Active Nitric Oxide Produced in the Red Cell under Hypoxic Conditions by Deoxyhemoglobin-mediated Nitrite Reduction

Abstract

Recent studies have generated a great deal of interest in a possible role for red blood cells in the transport of nitric oxide (NO) to the microcirculation and the vascular effect of this nitric oxide in facilitating the flow of blood through the microcirculation. Many questions have, however, been raised regarding such a mechanism. We have instead identified a completely new mechanism to explain the role of red cells in the delivery of NO to the microcirculation. This new mechanism results in the production of NO in the microcirculation where it is needed. Nitrite produced when NO reacts with oxygen in arterial blood is reutilized in the arterioles when the partial pressure of oxygen decreases and the deoxygenated hemoglobin formed reduces the nitrite regenerating NO. Nitrite reduction by hemoglobin results in a major fraction of the NO generated retained in the intermediate state where NO is bound to Hb(III) and in equilibrium with the nitrosonium cation bound to Hb(II). This pool of NO, unlike Hb(II)NO, is weakly bound and can be released from the heme. The instability of Hb(III)NO in oxygen and its displacement when flushed with argon requires that reliable determinations of red blood cell NO must be performed on freshly lysed samples without permitting the sample to be oxygenated. In fresh blood samples Hb(III)NO accounts for 75% of the red cell NO with appreciably higher values in venous blood than arterial blood. These findings confirm that nitrite reduction at reduced oxygen pressures is a major source for red cell NO. The formation and potential release from the red cell of this NO could have a major impact in regulating the flow of blood through the microcirculation.Nitric oxide (NO) 1 synthesized by endothelial nitric-oxide synthase diffuses both to the smooth muscle cells as well as the lumen (1, 2). The reaction of NO with smooth muscle cell guanylate cyclase plays a crucial role in the regulation of vascular tone and blood flow (3). In the lumen NO is either oxidized by oxygen resulting in the formation of nitrite (4) or is taken up by red blood cells (5). In red blood cells, NO rapidly reacts with oxyhemoglobin to form methemoglobin (Hb(III)) and nitrate and with deoxyhemoglobin (deoxyHb) to form heme-nitrosylated hemoglobin (Hb(II)NO) (6, 7). Earlier studies, therefore, assumed that the only effect of red blood cells on the bioactivity of nitric oxide was to scavenge and inactivate NO limiting its availability for vasodilation. This perspective was challenged by studies of Stamler and co-workers (8 -13) who proposed an active role for red blood cells in the delivery of NO. Their model is based on the potential binding of NO released from arterial endothelial cells to the few vacant deoxyhemes to form Hb(II)NO and to the ␤-93 cysteine to form S-nitrosohemoglobin (SNOHb) (11). The relative affinity for these two hemoglobin sites was assumed to depend on the hemoglobin quaternary conformation with heme-bound NO favored by the T-state present in deoxygenated hemoglobin transferred to the cysteine in ...

An S -nitrosothiol (SNO) synthase function of hemoglobin that utilizes nitrite as a substrate

“…1). Subsequent work examining the kinetics and product distribution of this reaction under a range of conditions (12,25,33) has continued to employ nitrite concentrations and [NaNO 2 ]͞ [deoxy-heme] ratios well above physiological values. A clue to the importance of reactant concentrations on this chemistry emerged from EPR studies, which revealed, in the aftermath of mixing very high concentrations of nitrite and deoxyHb, essentially equal subunit populations of nitrosyl heme [␣Fe(II)NO Ϸ ␤Fe(II)NO], whereas spectra obtained under conditions that simulate key aspects of the in vivo situation exhibit substantial ␤Fe(II)NO preference (16).…”

“…However, our kinetic analysis (12), like other work on nitrite͞Hb interactions (25)(26)(27), was performed at supraphysiological nitrite͞Hb ratios (typically nitrite:Hb 1:10-10:1 vs. Ϸ1:1,000 in vivo), with a global spectral deconvolution approach that was not adapted to recognize ''minority species.'' We now examine the product distribution of reactions of nitrite and deoxyhemoglobin by using a modified approach in which spectral deconvolution is used to test for the presence of minority species formed at physiologically relevant concentrations, whereas studies of the chemical reactivity of these species are used to verify their identity.…”

Fe(II) Oxidation Is an Innate Capability of Nitrate-Reducing Bacteria That Involves Abiotic and Biotic Reactions

“…For headspace NO analysis, culture headspace was anaerobically sampled and injected into the purge cell of a 280i NO analyzer (Sievers). For solution NO, samples of culture solution were injected into a purge solution in anoxic vials (31). The headspace from these vials was injected into the NO analyzer.…”

Bioanalytical profile of the l-arginine/nitric oxide pathway and its evaluation by capillary electrophoresis

“…High-capacity NO production in eukaryotic systems has two dominant but not unique routes: 1) the primary production via enzymatic oxygenation of the guanidine group of L-arginine to form NO and L-citrulline by a specific enzyme, NO synthase (NOS) [17]; and 2) the secondary production by enzymatic reduction of nitrite [18,19] via nitrite reductase activity (NRA) of xanthine oxidase [20,21], mitochondrial cytochrome complexes [22,23], deoxyhemoglobin [24][25][26], and some NOS isozymes under anoxic conditions [27]. It is generally assumed that the primary NO synthesis from L-arginine oxidation by NOS and following retrograde diffusion of the gaseous messenger is responsible for focal "neurotransmitter-like" actions of NO [28], as well as for pathogen-induced escalation of cytotoxic defense [29].…”