Intravascular flow decreases erythrocyte consumption of nitric oxide

Abstract

Nitric oxide (NO) produced by the endothelium diffuses both into the lumen and to the smooth muscle cells according to the concentration gradient in each direction. The extremely high reaction rate between NO and hemoglobin (Hb), k(Hb)= 3-5 x 10(7) M(-1).s(-1), suggests that most of the NO produced would be consumed by Hb in the red blood cells (RBCs), which then would block the biological effect of NO. Therefore, specific mechanisms must exist under physiological conditions to reduce the NO consumption by RBCs, in which the Hb concentration is very high (24 mM heme). By using isolated microvessels as a bioassay, here we show that physiological concentrations of RBCs in the presence of intravascular flow does not inhibit NO-mediated vessel dilation, suggesting that RBCs under this condition are not an NO scavenger. On the other hand, RBCs (50% hematocrit) without intravascular flow reduce NO-mediated dilation to serotonin by 30%. In contrast, free Hb (10 microM) completely inhibits NO-mediated dilation with or without intravascular flow. The effect of flow on NO consumption by RBCs may be attributed to the formation of an RBC-free zone near the vessel wall, which is caused by hydrodynamic forces on particles. Intravascular flow does not affect the reaction rate between NO and free Hb in the lumen, because the latter forms a homogeneous solution and is not subject to the hydrodynamic separation. However, intravascular flow only partially contributes to the reduced consumption of NO by RBCs, because without the flow, the NO consumption by RBCs is already about 3 orders of magnitude slower than free Hb.

Figure 1

NO mediated vasodilation to serotonin was attenuated by 10 μM oxyHb, giving a response identical to that of NOS inhibitor l-NMMA (10 μM). Basal diameter = 76 ± 2 μm, maximum diameter = 98 ± 3 μm; P < 0.05 versus control.

Figure 2

Flow-induced vasodilation was attenuated by oxyHb in a dose-dependent manner. Vasodilatory response to the increased flow was completely abolished by 10 μM oxyHb, identical to that of NOS inhibitor l-NMMA (10 μM). n = 5, basal diameter = 74 ± 4 μm; maximum diameter = 112 ± 3 μm; ∗, P < 0.05, all interventions versus control.

Figure 3

The vasodilation in response to serotonin (0.1 μM) was significantly inhibited by free oxyHb and RBCs with 50% or 90% hematocrit (corresponding to 3 and 5.6 mM overall Hb, respectively). Fifty percent hematocrit (3.1 mM Hb) had less inhibition to serotonin-induced dilation than 10 μM free oxyHb. n = 5, basal diameter = 76 ± 2 μm, maximum diameter = 98 ± 3 μm; ∗, P < 0.05, versus control; +, P < 0.05, versus 50% hematocrit.

Figure 4

The vasodilatory response to the increased flow (i.e., pressure gradient) during perfusion with a PSS or RBCs. The flow-induced vasodilation, especially at the lower flow rates, was slightly inhibited by RBCs. n = 5, basal diameter = 78 ± 4 μm, maximum diameter = 104 ± 3 μm.

Figure 5

Summary of the effect of flow on inhibition of vasodilation by free Hb and RBCs from Figs. 1–4. The presence of flow reduced the inhibitory effect of RBCs (50–60% hematocrit) on NO-mediated vessel dilation [+, P < 0.05, serotonin (5-HT) versus flow, ΔP = 60 cm H₂O]. In contrast, flow did not change the effect of free Hb. Free Hb (10 μM) effectively inhibited NO-mediated dilation in response to either serotonin (0.1 μM) or shear stress (ΔP = 60 cm H₂O), but RBCs (50–60% hematocrit) had significantly less effect. ∗, P < 0.05, RBCs versus free Hb.