Postprandial lipids accelerate and redirect nitric oxide consumption in plasma.

Nitric oxide (NO) and O2 are both three-to four-fold more soluble in biological lipids than in aqueous solutions. Their higher concentration within plasma lipids accelerates NO autoxidation to an extent that may be of importance to overall NO bioactivity. This study was undertaken to test the hypothesis that increased plasma lipids after a high-fat meal appreciably accelerate NO metabolism and alter the byproducts formed. We found that plasma collected from subjects after consumption of a single high-fat meal had a higher capacity for NO consumption and consumed NO more rapidly compared to fasting plasma. This increased NO consumption showed a direct correlation with plasma triglyceride concentrations (p = 0.006). The accelerated NO consumption in postprandial plasma was reversed by removal of the lipids from the plasma, was mimicked by the addition of hydrophobic micelles to aqueous buffer, and could not be explained by the presence of either free hemoglobin or ceruloplasmin. The products of NO consumption were shifted in postprandial plasma, with 55% more nitrite (n = 12, p = 0.002) but 50% less SNO (n = 12, p = 0.03) production compared to matched fasted plasma. Modeling calculations indicated that NO autoxidation was accelerated by about 48-fold in the presence of plasma lipids. We conclude that postprandial triglyceride-rich lipoproteins exert a significant influence on NO metabolism in plasma.

S-nitrosothiols dilate the mesenteric artery more potently than the femoral artery by a cGMP and L-type calcium channel-dependent mechanism.

S-nitrosothiols (SNOs) are metabolites of NO with potent vasodilatory activity. Our previous studies in sheep indicated that intra-arterially infused SNOs dilate the mesenteric vasculature more than the femoral vasculature. We hypothesized that the mesenteric artery is more responsive to SNO-mediated vasodilation, and investigated various steps along the NO/cGMP pathway to determine the mechanism for this difference. In anesthetized adult sheep, we monitored the conductance of mesenteric and femoral arteries during infusion of S-nitroso-l-cysteine (L-cysNO), and found mesenteric vascular conductance increased (137 ± 3%) significantly more than femoral conductance (26 ± 25%). Similar results were found in wire myography studies of isolated sheep mesenteric and femoral arteries. Vasodilation by SNOs was attenuated in both vessel types by the presence of ODQ (sGC inhibitor), and both YC-1 (sGC agonist) and 8-Br-cGMP (cGMP analog) mediated more potent relaxation in mesenteric arteries than femoral arteries. The vasodilatory difference between mesenteric and femoral arteries was eliminated by antagonists of either protein kinase G or L-type Ca(2+) channels. Western immunoblots showed a larger L-type Ca(2+)/sGC abundance ratio in mesenteric arteries than in femoral arteries. Fetal sheep mesenteric arteries were more responsive to SNOs than adult mesenteric arteries, and had a greater L-Ca(2+)/sGC ratio (p = 0.047 and r = -0.906 for correlation between Emax and L-Ca(2+)/sGC). These results suggest that mesenteric arteries, especially those in fetus, are more responsive to SNO-mediated vasodilation than femoral arteries due to a greater role of the L-type calcium channel in the NO/cGMP pathway.

Role of blood and vascular smooth muscle in the vasoactivity of nitrite.

Recent evidence from humans and rats indicates that nitrite is a vasodilator under hypoxic conditions by reacting with metal-containing proteins to produce nitric oxide (NO). We tested the hypothesis that near-physiological concentrations of nitrite would produce vasodilation in a hypoxia- and concentration-dependent manner in the hind limb of sheep. Anesthetized sheep were instrumented to measure arterial blood pressure and femoral blood flows continuously in both hind limbs. Nitrite was infused into one femoral artery to raise the nitrite concentration in the femoral vein by 10 to 15-fold while the sheep breathed 50%, 14% or 12% oxygen in inspired air. In contrast to reports in humans and rats, the nitrite infusion had no measurable effect on mean femoral blood flows or vascular conductances, regardless of inspired O2 levels. In vitro experiments showed no significant difference in the release of NO from nitrite in sheep and human red blood cells. Further experiments demonstrated nitrite is converted to NO in rat artery homogenates faster than sheep arteries, and that this source of NO production is attenuated in the presence of a heme oxidizer. Finally, western blots indicate that concentrations of the heme-containing protein cytoglobin, but not myoglobin, are markedly lower in sheep arteries compared with rats. Overall, the results demonstrate that nitrite is not a physiological vasodilator in sheep. This is likely due to a lack of conversion of nitrite to NO within the vascular smooth muscle, perhaps due to deficient amounts of the heme-containing protein cytoglobin.

