3-ammoniopropyl)-N-[4-(3-aminopropylammonio)butyl]-amino]diazen-1-ium-1,2-diolate, an NO donor, is much less effective at intracellular S-nitrosothiol formation in the presence of L-cystine or L-cysteine, suggesting that the biochemical changes that occur after exposure of cells to S-nitrosothiol, with respect to thiol chemistry, are distinctly different from those observed with NO.
Biological Transport↗
S-nitrosohemoglobin: a biochemical perspective.
It has been suggested that S-nitrosohemoglobin (HbSNO) is an oxygen-dependent mediator of nitric oxide delivery to vascular smooth muscle cells, thus regulating vascular tone and blood flow. Central to this much-debated hypothesis is the concept that our previous understanding of the interaction between nitric oxide and ferrous hemoglobin was deficient. In this review we will examine the chemical and biochemical mechanisms for the formation of HbSNO, the properties of HbSNO, and the release of nitric oxide from HbSNO. This review concludes that although novel reactions of nitric oxide, nitrite, and S-nitrosothiols with hemoglobin have been uncovered, there is little evidence to support the notion that the interaction of nitric oxide with ferrous hemoglobin is more complex than had been previously established.
Animals↗
Formation and stability of S-nitrosothiols in RAW 264.7 cells.
S-Nitrosothiols have been suggested to be mediators of many nitric oxide-dependent processes, including apoptosis and vascular relaxation. Thiol nitrosation is a poorly understood process in vivo, and the mechanisms by which nitric oxide can be converted into a nitrosating agent have not been established. There is a discrepancy between the suggested biological roles of nitric oxide and its known chemical and physical properties. In this study, we have examined the formation of S-nitrosothiols in lipopolysaccharide-treated RAW 264.7 cells. This treatment generated 17.4 +/- 1.0 pmol/mg of protein (means +/- SE, n =27) of intracellular S-nitrosothiol that slowly decayed over several hours. S-Nitrosothiol formation depended on the formation of nitric oxide and not on the presence of nitrite. Extracellular thiols were nitrosated by cell-generated nitric oxide. Oxygenated ferrous hemoglobin inhibited the formation of S-nitrosothiol, indicating the nitrosation occurred more slowly than diffusion. We discuss several mechanisms for S-nitrosothiol formation and conclude that the nitrosation propensity of nitric oxide is a freely diffusible element that is not constrained within an individual cell and that both nitric oxide per se and nitric oxide-derived nitrosating agents are able to diffuse across cell membranes. To achieve intracellular localization of the nitrosation reaction, mechanisms must be invoked that do not involve the formation of nitric oxide as an intermediate.
Animals↗
Oxidation and nitrosylation of oxyhemoglobin by S-nitrosoglutathione via nitroxyl anion.
The reaction between low molecular weight S-nitrosothiols and hemoglobin is often used to synthesize S-nitrosohemoglobin, a form of hemoglobin suggested to be involved in the regulation of vascular oxygen delivery. However, this reaction has not been studied in detail, and several groups have reported a variable co-formation of oxidized methemoglobin (metHb) during synthesis. This study examines the mechanism of metHb formation and shows that nitrosylhemoglobin (HbNO) can also be formed. Generation of metHb and HbNO is largely dependent on the presence of protein thiol groups. We present evidence for a mechanism for the formation of metHb and HbNO involving the intermediacy of nitroxyl anion. Specifically, the reaction of nitroxyl with S-nitrosothiols to liberate nitric oxide and reduced thiol is proposed to be central to the reaction mechanism.
Anions↗
Comparative investigation of superoxide trapping by cyclic nitrone spin traps: the use of singular value decomposition and multiple linear regression analysis.
