Effects of inhaled nitric oxide on regional blood flow are consistent with intravascular nitric oxide delivery

Abstract

Nitric oxide (NO) may be stabilized by binding to hemoglobin, by nitrosating thiol-containing plasma molecules, or by conversion to nitrite, all reactions potentially preserving its bioactivity in blood. Here we examined the contribution of blood-transported NO to regional vascular tone in humans before and during NO inhalation. While breathing room air and then room air with NO at 80 parts per million, forearm blood flow was measured in 16 subjects at rest and after blockade of forearm NO synthesis with N(G)-monomethyl-L-arginine (L-NMMA) followed by forearm exercise stress. L-NMMA reduced blood flow by 25% and increased resistance by 50%, an effect that was blocked by NO inhalation. With NO inhalation, resistance was significantly lower during L-NMMA infusion, both at rest and during repetitive hand-grip exercise. S-nitrosohemoglobin and plasma S-nitrosothiols did not change with NO inhalation. Arterial nitrite levels increased by 11% and arterial nitrosyl(heme)hemoglobin levels increased tenfold to the micromolar range, and both measures were consistently higher in the arterial than in venous blood. S-nitrosohemoglobin levels were in the nanomolar range, with no significant artery-to-vein gradients. These results indicate that inhaled NO during blockade of regional NO synthesis can supply intravascular NO to maintain normal vascular function. This effect may have application for the treatment of diseases characterized by endothelial dysfunction.

Figure 1

Forearm blood flow (a) and vascular resistance (b) at baseline and during intra-arterial infusion of L-NMMA (to inhibit forearm NO synthesis), followed by forearm exercise. Initial measurements were made on room air, then repeated during NO breathing at 80 ppm. Data are mean ± SEM. *P < 0.001 compared with baseline.

Figure 2

Forearm blood flow (a) and vascular resistance (b), expressed as the relative change from baseline during intra-arterial infusion of L-NMMA, followed by forearm exercise (c and d). Initial measurements were made on room air (open bars), then repeated during NO breathing at 80 ppm (filled bars). Data are mean ± SEM.

Figure 3

Forearm artery-to-vein (A-V) differences in pH (a), pO₂ (b), and pCO₂ (c) expressed as change from baseline during intra-arterial infusion of L-NMMA (left column), followed by forearm exercise (right column). Initial measurements were made on room air, then repeated during NO breathing at 80 ppm. Data are mean ± SEM.

Figure 4

Arterial (A) and venous (V) plasma levels of nitrite (a) and nitrate (b) at baseline and during infusion of L-NMMA, followed by forearm exercise. Initial measurements were made on room air, then repeated during NO breathing at 80 ppm. Data are mean ± SEM.

Figure 5

Arterial (A) and venous (V) levels of red cell S-nitrosohemoglobin (SNO-Hb) (a) and nitrosyl(heme)hemoglobin (HbFe^IINO) (b) measured during NO breathing at 80 ppm at baseline and during infusion of L-NMMA, followed by forearm exercise. Data are mean ± SEM.

Figure 6

A model of the mechanism of vascular transport of bioactive NO by red cells and plasma during NO inhalation (based on refs. –15). (a) During NO inhalation, NO and oxygen in the pulmonary vasculature react to form nitrite (NO₂^–). NO also binds to deoxyheme groups of hemoglobin to form nitrosyl(heme)hemoglobin (Fe^II-NO) and possibly with oxyhemoglobin β-globin cysteine-93 to form S-nitrosohemoglobin (β-cys93-S-NO). The major reaction of NO with oxyhemoglobin to form methemoglobin and nitrate (NO₃^–) is not shown here but accounts for the rise of methemoglobin from approximately 0.2% to 1% during NO inhalation. (b) In the partially deoxygenated red cell some NO of nitrosyl(heme)hemoglobin will react with oxygen or with oxyhemoglobin to form nitrate and methemoglobin. (c) When hemoglobin saturation and tissue pO₂ are very low, these reactions are significantly reduced and NO release from the red cell becomes possible. The hemoglobin structural transition from the oxy-state (R) to the deoxy-state (T) destabilizes the remaining NO ligand. This rate is further accelerated by heterotropic effectors, such as protons and 2,3-diphosphoglycerate, and requires a high-affinity acceptor for NO. It is possible that transfer of NO (NO+) from heme to the hemoglobin β-chain cysteine-93 occurs to form a S-nitrosohemoglobin intermediate that then releases NO by transnitrosation with glutathione (GSH). In addition, plasma nitrite may be converted to NO by disproportionation or by metal- or enzyme-catalyzed (xanthine oxidoreductase) processes. Finally, plasma S-nitrosothiol proteins could bind and deliver NO. Most of these pathways will occur preferentially in regions with low O₂ tension and pH, resulting in delivery of NO to these sites.