Role of ceruloplasmin in nitric oxide metabolism in plasma of humans and sheep: a comparison of adults and fetuses.

Nitric oxide (NO) is metabolized in plasma, in part by the ferroxidase ceruloplasmin (Cp), to form nitrite and nitrosothiols (SNOs), which are proposed to mediate protective responses to hypoxia and ischemia. We hypothesized that NO metabolism would be attenuated in fetal plasma due to low Cp activity. We measured Cp concentrations and activity in plasma samples collected from adults and fetuses of humans and sheep. We then added NO ([NO]: 1.5 or 100 µM) to plasma and aqueous buffer and measured rates of NO disappearance and the production of nitrite and SNO. Cp concentrations in fetal plasma were <15% of adult levels. In aqueous buffer, 1.5 µM NO disappeared with a half-life of 347 ± 64 s (means ± SE) but in plasma of humans the half-life was 19 ± 2 s (adult) and 11 ± 1 s (fetus, P = 0.004) and in sheep it was 31 ± 3 s (adult) and 43 ± 5 s (fetus, P = 0.04). Cp activity was not correlated with the overall elimination half-life of NO or with the amount of SNO ([NO]: 100 µM) or nitrite ([NO]: 1.5 or 100 µM) produced but correlated with SNO yields at 1.5 µM [NO] (r = 0.92, P = 0.04). Our data demonstrate that Cp is not essential to the increased rate of metabolism of NO in plasma relative to aqueous buffers and that it is not essential to the production of nitrite from NO. Cp may be involved in the conversion of NO to SNO in plasma under near-physiological concentrations of NO.

Inhaled nitrite reverses hemolysis-induced pulmonary vasoconstriction in newborn lambs without blood participation.

BACKGROUND: Nitrite can be converted to nitric oxide (NO) by a number of different biochemical pathways. In newborn lambs, an aerosol of inhaled nitrite has been found to reduce pulmonary blood pressure, possibly acting via conversion to NO by reaction with intraerythrocytic deoxyhemoglobin. If so, the vasodilating effects of nitrite would be attenuated by free hemoglobin in plasma that would rapidly scavenge NO. METHODS AND RESULTS: Pulmonary vascular pressures and resistances to flow were measured in anesthetized newborn lambs. Plasma hemoglobin concentrations were then elevated, resulting in marked pulmonary hypertension. This effect was attenuated if infused hemoglobin was first oxidized to methemoglobin, which does not scavenge NO. These results further implicate NO as a tonic pulmonary vasodilator. Next, while free hemoglobin continued to be infused, the lambs were given inhaled NO gas (20 ppm), inhaled sodium nitrite aerosol (0.87 mol/L), or an intravascular nitrite infusion (3 mg/h bolus, 5 mg · kg⁻¹ · h⁻¹ infusion). Inhaled NO and inhaled nitrite aerosol both resulted in pulmonary vasodilation. Intravascular infusion of nitrite, however, did not. Increases in exhaled NO gas were observed in lambs while breathing the nitrite aerosol (≈ 20 ppb NO) but not during intravascular infusion of nitrite. CONCLUSIONS: We conclude that the pulmonary vasodilating effect of inhaled nitrite results from its conversion to NO in airway and parenchymal lung tissue and is not dependent on reactions with deoxyhemoglobin in the pulmonary circulation. Inhaled nitrite aerosol remains a promising candidate to reduce pulmonary hypertension in clinical application.

Fetal-maternal nitrite exchange in sheep: Experimental data, a computational model and an estimate of placental nitrite permeability.