The kinetics of the reaction between superoxide and the spin trapping agents 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO), and 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO) were re-examined in the superoxide-generating xanthine/xanthine oxidase system, by competition with spontaneous dismutation. The approach used singular value decomposition (SVD), multiple linear regression, and spectral simulation. The experiments were carried out using a two-syringe mixing arrangement with fast scan acquisition of 100 consecutive EPR spectra. Using SVD analysis, the extraction of both temporal and spectral information could be obtained from in a single run. The superoxide spin adduct was the exclusive EPR active species in the case of DEPMPO and BMPO, and the major component when DMPO was used. In the latter case a very low concentration of hydroxyl adduct was also observed, which did not change during the decay of the DMPO-superoxide adduct. This indicates that the hydroxyl radical adduct is not formed from the spontaneous decay of the superoxide radical adduct, as has been previously suggested [correction]. It was established that in short-term studies (up to 100 s) DMPO was the superior spin trapping agent, but for reaction times longer than 100 s the other two spin traps were more advantageous. The second order rate constants for the spin trapping reaction were found to be DMPO (2.4 M(-1)s(-1)), DEPMPO (0.53 M(-1)s(-1)), and BMPO (0.24 M(-1)s(-1)) determined through competition with spontaneous dismutation of superoxide, at pH 7.4 and 20 degrees C.
Cyclic N-Oxides↗
Measurements of nitric oxide on the heme iron and beta-93 thiol of human hemoglobin during cycles of oxygenation and deoxygenation.
Nitric oxide has been proposed to be transported by hemoglobin as a third respiratory gas and to elicit vasodilation by an oxygen-linked (allosteric) mechanism. For hemoglobin to transport nitric oxide bioactivity it must capture nitric oxide as iron nitrosyl hemoglobin rather than destroy it by dioxygenation. Once bound to the heme iron, nitric oxide has been reported to migrate reversibly from the heme group of hemoglobin to the beta-93 cysteinyl residue, in response to an oxygen saturation-dependent conformational change, to form an S-nitrosothiol. However, such a transfer requires redox chemistry with oxidation of the nitric oxide or beta-93 cysteinyl residue. In this article, we examine the ability of nitric oxide to undergo this intramolecular transfer by cycling human hemoglobin between oxygenated and deoxygenated states. Under various conditions, we found no evidence for intramolecular transfer of nitric oxide from either cysteine to heme or heme to cysteine. In addition, we observed that contaminating nitrite can lead to formation of iron nitrosyl hemoglobin in deoxygenated hemoglobin preparations and a radical in oxygenated hemoglobin preparations. Using 15N-labeled nitrite, we clearly demonstrate that nitrite chemistry could explain previously reported results that suggested apparent nitric oxide cycling from heme to thiol. Consistent with our results from these experiments conducted in vitro, we found no arterial/venous gradient of iron nitrosyl hemoglobin detectable by electron paramagnetic resonance spectroscopy. Our results do not support a role for allosterically controlled intramolecular transfer of nitric oxide in hemoglobin as a function of oxygen saturation.
Heme↗
Pulmonary arterial endothelial cells affect the redox status of coenzyme Q0.
The pulmonary endothelium is capable of reducing certain redox-active compounds as they pass from the systemic venous to the arterial circulation. This may have important consequences with regard to the pulmonary and systemic disposition and biochemistry of these compounds. Because quinones comprise an important class of redox-active compounds with a range of physiological, toxicological, and pharmacological activities, the objective of the present study was to determine the fate of a model quinone, coenzyme Q0 (Q), added to the extracellular medium surrounding pulmonary arterial endothelial cells in culture, with particular attention to the effect of the cells on the redox status of Q in the medium. Spectrophotometry, electron paramagnetic resonance (EPR), and high-performance liquid chromatography (HPLC) demonstrated that, when the oxidized form Q is added to the medium surrounding the cells, it is rapidly converted to its quinol form (QH2) with a small concentration of semiquinone (Q*-) also detectable. The isolation of cell plasma membrane proteins revealed an NADH-Q oxidoreductase located on the outer plasma membrane surface, which apparently participates in the reduction process. In addition, once formed the QH2 undergoes a cyanide-sensitive oxidation by the cells. Thus, the actual rate of Q reduction by the cells is greater than the net QH2 output from the cells.
Animals↗
Transmembrane nitration of hydrophobic tyrosyl peptides. Localization, characterization, mechanism of nitration, and biological implications.
