Erythrocytes are the major intravascular storage sites of nitrite in human blood

Dejam, André; Hunter, Christian J.; Pelletier, Mildred M.; Hsu, Lewis L.; Machado, Roberto F.; Shiva, Shruti; Power, Gordon G.; Kelm, Malte; Gladwin, Mark T.; Schechter, Alan N.

doi:10.1182/blood-2005-02-0567

Cited by 303 publications

(271 citation statements)

References 47 publications

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“…The reaction of nitrite with RBC Hb is not significantly impeded by RBC uptake [103]. When we reacted 50 μM nitrite (which is much more than the basal amount found in RBCs (< 1 μM) [103,104]) with RBCs for 30 minutes, 16 μM metHb formed (data not shown). Note that this is significantly more than would form from nitrite derived from 50 μM AS after 30 minutes since the AS does not decompose immediately.…”

Section: Discussionmentioning

confidence: 82%

The potential of Angeli's salt to decrease nitric oxide scavenging by plasma hemoglobin

Azarov

Jeffers

et al. 2008

Free Radical Biology and Medicine

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Release of hemoglobin from the erythrocyte during intravascular hemolysis contributes to the pathology of a variety of diseased states. This effect is partially due to the enhanced ability of cellfree plasma hemoglobin, which is primarily found in the ferrous, oxygenated state, to scavenge nitric oxide. Oxidation of the cell-free hemoglobin to methemoglobin, which does not effectively scavenge nitric oxide, using inhaled nitric oxide has been shown to be effective in limiting pulmonary and systemic vasoconstriction. However, the ferric heme species may be reduced back to ferrous hemoglobin in plasma and has the potential to drive injurious redox chemistry. We propose that compounds that selectively convert cell-free hemoglobin to ferric, and ideally iron-nitrosylated heme species that do not actively scavenge nitric oxide would effectively treat intravascular hemolysis. We show here that nitroxyl, generated by Angeli's salt (Sodium α-oxyhyponitrite, Na 2 N 2 O 3 ), preferentially reacts with cell-free hemoglobin compared to that encapsulated in the red blood cell under physiologically relevant conditions. Nitroxyl oxidizes oxygenated ferrous hemoglobin to methemoglobin and can convert the methemoglobin to a more stable, less toxic species, iron-nitrosyl hemoglobin. These results support the notion that Angeli's salt or a similar compound could be used to effectively treat conditions associated with intravascular hemolysis.

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Section: Discussionmentioning

confidence: 82%

The potential of Angeli's salt to decrease nitric oxide scavenging by plasma hemoglobin

Azarov

Jeffers

et al. 2008

Free Radical Biology and Medicine

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“…Because the nitrite level in red blood cells and in plasma is 300 -500 nM (32,33) under physiological conditions, there is little chance of autocatalysis and NO 2 formation. Consequently the rate of metHb formed will be limited by the slow first step of the reaction scheme with a rate constant of about 0.2-0.4 M Ϫ1 s Ϫ1 .…”

Section: Discussionmentioning

confidence: 99%

The Reaction between Nitrite and Oxyhemoglobin

Keszler

Piknová

Schechter

et al. 2008

Journal of Biological Chemistry

101

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The nitrite anion (NO 2 ؊ ) has recently received much attention as an endogenous nitric oxide source that has the potential to be supplemented for therapeutic benefit. One major mechanism of nitrite reduction is the direct reaction between this anion and the ferrous heme group of deoxygenated hemoglobin. However, the reaction of nitrite with oxyhemoglobin (oxyHb) is well established and generates nitrate and methemoglobin (metHb). Several mechanisms have been proposed that involve the intermediacy of protein-free radicals, ferryl heme, nitrogen dioxide (NO 2 ), and hydrogen peroxide (H 2 O 2 ) in an autocatalytic free radical chain reaction, which could potentially limit the usefulness of nitrite therapy. In this study we show that none of the previously published mechanisms is sufficient to fully explain the kinetics of the reaction of nitrite with oxyHb. Based on experimental data and kinetic simulation, we have modified previous models for this reaction mechanism and show that the new model proposed here is consistent with experimental data. The important feature of this model is that, whereas previously both H 2 O 2 and NO 2 were thought to be integral to both the initiation and propagation steps, H 2 O 2 now only plays a role as an initiator species, and NO 2 only plays a role as an autocatalytic propagatory species. The consequences of uncoupling the roles of H 2 O 2 and NO 2 in the reaction mechanism for the in vivo reactivity of nitrite are discussed.Elevated levels of nitrite in blood can trigger the oxidation of hemoglobin, leading to methemoglobinemia (1, 2). The mechanism of nitrite-dependent oxidation of deoxyhemoglobin (deoxyHb) 2 under anaerobic conditions, and its physiological potential in hypoxic vasodilation has recently been established (3-5). However, the mechanism of reaction between oxyhemoglobin (oxyHb) and nitrite has not yet been fully elucidated. Existing mechanistic approaches, starting with the earliest observations of Gamgee in 1868 (6), were recently reviewed (7). The end products of the reaction are methemoglobin (metHb) and nitrate. The kinetics of this reaction, when nitrite is in excess, are complex and exhibit an initial slow phase (often referred to as the lag phase) that accelerates into a rapid phase (referred to as the propagation phase) of oxidation. This reaction profile has been modeled as an autocatalytic, free radical chain reaction. The chain carrier radical and the autocatalytic steps have been variously hypothesized. Doyle et al. (8) found that hydrogen peroxide (H 2 O 2 ) accelerated oxyHb decay, whereas both catalase and superoxide dismutase elongated the half-time of the reaction. Based on these observations they suggested a mechanism according to which H 2 O 2 and superoxide played crucial roles. At the same time, Kosaka et al. (9) observed that catalase could extend the slow phase, but observed no effect of superoxide dismutase. In addition, they detected a transient protein free radical by EPR spectroscopy, and proposed a model with the intermediacy of H 2...

