John R. Pawloski

Export by red blood cells of nitric oxide bioactivity.

Previous studies support a model in which the physiological O2 gradient is transduced by haemoglobin into the coordinate release from red blood cells of O2 and nitric oxide (NO)-derived vasoactivity to optimize oxygen delivery in the arterial periphery. But whereas both O2 and NO diffuse into red blood cells, only O2 can diffuse out. Thus, for the dilation of blood vessels by red blood cells, there must be a mechanism to export NO-related vasoactivity, and current models of NO-mediated intercellular communication should be revised. Here we show that in human erythrocytes haemoglobin-derived S-nitrosothiol (SNO), generated from imported NO, is associated predominantly with the red blood cell membrane, and principally with cysteine residues in the haemoglobin-binding cytoplasmic domain of the anion exchanger AE1. Interaction with AE1 promotes the deoxygenated structure in SNO–haemoglobin, which subserves NO group transfer to the membrane. Furthermore, we show that vasodilatory activity is released from this membrane precinct by deoxygenation. Thus, the oxygen-regulated cellular mechanism that couples the synthesis and export of haemoglobin-derived NO bioactivity operates, at least in part, through formation of AE1–SNO at the membrane–cytosol interface.

Nitric oxide in the human respiratory cycle

Interactions of nitric oxide (NO) with hemoglobin (Hb) could regulate the uptake and delivery of oxygen (O2) by subserving the classical physiological responses of hypoxic vasodilation and hyperoxic vasconstriction in the human respiratory cycle. Here we show that in in vitro and ex vivo systems as well as healthy adults alternately exposed to hypoxia or hyperoxia (to dilate or constrict pulmonary and systemic arteries in vivo), binding of NO to hemes (FeNO) and thiols (SNO) of Hb varies as a function of HbO2 saturation (FeO2). Moreover, we show that red blood cell (RBC)/SNO-mediated vasodilator activity is inversely proportional to FeO2 over a wide range, whereas RBC-induced vasoconstriction correlates directly with FeO2. Thus, native RBCs respond to changes in oxygen tension (pO2) with graded vasodilator and vasoconstrictor activity, which emulates the human physiological response subserving O2 uptake and delivery. The ability to monitor and manipulate blood levels of NO, in conjunction with O2 and carbon dioxide, may therefore prove useful in the diagnosis and treatment of many human conditions and in the development of new therapies. Our results also help elucidate the link between RBC dyscrasias and cardiovascular morbidity.

Cell-Free and Erythrocytic S-Nitrosohemoglobin Inhibits Human Platelet Aggregation

BACKGROUND Nitric oxide (NO) and related molecules are thought to inhibit human platelet aggregation by raising levels of cGMP. METHODS AND RESULTS Both oxidative stress (reactive oxygen species) and hemoglobin (Hb) seem to oppose NO effects. A major fraction of NO in the blood is bound to thiols of Hb, forming S-nitrosohemoglobin (SNO-Hb), which releases the NO group on deoxygenation in the microcirculation. Here we show that (1) both cell-free and intraerythrocytic SNO-Hb (SNO-RBC) inhibit platelet aggregation, (2) the oxidation state of the hemes in Hb influences the response--SNO-metHb (which is functionally similar to SNO-deoxyHb) has greater platelet inhibitory effects than SNO-oxyHb, and (3) the mechanism of platelet inhibition by SNO-Hb is cGMP independent. CONCLUSIONS We suggest that the RBC has evolved a means to counteract platelet activation in small vessels and the proaggregatory effects of oxidative stress by forming SNO-Hb.

In Vivo Gene Transfer of Nitric Oxide Synthase Enhances Vasomotor Function in Carotid Arteries From Normal and Cholesterol-Fed Rabbits

BACKGROUND The vascular endothelium is anatomically intact but functionally abnormal in preatherosclerotic states, and an early deficit in the bioavailability of nitric oxide (NO) or related molecules has been described in both humans and animal models. We hypothesized that the targeted gene transfer of NO synthase (NOS) isoforms might ameliorate or reverse the deficit. METHODS AND RESULTS We constructed a recombinant adenovirus, Ad.nNOS, that expresses the neuronal isoform of NOS (nNOS) and used it for in vivo endovascular gene transfer to carotid arteries (CA) from normal and cholesterol-fed rabbits. Vessels were harvested 3 days after gene transfer. In CA from normal rabbits, Ad.nNOS generated high levels of functional nNOS protein predominantly in endothelial cells and increased vascular NOS activity by 3.4-fold relative to sham-infected control CA. Ad.nNOS gene transfer also significantly enhanced endothelium-dependent vascular relaxation to acetylcholine; at 3 micromol/L acetylcholine, Ad.nNOS-treated arteries showed an 86+/-4% reduction in precontracted tension, whereas control CA showed a 47+/-6% reduction in tension. Contraction in response to phenylephrine and relaxation in response to nitroprusside were unaffected in both control and Ad.nNOS-treated CA. To determine the effect of Ad.nNOS in atherosclerotic arteries, 10 male New Zealand White rabbits maintained on a 1% cholesterol diet for 10 to 12 weeks underwent gene transfer according to the same protocol used in normal rabbits. Ad.nNOS-treated arteries showed a 2-fold increase in NADPH-diaphorase staining intensity relative to sham-infected and Ad. betaGal-treated arteries. The CA from cholesterol-fed rabbits showed impaired acetylcholine-induced relaxation, but this abnormality was almost entirely corrected by Ad.nNOS gene transfer. CONCLUSIONS In vivo adenovirus-mediated endovascular delivery of nNOS markedly enhances vascular NOS activity and can favorably influence endothelial physiology in the intact and atherosclerotic vessel wall.