We have shown previously that peroxynitrite-induced nitration of a hydrophobic tyrosyl probe is greater than that of tyrosine in the aqueous phase (Zhang, H., Joseph, J., Feix, J., Hogg, N., and Kalyanaraman, B. (2001) Biochemistry 40, 7675-7686). In this study, we have tested the hypothesis that the extent of tyrosine nitration depends on the intramembrane location of tyrosyl probes and on the nitrating species. To this end, we have synthesized membrane spanning 23-mer containing a single tyrosyl residue at positions 4, 8, and 12. The location of the tyrosine residues in the phospholipid membrane was determined by fluorescence and electron spin resonance techniques. Nitration was initiated by slow infusion of peroxynitrite, co-generated superoxide and nitric oxide ((.)NO), or a myeloperoxidase/hydrogen peroxide/nitrite anion (MPO/H(2)O(2)/NO(2)(-)) system. Results indicate that with slow infusion of peroxynitrite, nitration of transmembrane tyrosyl peptides was much higher (10-fold or more) than tyrosine nitration in aqueous phase. Peroxynitrite-dependent nitration of tyrosyl-containing peptides increased with increasing depth of the tyrosyl residue in the bilayer. In contrast, MPO/H(2)O(2)/ NO(2)(-)-induced tyrosyl nitration decreased with increasing depth of tyrosyl residues in the membrane. Transmembrane nitrations of tyrosyl-containing peptides induced by both peroxynitrite and MPO/H(2)O(2)/NO(2)(-) were totally inhibited by (.)NO that was slowly released from spermine NONOate. Nitration of peptides in both systems was concentration-dependently inhibited by unsaturated fatty acid. Concomitantly, an increase in lipid oxidation was detected. A mechanism involving (.)NO(2) radical is proposed for peroxynitrite and MPO/H(2)O(2)/NO(2)(-)-dependent transmembrane nitration reactions.
Chromatography, High Pressure Liquid↗
Ischemic preconditioning: triggering role of nitric oxide-derived oxidants in isolated hearts.
There is evidence that oxidants generated during ischemic preconditioning (IPC) trigger or mediate cardioprotection. We examined whether a causal relationship exists between oxidant formation during ischemic preconditioning and cardioprotection. We monitored formation of dityrosine in crystalloid-perfused guinea pig isolated hearts after a preconditioning protocol and after prolonged ischemia. Superoxide dismutase, catalase, and glutathione (SCG), or the L-arginine analogue NGnitro L-arginine methyl ester (L-NAME) were given during preconditioning. Dityrosine was observed in the coronary effluent immediately after both stimuli, but not after bracketing with SCG or L-NAME. After prolonged ischemia, dityrosine was significantly lower in the IPC group than in other groups. IPC was evidenced by improved mechanical and metabolic function on reperfusion, and by reduced infarction. These effects were abrogated by either SCG or L-NAME. These data support the hypothesis that the formation of nitric oxide-derived oxidants during ischemic preconditioning is causally related to myocardial adaptation to reperfusion injury.
Animals↗
Cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease.
Although the deleterious vasoconstrictive effects of cell-free, hemoglobin-based blood substitutes have been appreciated, the systemic effects of chronic hemolysis on nitric oxide bioavailability have not been considered or quantified. Central to this investigation is the understanding that nitric oxide reacts at least 1,000 times more rapidly with free hemoglobin solutions than with erythrocytes. We hypothesized that decompartmentalization of hemoglobin into plasma would divert nitric oxide from homeostatic vascular function. We demonstrate here that plasma from patients with sickle-cell disease contains cell-free ferrous hemoglobin, which stoichiometrically consumes micromolar quantities of nitric oxide and abrogates forearm blood flow responses to nitric oxide donor infusions. Therapies that inactivate plasma hemoglobin by oxidation or nitric oxide ligation restore nitric oxide bioavailability. Decompartmentalization of hemoglobin and subsequent dioxygenation of nitric oxide may explain the vascular complications shared by acute and chronic hemolytic disorders.
