Hypoxic Vasodilation Research Articles

Impaired vasodilation by red blood cells in sickle cell disease

Red blood cells (RBCs) have been ascribed a unique role in dilating blood vessels, which requires O2-regulated binding and bioactivation of NO by Hb and transfer of NO equivalents to the RBC membrane. Vasoocclusion in hypoxic tissues is the hallmark of sickle cell anemia. Here we show that sickle cell Hb variant S (HbS) is deficient both in the intramolecular transfer of NO from heme iron (iron nitrosyl, FeNO) to cysteine thiol (S-nitrosothiol, SNO) that subserves bioactivation, and in transfer of the NO moiety from S-nitrosohemoglobin (SNO-HbS) to the RBC membrane. As a result, sickle RBCs are deficient in membrane SNO and impaired in their ability to mediate hypoxic vasodilation. Further, the magnitudes of these impairments correlate with the clinical severity of disease. Thus, our results suggest that abnormal RBC vasoactivity contributes to the vasoocclusive pathophysiology of sickle cell anemia, and that the phenotypic variation in expression of the sickle genotype may be explained, in part, by variable deficiency in RBC processing of NO. More generally, our findings raise the idea that defective NO processing may characterize a new class of hemoglobinopathy.

Does endogenous fatty acid metabolism allow cancer cells to sense hypoxia and mediate hypoxic vasodilatation? Characterization of a novel molecular connection between fatty acid synthase (FAS) and hypoxia‐inducible factor‐1α (HIF‐1α)‐related expression of vascular endothelial growth factor (VEGF) in cancer cells overexpressing her‐2/neu oncogene

Her-2/neu (erbB-2) oncogene overexpression is associated with increased tumor progression and metastasis. Fatty acid synthase (FAS), the key lipogenic enzyme responsible for the endogenous synthesis of fatty acids, has been shown to be one of the genes regulated by Her-2/neu at the level of transcription, translation, and biosynthetic activity. Interestingly, we recently established that both pharmacological inhibition of FAS activity and silencing of FAS gene expression specifically suppress Her-2/neu oncoprotein expression and tyrosine-kinase activity in breast and ovarian Her-2/neu overexpressors. Unraveling the functional organization of this novel bi-directional molecular connection between Her-2/neu and FAS-dependent neoplastic lipogenesis is a major challenge that the field is only beginning to take on. Considering that Her-2/neu overexpression correlates with increased expression of the hypoxia inducible factor-1alpha (HIF-1alpha), which, in a mitogen-activated protein kinase (MAPK)-dependent manner, plays a key role in the expression of several genes including cytokines such as vascular endothelial growth factor (VEGF), we hypothesized that FAS blockade should result in a concomitant down-regulation of VEGF. Unexpectedly, the specific inhibition of the de novo fatty acid synthesis with the small-molecule inhibitor of FAS activity C75 resulted in a dramatic dose-dependent enhancement (up to 500% increase) of VEGF secretion in Her-2/neu-overexpressing SK-Br3, BT-474, and SKOV3 cancer cells. Concurrently, FAS blockade drastically activated MAPK and promoted further a prominent accumulation of HIF-1alpha in Her-2/neu overexpressors. Moreover, U0126-induced inhibition of MAPK activity completely abolished C75-induced up-regulation of HIF-1alpha expression and VEGF secretion, whereas it did not modulate C75-induced down-regulation of Her-2/neu oncogene. Importantly, RNA interference (RNAi)-mediated silencing of the FAS gene recapitulated C75's effects by up-regulating VEGF secretion, MAPK activation and HIF-1alpha expression. Therefore, it appears that perturbation of cancer-associated endogenous fatty metabolism triggers a "hypoxia-like" (oxygen-independent) condition that actively rescues Her-2/neu-dependent MAPK --> HIP-1alpha --> VEGF cascade. It is tempting to suggest that an intact FAS-catalyzed endogenous fatty acid metabolism is a necessary metabolic adaptation to support the enhanced ability of Her-2/neu-overexpressing cancer cells to survive cellular hypoxia in a HIF-alpha-dependent manner.

Assessments of the chemistry and vasodilatory activity of nitrite with hemoglobin under physiologically relevant conditions

Hypoxic vasodilation involves detection of the oxygen content of blood by a sensor, which rapidly transduces this signal into vasodilatory bioactivity. Current perspectives on the molecular mechanism of this function hold that hemoglobin (Hb) operates as both oxygen sensor and a condition-responsive NO reactor that regulates the dispensing of bioactivity through release of the NO group from the β-cys93 S-nitroso derivative of Hb, SNO-Hb. A common path to the formation of SNO-Hb involves oxidative transfer of the NO-group from heme to thiol. We have previously reported that the reaction of nitrite with deoxy-Hb, which furnishes heme-Fe(II)NO, represents one attractive route for the formation of SNO-Hb. Recent literature, however, posits that the nitrite-reductase reaction of Hb might produce physiological vasodilatory effects through NO that evades trapping on heme-Fe(II) and may be stored before release as Fe(III)NO. In this article, we briefly review current perspectives in NO biology on the nitrite-reductase reaction of Hb. We report in vitro spectroscopic (UV/Vis, EPR) studies that are difficult to reconcile with suggestions that this reaction either generates a heme-Fe(III)NO reservoir or significantly liberates NO. We further show in bioassay experiments that combinations of nitrite and deoxy-Hb – under conditions that suppress SNO-Hb formation – exhibit no direct vasodilatory activity. These results help underscore the differences between physiological, RBC-regulated, hypoxic vasodilation versus pharmacological effects of exogenous nitrite.