INTRODUCTION: Nitrite conveys NO-bioactivity that may contribute to the high-flow, low-resistance character of the fetal circulation. Fetal blood nitrite concentrations depend partly on placental permeability which has not been determined experimentally. We aimed to extract the placental permeability-surface (PS) product for nitrite in sheep from a computational model. METHODS: An eight-compartment computational model of the fetal-maternal unit was constructed (Matlab(®) (R2013b (8.2.0.701), MathWorks Inc., Natick, MA). Taking into account fetal and maternal body weights, four variables (PS, the rate of nitrite metabolism within red cells, and two nitrite distribution volumes, one with and one without nitrite metabolism), were varied to obtain optimal fits to the experimental plasma nitrite profiles observed following the infusion of nitrite into either the fetus (n = 7) or the ewe (n = 8). RESULTS: The model was able to replicate the average and individual nitrite-time profiles (r(2) > 0.93) following both fetal and maternal nitrite infusions with reasonable variation of the four fitting parameters. Simulated transplacental nitrite fluxes were able to predict umbilical arterial-venous nitrite concentration differences that agreed with experimental values. The predicted PS values for a 3 kg sheep fetus were 0.024 ± 0.005 l∙min(-1) in the fetal-maternal direction and 0.025 ± 0.003 l∙min(-1) in the maternal-fetal direction (mean ± SEM). These values are many-fold higher than the reported PS product for chloride anions across the sheep placenta. CONCLUSION: The result suggests a transfer of nitrite across the sheep placenta that is not exclusively by simple diffusion through water-filled channels.

Local and systemic vasodilatory effects of low molecular weight S-nitrosothiols.

S-nitrosothiols (SNOs) such as S-nitroso-L-cysteine (L-cysNO) are endogenous compounds with potent vasodilatory activity. During circulation in the blood, the NO moiety can be exchanged among various thiol-containing compounds by S-transnitrosylation, resulting in SNOs with differing capacities to enter the cell (membrane permeability). To determine whether the vasodilating potency of SNOs is dependent upon membrane permeability, membrane-permeable L-cysNO and impermeable S-nitroso-D-cysteine (D-cysNO) and S-nitroso-glutathione (GSNO) were infused into one femoral artery of anesthetized adult sheep while measuring bilateral femoral and systemic vascular conductances. L-cysNO induced vasodilation in the infused hind limb, whereas D-cysNO and GSNO did not. L-cysNO also increased intracellular NO in isolated arterial smooth muscle cells, whereas GSNO did not. The infused SNOs remained predominantly in a low molecular weight form during first-passage through the hind limb vasculature, but were converted into high molecular weight SNOs upon systemic recirculation. At systemic concentrations of ~0.6 µmol/L, all three SNOs reduced mean arterial blood pressure by ~50%, with pronounced vasodilation in the mesenteric bed. Pharmacokinetics of L-cysNO and GSNO were measured in vitro and in vivo and correlated with their hemodynamic effects, membrane permeability, and S-transnitrosylation. These results suggest local vasodilation by SNOs in the hind limb requires membrane permeation, whereas systemic vasodilation does not. The systemic hemodynamic effects of SNOs occur after equilibration of the NO moiety amongst the plasma thiols via S-transnitrosylation.

Fetal sheep with tracheal occlusion: monitoring lung development with MR imaging and B-mode US.

PURPOSE: To assess the accuracy of magnetic resonance (MR) imaging in determining fetal lung volume (FLV) and to observe fetal lung development with B-mode ultrasonography (US) and MR imaging. MATERIALS AND METHODS: Seven sheep fetuses between 92 and 141 gestational days (term, 145 days) with and without tracheal occlusion (controls) underwent serial MR imaging and US. FLV at MR imaging was measured with true fast imaging with steady-state precession in coronal and transverse planes. The combined cross-sectional left- and right-lung area was measured with US at three transverse levels. FLV was measured at autopsy. Statistical evaluations included linear regression analysis and calculation of the mean and 95% CI. RESULTS: No differences in FLV were observed on coronal or transverse MR images (r2 = 0.98; slope = 0.91; 95% CI: 0.82, 1.01). FLV at MR imaging at termination of the experiment was significantly related to FLV at autopsy (r2 = 0.96; slope = 1.27; 95% CI: 0.97, 1.57; n = 6). FLV at MR imaging increased more rapidly with gestational age in fetuses with tracheal occlusion (21.0 mL/d; 95% CI: 10.7, 31.3) than in controls (4.7 mL/d; 95% CI: 1.7, 7.7). Increase in left- and right-lung area at US was accelerated in fetuses with tracheal occlusion (1.60 cm2/d; 95% CI: 1.3, 1.9) compared with controls (0.38 cm2/d; 95% CI: 0.23, 0.53). Left- and right-lung area at US and FLV at MR imaging were significantly correlated (r2 = 0.82). CONCLUSION: FLV can be measured with moderate accuracy at MR imaging on both coronal and transverse images. MR imaging and B-mode US are useful tools for monitoring and quantifying tracheal occlusion-stimulated fetal lung growth in sheep fetuses.