Role of circulating nitrite and S-nitrosohemoglobin in the regulation of regional blood flow in humans

Abstract

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-nitroso-albumin 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.

Figures

Figure 1

Physiological effect of NO synthase inhibition and exercise on forearm blood flow. Forearm blood flow measurements, expressed as ml/min/100 g tissue, were performed at baseline, during L-NMMA infusion, and during L-NMMA infusion with hand-grip exercise. Basal forearm blood flow was significantly reduced from 2.79 ± 0.34 ml/min per 100 ml of tissue to 2.04 ± 0.22 ml/min per 100 ml of tissue (P = 0.001) during L-NMMA infusion (data shown represent the average of measurements made during the morning and the repeated afternoon study in 10 individuals). Exercise, during L-NMMA infusion, significantly increased blood flow to 14.50 ± 1.71 ml/min per 100 ml of tissue (P < 0.001). Data are expressed as the mean ± SEM.

Figure 2

Plasma circulatory levels of nitrite, HMW-SNO, and SNO-Hb at rest and during NO synthase inhibition followed by exercise. SNO-Hb: S-nitrosohemoglobin in arterial (empty bars) and venous (hatched bars) red blood cells at rest and during L-NMMA infusion, followed by exercise. The arterial and venous levels were close to the limits of detection by chemiluminescent technology at 1.6 × 10−5 mol NO/mol heme subunit (161 nM in whole blood) and 1.4 × 10−5 mol of NO/mol of heme subunit (141 nM in whole blood), respectively. We observed an insignificant arterial-venous gradient in SNO-Hb (P = 0.53 at baseline, P = 0.17 during L-NMMA infusion, and P = 0.31 during exercise, by paired t test, and P = 0.24 by repeated-measures ANOVA). HMW-SNO: plasma passed through a G25 Sephadex sizing column was reacted in I3− to measure HMW-SNO in arterial and venous plasma. HMW-SNO displayed a reversed gradient with venous levels (63.4 ± 13 nM) greater than arterial levels (44.9 ± 14 nM) (P values for paired t tests shown and P < 0.05 for venous-arterial differences by repeated-measures ANOVA). Nitrite: filtered plasma was reacted in KI in acetic acid to measure nitrite in arterial and venous plasma. Although nitrite levels varied widely between individuals, within individuals the arterial levels of nitrite (540 ± 74 nM) were significantly higher than venous levels (466 ± 79 nM), suggesting delivery or metabolism in the peripheral circulation (P < 0.05 for all experiments by paired t test).

Figure 3

Consumption of nitrogen oxide species at baseline, during NO synthase inhibition, and during exercise with NO synthase inhibition. Arterial and venous nitrite and S-nitrosothiol gradients were multiplied by flow measurements to determine consumption (positive value on the y axis) or, in the case of HMW-SNO, production (negative value on the y axis). SNO-Hb: Basal SNO-Hb consumption was 92 ± 86 pmol/ml/min/100 ml of tissue. SNO-Hb consumption increased nonsignificantly from 143 ± 83 pmol/ml/min/100 ml of tissue during L-NMMA infusion to 366 ± 471 pmol/ml/min/100 ml of tissue during L-NMMA infusion with exercise. Data are expressed as the mean ± SEM. HMW-SNO: At baseline, HMW-SNO forms (negative value for consumption) in the peripheral circulation at a rate of 23 ± 14 pmol/ml/min/100 ml of tissue. Although there was no significant change in the formation of HMW-SNO during L-NMMA infusion (25 ± 8 pmol/ml/min/100 ml of tissue), a trend toward increased formation of HMW-SNO was observed during exercise and L-NMMA infusion (127 ± 117 pmol/ml/min/100 ml of tissue). Nitrite: Nitrite consumption significantly increased with exercise, from 156 ± 59 pmol/ml/min/100 ml of tissue during L-NMMA infusion to 1583 ± 517 pmol/ml/min/100 ml of tissue during L-NMMA infusion with exercise, suggesting that intravascular nitrite is used even during physiological stress.