A nitric oxide processing defect of red blood cells created by hypoxia: Deficiency of S-nitrosohemoglobin in pulmonary hypertension

Abstract

The mechanism by which hypoxia [low partial pressure of O2 (pO2)] elicits signaling to regulate pulmonary arterial pressure is incompletely understood. We considered the possibility that, in addition to its effects on smooth muscle, hypoxia may influence pulmonary vascular tone through an effect on RBCs. We report that exposure of native RBCs to sustained hypoxia is accompanied by a buildup of heme iron-nitrosyl (FeNO) species that are deficient in pO 2-governed intramolecular transfer of NO to cysteine thiol, yielding a deficiency in the vasodilator S-nitrosohemoglobin (SNO-Hb). hemoglobin ͉ red blood cell vasodilation ͉ S-nitrosylation I n the systemic microcirculation, blood flow is regulated by physiological O 2 gradients that couple the O 2 content of blood to regulated vasodilation and vasoconstriction (1-3). Blood flow is thereby matched to tissue O 2 demand. An analogous mechanism operates in the lungs, where O 2 uptake (ventilation) is optimized through regulated vasodilation and vasoconstriction (perfusion). Blood flow is thereby matched to alveolar ventilation (2). Because it is Hb O 2 saturation, not the partial pressure of O 2 (pO 2 ), that is coupled to blood flow in vivo (1, 3) it has been deduced that RBCs may serve as O 2 sensors within the integrated vascular system. In support of this idea, it has been shown recently that RBCs can act as O 2 -responsive transducers of vasodilator and vasoconstrictor activity (4-10), at least partly by modulating the availability of [6][7][8]10,11). According to these studies, RBCs release NO bioactivity under hypoxia and sequester it at hyperoxia. The release of NO bioactivity would facilitate hypoxic vasodilation in peripheral tissues and oppose hypoxic pulmonary vasoconstriction (HPV) in the lungs. S-nitrosothiol (SNO)-deficientThe mechanism by which NO bioactivity escapes from RBCs is incompletely understood. It is generally accepted that the rapid reaction of NO with the hemes of Hb produces a heme-iron nitrosyl adduct (Hb [FeNO]) that exhibits no vasodilator activity (4,7,12). Hb also sustains S-nitrosylation at two cysteine residues conserved in all mammals and birds. Biochemical and mutational analyses (␤93Cys3Ala) indicate that S-nitrosohemoglobin (SNO-Hb) is formed upon oxygenation of Hb [FeNO] by means of heme-to-Cys NO transfer (13-15) and by transnitrosylative transfer from low-mass S-nitrosothiols (SNOs) (16,17). SNO-Hb is very stable in the oxygenated (or R) structure and thus cannot effectively dilate blood vessels (5, 10, 18). However, upon deoxygenation [or with change in the spin state of the hemes (3)], the vasodilator potency of SNO-Hb is markedly potentiated (5,16,18). Crystal structures and molecular models show that the ␤-Cys NO gains solvent access in the deoxygenated (or T) state (3, 19). Solvent-exposed NO can exchange with acceptor thiols within the N-terminal cytoplasmic domain of the RBC membrane anion exchange protein (AE1; band 3) (4, 15). Transnitrosylation of AE1 by SNO-Hb involves a direct protein-protein interaction. The st...

Assessment of nitric oxide signals by triiodide chemiluminescence

“…These latter assays involved removal of protein to avoid the potential errors introduced by side reactions with proteins. Early concerns that photolysis directly detects nitrite and nitrate (in the added presence of thiol) proved unfounded (47). Furthermore, we have shown that the amounts of SNO measured by photolysis directly predict vasodilatory activity of RBCs (47,49,50).…”

“…Moreover, few NO standards have actually been tested, and no basis has been provided for asserting that response of these standards captures the general behavior. Recovery of certain FeNO standards is reported to be as low as zero (40), and the one SNO-Hb standard that has been widely used (an R-structured Hb that contains Ϸ2 NO per tetramer) (40)(41)(42)(43)(44)(45)(46) is neither characteristic of general SNO-Hb reactivity nor of the reactivity of the micropopulation found in RBCs (a valency hybrid, estimated 1 NO per tetramer) (5,47). Furthermore, the claimed effects of added reagents in triiodide assays, including FeCN and NEM (alone and in combination), are not supported, and they, along with the acidic and denaturing conditions of the triiodide assay, can alter the reactivities of SNO and FeNO as well as disrupt the partitioning of NO species within hydrophobic compartments and thus lead to their misidentification.…”

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

“…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). In addition, the importance of duration of reaction on product distribution was recognized: Aging of NO-deoxyHb samples that is incurred over the lengthy course of the nitrite reaction enables competing chemistry, including redistribution of NO from ␤-to ␣-chains, reductive loss of NO to HNO and quenching of radicals in Hb (4,7,(13)(14)(15)34). Because previous work has suggested collectively that the efficacy of transfer of NO groups from heme to ␤Cys-93 (to form a bioactive SNO) might require not only physiological NO͞Hb ratios and HbNO concentrations (Ͻ 1 M) but also preferential processing of NO within the ␤-chain (14-17), the conditions used in prior work were not optimized for the study of SNO formation.…”

“…We subsequently demonstrated that vasoconstriction by RBCs is seen only at relatively high partial pressures of O 2 (pO 2 ); at lower pO 2 s characteristic of tissues (5-20 mm Hg), RBCs dilate blood vessels (2,3). Vasodilation (of aortic or pulmonary artery rings) by RBCs in bioassay is rapid, in keeping with the temporal requirements of arterial-venous transit (in seconds) (3,4). Moreover, when infused into animals, RBCs increase blood flow and improve oxygenation, an indication that RBCs elicit vasodilation in both the systemic and pulmonary circulations (1,(4)(5)(6).…”

“…Vasodilation (of aortic or pulmonary artery rings) by RBCs in bioassay is rapid, in keeping with the temporal requirements of arterial-venous transit (in seconds) (3,4). Moreover, when infused into animals, RBCs increase blood flow and improve oxygenation, an indication that RBCs elicit vasodilation in both the systemic and pulmonary circulations (1,(4)(5)(6). Collectively, the observation of vasoconstriction by RBCs at higher pO 2 but graded vasodilation with increasing hypoxia appears to provide a mechanism for matching blood flow to metabolic demand in the peripheral tissues and for matching ventilation to perfusion in the lungs (7).…”