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“…Oxygenated HGB takes up NO, whereas deoxygenated HGB releases NO (37,40,41). Most recently, NO donors have been shown to counteract eryptosis, an effect partially mimicked by dibutyryl-cGMP (42).…”

Section: Figmentioning

confidence: 99%

“…A major stimulator of cGKI is NO (4,34,35), which can be released or removed by erythrocytes (36)(37)(38)(39). Oxygenated HGB takes up NO, whereas deoxygenated HGB releases NO (37,40,41).…”

Section: Figmentioning

confidence: 99%

Anemia and splenomegaly in cGKI-deficient mice

Föller

Feil²,

Ghoreschi³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

133

114

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To explore the functional significance of cGMP-dependent protein kinase type I (cGKI) in the regulation of erythrocyte survival, gene-targeted mice lacking cGKI were compared with their control littermates. By the age of 10 weeks, cGKI-deficient mice exhibited pronounced anemia and splenomegaly. Compared with control mice, the cGKI mutants had significantly lower red blood cell count, packed cell volume, and hemoglobin concentration. Anemia was associated with a higher reticulocyte number and an increase of plasma erythropoietin concentration. The spleens of cGKI mutant mice were massively enlarged and contained a higher fraction of Ter119 ؉ erythroid cells, whereas the relative proportion of leukocyte subpopulations was not changed. The Ter119 ؉ cGKI-deficient splenocytes showed a marked increase in annexin V binding, pointing to phosphatidylserine (PS) exposure at the outer membrane leaflet, a hallmark of suicidal erythrocyte death or eryptosis. Compared with control erythrocytes, cGKI-deficient erythrocytes exhibited in vitro a higher cytosolic Ca 2؉ concentration, a known trigger of eryptosis, and showed increased PS exposure, which was paralleled by a faster clearance in vivo. Together, these results identify a role of cGKI as mediator of erythrocyte survival and extend the emerging concept that cGMP/cGKI signaling has an antiapoptotic/prosurvival function in a number of cell types in vivo.apoptosis ͉ Ca2ϩ channels ͉ phosphatidylserine ͉ spleen N itric oxide (NO) has been shown to be a powerful regulator of cell survival (1, 2). Depending on the source or concentration of NO and the influence of additional regulators, NO may stimulate or inhibit apoptosis (3). NO exerts its effects in part through S-nitrosylation of target proteins. However, the effect of NO donors on Ca 2ϩ -induced phosphatidylserine (PS) exposure, a hallmark of apoptosis, could be mimicked by cGMP analogs (4), suggesting the involvement of soluble guanylyl cyclase, cGMP, and cGMP-dependent protein kinase type I (cGKI), a well known signaling cascade downstream of NO (5, 6).Recent studies indicated that erythroid cells possess a functional NO/cGMP pathway (7-9), which may be involved in the regulation of eryptosis, the suicidal death of erythrocytes (10). Erythrocyte cGMP production might also be stimulated by NO generated in the endothelium. The cGKI can also be activated independent of cGMP in response to oxidative stress (11). Eryptosis may follow osmotic shock, energy depletion, and oxidative stress (10), which activate Ca 2ϩ -permeable cation channels (12). Subsequent Ca 2ϩ entry leads to activation of Ca 2ϩ -sensitive K ϩ channels, exit of KCl with osmotically obliged water, and thus cell shrinkage (13). In addition, Ca 2ϩ entry triggers Ca 2ϩ -sensitive scrambling of the cell membrane (14, 15) with subsequent exposure of PS at the erythrocyte surface (12). Cell membrane scrambling may further be triggered by ceramide (16) and activation of protein kinase C (17). PS-exposing erythrocytes bind to PS receptors (18) and are recognized, ...

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Erythrocytes are the major intravascular storage sites of nitrite in human blood

Cited by 303 publications

References 47 publications

The potential of Angeli's salt to decrease nitric oxide scavenging by plasma hemoglobin

The potential of Angeli's salt to decrease nitric oxide scavenging by plasma hemoglobin

The Reaction between Nitrite and Oxyhemoglobin

Anemia and splenomegaly in cGKI-deficient mice

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