Pulmonary hypertension, diagnosed by Doppler echocardiography, is common in adults with sickle cell disease. It appears to be a complication of chronic hemolysis, is resistant to hydroxyurea therapy, and confers a high risk of death. Therapeutic trials targeting this population of patients are indicated.

To determine the relative contributions of endothelial-derived nitric oxide (NO) vs. intravascular nitrogen oxide species in the regulation of human blood flow, we simultaneously measured forearm blood flow and arterial and venous levels of plasma nitrite, LMW-SNOs and HMW-SNOs, and red cell S-nitrosohemoglobin (SNO-Hb). Measurements were made at rest and during regional inhibition of NO synthesis, followed by forearm exercise. Surprisingly, we found significant circulating arterial-venous plasma nitrite gradients, providing a novel delivery source for intravascular NO. Further supporting the notion that circulating nitrite is bioactive, the consumption of nitrite increased significantly with exercise during the inhibition of regional endothelial synthesis of NO. The role of circulating S-nitrosothiols and SNO-Hb in the regulation of basal vascular tone is less certain. We found that low-molecular-weight S-nitrosothiols were undetectable and S-nitroso-albumin levels were two logs lower than previously reported. In fact, S-nitroso-albumin primarily formed in the venous circulation, even during NO synthase inhibition. Whereas SNO-Hb was measurable in the human circulation (brachial artery levels of 170 nM in whole blood), arterial-venous gradients were not significant, and delivery of NO from SNO-Hb was minimal. In conclusion, we present data that suggest (i) circulating nitrite is bioactive and provides a delivery gradient of intravascular NO, (ii) S-nitrosoalbumin does not deliver NO from the lungs to the tissue but forms in the peripheral circulation, and (iii) SNO-Hb and S-nitrosothiols play a minimal role in the regulation of basal vascular tone, even during exercise stress. N itric oxide (NO) is a soluble gas synthesized in endothelial cells from the amino acid L-arginine by the constitutive calcium and calmodulin-dependent enzyme NO synthase (1). In their seminal experiment, Furchgott and Zawadzki (2) found that strips of rabbit aorta with intact endothelium relaxed in response to acetylcholine but constricted in response to the same agonist when the endothelium had been rubbed off. The substance responsible for acetylcholine-stimulated relaxation was initially called endothelium-derived relaxant factor but was subsequently found to include NO (3, 4). The importance of endothelium-derived NO in the regulation of coronary and systemic vasodilator tone has been demonstrated experimentally by regional inhibition of its synthesis with N G -monomethyl-Larginine (L-NMMA), which competes with L-arginine as the substrate for NO synthase (5, 6).Because of the instability and short half-life of NO, there has been considerable interest in the role of more stable NO adducts and metabolites that could circulate and regulate vascular tone in vivo. It has recently been proposed that NO is stabilized by covalent bonding with thiols such as glutathione, cysteine, albumin, and hemoglobin (7-10). These low-and highmolecular-weight S-nitrosothiols (LMW-SNOs and HMWSNOs) are believed to play a role in the stabilization and deli...

Background-Although reduced endothelial nitric oxide (NO) bioavailability has been demonstrated in arteriosclerotic vascular disease, the integrity of this system in sickle cell disease remains uncertain. Methods and Results-We measured forearm blood flow in 21 patients with sickle cell disease (hemoglobin SS genotype) and 18 black control subjects before and after intra-arterial infusions of acetylcholine, nitroprusside, and the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA). Endothelium-dependent vasodilation, measured by the percent increase in flow induced by acetylcholine infusion, was significantly greater than in controls (252Ϯ37% for patients versus 134Ϯ24% for controls; PϽ0.0001). However, there was a large sex difference in blood flow responses between female and male patients (340Ϯ46% versus 173Ϯ41%; Pϭ0.035). Similarly, basal NO bioactivity, as measured by the percent decrease in flow induced by L-NMMA, was depressed in male compared with female patients (Ϫ17Ϯ5% versus Ϫ34Ϯ4%; Pϭ0.01), as was the response to nitroprusside (86Ϯ21% versus 171Ϯ22%; Pϭ0.008). L-NMMA reduced the blood flow response to acetylcholine in women, but not in men. Sex differences in vascular cell adhesion molecule-1 were appreciated, with significant correlations between levels of soluble vascular cell adhesion molecule-1 and blood flow responses to L-NMMA and nitroprusside (rϭ0.53, Pϭ0.004 and rϭϪ0.66, PϽ0.001, respectively). Conclusions-NO bioavailability and NO responsiveness are greater in women than in men with sickle cell disease and determines adhesion molecule expression. Endothelium-dependent blood flows are largely non-NO mediated in male patients. These results provide a possible mechanism for reported sex differences in sickle cell disease morbidity and mortality and provide a basis for novel pharmacological interventions.

To quantify the reactions of nitric oxide (NO) with hemoglobin under physiological conditions and to test models of NO transport on hemoglobin, we have developed an assay to measure NOhemoglobin reaction products in normal volunteers, under basal conditions and during NO inhalation. NO inhalation markedly raised total nitrosylated hemoglobin levels, with a significant arterial-venous gradient, supporting a role for hemoglobin in the transport and delivery of NO. The predominant species accounting for this arterial-venous gradient is nitrosyl(heme)hemoglobin. NO breathing increases S-nitrosation of hemoglobin ␤-chain cysteine 93, however only to a fraction of the level of nitrosyl(heme)hemoglobin and without a detectable arterial-venous gradient. A strong correlation between methemoglobin and plasma nitrate formation was observed, suggesting that NO metabolism is a primary physiological cause of hemoglobin oxidation. Our results demonstrate that NO-heme reaction pathways predominate in vivo, NO binding to heme groups is a rapidly reversible process, and S-nitrosohemoglobin formation is probably not a primary transport mechanism for NO but may facilitate NO release from heme. In the last several years, the interactions of nitric oxide (NO) and hemoglobin have become the subject of much interest. A model has been recently proposed by Stamler and colleagues suggesting that NO is transported on hemoglobin by binding to the highly conserved ␤-chain cysteine 93 residue, forming Snitrosohemoglobin (SNO-Hb) (1-4). According to this theory, NO carried on hemoglobin ␤-cysteine 93 is delivered from the lungs to the microvasculature where, on deoxygenation in the tissues, the S-nitroso linkage is weakened. This allows the NO molecules, free or complexed to small thiols, to diffuse through the erythrocytes to the vascular walls, causing vasodilation of the microvasculature and subsequent recruitment of more erythrocytes. The model also posits that in the venous circulation, deoxygenated hemoglobin preferentially binds NO at the heme group to form nitrosyl(heme)hemoglobin [Hb(FeII)NO], as total NO-modified hemoglobin levels were found to be equal in the arterial and venous blood of rats (2). However, recent studies argue that NO either is not released from SNO-Hb during deoxygenation (5) or that any NO released from SNO-Hb is actively scavenged by oxyhemoglobin and fails to affect vascular tone (6). Furthermore, to date in vivo SNO-Hb measurements have been performed only in the Sprague-Dawley rat, thus the physiological significance in humans has been questioned.The reaction of NO with hemoglobin to form SNO-Hb is in addition to two other well-characterized NO reactions: with oxyhemoglobin to form methemoglobin (FeIII) and nitrate ion and with deoxyhemoglobin to form Hb(FeII)NO. This second reaction is also the subject of much current research. Yonetani and his research group and Kosaka and colleagues have shown that Hb(FeII)NO is a six-coordinate species (iron binds four nitrogens, a proximal histidine, and NO) and that on d...