Adult↗
Mixing artifacts from the bolus addition of nitric oxide to oxymyoglobin: implications for S-nitrosothiol formation.
The addition of nitric oxide (NO) solution to oxygenated heme proteins has been used to measure NO concentration and as an experimental model to investigate the biochemical mechanism of NO metabolism. In this paper we demonstrate that bolus addition of NO to oxymyoglobin (oxyMb) results in the artifactual formation of nitrosating intermediates. When NO is added as a bolus, using fully aerated oxyMb solutions, the measured NO concentration is half as much as that when the oxyMb solution is partially degassed (0.86 +/- 0.01 mM vs. 1.61 +/- 0.02 mM, mean +/- SD). Similar results are found when calibrating NO concentration using a nitronyl nitroxide-type NO scavenger. The apparent stoichiometry of NO to oxyMb increases when the solution oxygen concentration increases. A fraction of the added NO generates nitrite or, in the presence of glutathione (GSH), S-nitrosoglutathione (GSNO). When using an NO donor, which slowly releases NO, oxyMb oxidation shows no dependence on the presence of oxygen in solution, and no nitrosating intermediate is formed. Bolus NO addition causes a local high concentration of NO. Kinetic calculations under this condition using known rate constants indicate that both the NO/oxyMb reaction and the NO/O(2) reaction can occur before it is possible fully to mix the solution. Our results suggest that the presence of the NO/O(2) reaction is an artifact of bolus NO addition, and leads to the formation of nitrite, or GSNO in the presence of GSH.
Animals↗
The biochemistry and physiology of S-nitrosothiols.
S-nitrosothiols are biological metabolites of nitric oxide. It has often been suggested that they represent a more stable metabolite of nitric oxide that can either be stored, or transported, although the evidence for this is sparse. There are many unanswered questions concerning how S-nitrosothiols are formed, how they are metabolized and how they elicit biological responses. These questions are highlighted by the fact that the known chemistry of nitric oxide, thiols, and S-nitrosothiols cannot serve to explain their proposed biological activities. This review attempts to highlight the gulf between our chemical understanding of S-nitrosothiols and the proposed biological activities of these compounds with respect to guanylyl cyclase-independent nitric oxide bioactivity and also the control of vascular tone.
Animals↗
Anesthetic preconditioning: triggering role of reactive oxygen and nitrogen species in isolated hearts.
We postulated that anesthetic preconditioning (APC) is triggered by reactive oxygen/nitrogen species (ROS/RNS). We used the isolated guinea pig heart perfused with L-tyrosine, which reacts with ROS and RNS to form strong oxidants, principally peroxynitrite (ONOO(-)), and then forms fluorescent dityrosine. ROS scavengers superoxide dismutase, catalase, and glutathione (SCG) and NO. synthesis inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) were given 5 min before and after sevoflurane preconditioning stimuli. Drugs were washed out before 30 min of ischemia and 120 min of reperfusion. Groups were control (nontreated ischemia control), APC (two, 2-min periods of perfusion with 0.32 +/- 0.02 mM of sevoflurane; separated by a 6-min period of perfusion without sevoflurane), SCG, APC + SCG, L-NAME, and APC + L-NAME. Effluent dityrosine at 1 min reperfusion was 56 +/- 6 (SE), 15 +/- 5, 40 +/- 5(++), 39 +/- 4(++), 35 +/- 4(++) , and 33 +/- 5(++) units ((++)P< 0.05 vs. APC), respectively; left ventricular pressure (%baseline) at 60 min of reperfusion was 30 +/- 5(++), 60 +/- 4, 35 +/- 5(++), 37 +/- 5(++), 44 +/- 4, and 47 +/- 4; and infarct size (%total heart weight) was 50 +/- 5(++), 19 +/- 2, 48 +/- 3(++), 46 +/- 4(++), 42 +/- 4(++), and 45 +/- 2(++). Thus APC is initiated by ROS as shown by improved function, reduced infarct size, and reduced dityrosine on reperfusion; protective and ROS/RNS-reducing effect of APC were attenuated when bracketed by ROS scavengers or NO* inhibition.
Anesthetics↗