Nitric oxide in RBCs

S ubstantial evidence has accrued to support the view that nitric oxide (NO) is a key component of the respiratory cycle, a third gas transported (together with oxygen [O2] and carbon dioxide [CO2]) by RBCs.1,2 NO, a potent vasodilator and inhibitor of platelet activation produced by the vascular endothelium, is sequestered in RBCs via reactions with the heme prosthetic groups of Hb and with cysteine residues in the chain (Cys 93). Under low pO2 conditions (physiologic “hypoxia”), the RBC releases NO groups from Cys 93 to increase blood flow. Thus, the RBC dynamically processes pO2 into a regulated mechanism for control of blood vessel tone, constricting (hyperoxia) or dilating (hypoxia) blood vessels commensurate with the metabolic requirements of the tissues it moves through. NO bioactivity derived from RBCs hinges on newly described chemical interplay between Hb and the RBC membrane.

Role of Circulating S-Nitrosothiols in Control of Blood Pressure

The biological effects of nitric oxide (NO) are in large part mediated by S -nitrosylation of peptides and proteins to produce bioactive S -nitrosothiols (SNOs).1–3 The observation of abnormal SNO levels in numerous pathophysiological states2 suggests that dysregulation of SNO homeostasis may contribute to disease pathogenesis. For example, the hypotension of human sepsis is accompanied by increases in circulating levels of vasodilatory SNOs.3 Although such altered SNO levels may simply mirror NO production (eg, induction of inducible NO synthase in sepsis), they may also reflect changes specific to SNO biosynthesis and metabolism. Indeed, mice lacking a SNO-metabolizing enzyme are profoundly hypotensive under anesthesia.3 Thus, blood pressure is evidently regulated by both synthesis and turnover of SNOs. In this issue of Hypertension , Gandley et al4 extend this paradigm by proposing that a defect in SNO turnover contributes to the hypertension of preeclampsia. In the blood, S- nitrosoalbumin (SNO-albumin) and S -nitrosohemoglobin (SNO-Hb) constitute the major conduits for circulating NO bioactivity. Although both SNOs may influence blood pressure, they operate within distinct signaling circuits. SNO-Hb can be viewed as a principal regulator of SNO homeostasis, adaptively modulating NO chemistry to control NO bioactivity. SNO-Hb is formed by transfer of NO from heme-iron to Cysβ93 thiol on T to R structural transition (oxygenation) of the hemoglobin tetramer.5 SNO-Hb associates with the red blood cell (RBC) membrane via an interaction with the cytoplasmic domain of anion-exchanger 1 protein (CDAE1, Band 3); on deoxygenation (R→T) transfer of the NO group from SNO-Hb to a cysteine thiol within CDAE1 supports export of RBC vasodilatory activity.6 SNO-Hb thus serves as an O2 sensor and O2-dependent transducer of NO bioactivity. In contrast, it appears that rather than transducing a specific signal, albumin operates as a buffer to …

Oxygen Regulation of Tumor Perfusion by S-Nitrosohemoglobin Reveals a Pressor Activity of Nitric Oxide

In erythrocytes, S-nitrosohemoglobin (SNO-Hb) arises from S-nitrosylation of oxygenated hemoglobin (Hb). It has been shown that SNO-Hb behaves as a nitric oxide (NO) donor at low oxygen tensions. This property, in combination with oxygen transport capacity, suggests that SNO-Hb may have unique potential to reoxygenate hypoxic tissues. The present study was designed to test the idea that the allosteric properties of SNO-Hb could be manipulated to enhance oxygen delivery in a hypoxic tumor. Using Laser Doppler flowmetry, we showed that SNO-Hb infusion to animals breathing 21% O2 reduced tumor perfusion without affecting blood pressure and heart rate. Raising the pO2 (100% O2) slowed the release of NO bioactivity from SNO-Hb (ie, prolonged the plasma half-life of the SNO in Hb), preserved tumor perfusion, and raised the blood pressure. In contrast, native Hb reduced both tumor perfusion and heart rate independently of the oxygen concentration of the inhaled gas, and did not elicit hypertensive effects. Window chamber (to image tumor arteriolar reactivity in vivo) and hemodynamic measurements indicated that the preservation of tissue perfusion by micromolar concentrations of SNO-Hb is a composite effect created by reduced peripheral vascular resistance and direct inhibition of the baroreceptor reflex, leading to increased blood pressure. Overall, these results indicate that the properties of SNO-Hb are attributable to allosteric control of NO release by oxygen in central as well as peripheral issues.