Nitric Oxide (NO) in normal and hypoxic vascular regulation of the spiny dogfish, Squalus acanthias

Nitric oxide (NO) is a potent vasodilator in terrestrial vertebrates, but whether vascular endothelial-derived NO plays a role in vascular regulation in fish remains controversial. To explore this issue, a study was made of spiny dogfish sharks (Squalus acanthias) in normoxia and acute hypoxia (60 min exposure to seawater equilibrated with 3% oxygen) with various agents known to alter NO metabolism or availability. In normoxia, nitroprusside (a NO donor) reduced blood pressure by 20%, establishing that vascular smooth muscle responds to NO. L-arginine, the substrate for NO synthase, had no hemodynamic effect. Acetylcholine, which stimulates endothelial NO and prostaglandin production in mammals, reduced blood pressure, but also caused marked bradycardia. L-NAME, an inhibitor of all NO synthases, caused a small 10% rise in blood pressure, but cell-free hemoglobin (a potent NO scavenger and hypertensive agent in mammals) had no effect. Acute hypoxia caused a 15% fall in blood pressure, which was blocked by L-NAME and cell-free hemoglobin. Serum nitrite, a marker of NO production, rose with hypoxia, but not with L-NAME. Results suggest that NO is not an endothelial-derived vasodilator in the normoxic elasmobranch. The hypertensive effect of L-NAME may represent inhibition of NO production in the CNS and nerves regulating blood pressure. In acute hypoxia, there is a rapid up-regulation of vascular NO production that appears to be responsible for hypoxic vasodilation.

The reaction between nitrite and hemoglobin: the role of nitrite in hemoglobin-mediated hypoxic vasodilation

The reaction between nitrite and hemoglobin has been studied for over a century. However, recent evidence indicating nitrite is a latent vasodilatory agent that can be activated by its reaction with deoxyhemoglobin has led to renewed interest in this reaction. In this review we survey, in the context of our own recent studies, the chemical reactivity of nitrite with oxyhemoglobin, deoxyhemoglobin and methemoglobin, and place these reactions in both a physiological and pharmacological/therapeutic context.

Hypoxic Vasodilation by Red Blood Cells and Impairment in Vascular Disorders.

Physiological O2 gradients are principal regulators of blood flow in the microcirculation: position-to-position changes in hemoglobin (Hb) O2 saturation are coupled to regulated vasodilation (“hypoxic vasodilation”). The mechanism by which graded changes in O2 content of blood evoke this response has been a great challenge to understand. A new role for red blood cells (RBCs) in hypoxic dilation of blood vessels and inhibition of platelet activation involving release of nitric oxide (NO) bioactivity is described. We show that NO groups can be transferred within hemoglobin (Hb) from hemes to highly-conserved cysteine thiols (β-Cys93) to form bioactive S-nitrosohemoglobin (SNO-Hb), and that efficient production of SNO-Hb requires selective processing of NO within the β-subunits. Bioactive SNO-Hb is localized primarily to the RBC membrane through interaction with Band 3, the transmembrane anion-exchanger 1 protein (AE1). Upon deoxygenation, transfer of the NO group from β-Cys93 of Hb to a cysteine thiol within AE1 serves the RBC vasodilator activity. In this way, O2 binding in Hb modulates the release of NO bioactivity. We further show that RBC NO bioactivity is inversely proportional to pO2 and impaired in disease. In an aortic ring bioassay sparged with variable concentrations of O2, addition of normal human RBCs elicited graded responses from relaxation at tissue pO2 (~3–7 mm Hg, hypoxic vasodilation), to loss of relaxation and progressively greater contractions at pO2's of 10–63 mm Hg (hyperoxic vasoconstriction). Notably, RBC SNO-Hb levels and hypoxic vasodilation are impaired in several diseases characterized by vascular dysfunction. For example, in RBCs from patients with pulmonary arterial hypertension (PAH), we found decreased (13% of control) SNO-Hb content (assessed by photolysis-chemiluminescence) and impaired O2-dependent vasodilation (bioassay). RBCs from patients with other ischemic disorders have also been examined: RBCs demonstrate a pathogenesis-based impairment in their ability to mediate hypoxic vasodilation by NO. These results confirm the (patho)physiologic importance of RBC NO, and suggest that RBC dysfunction may contribute to impaired blood flow in diseases of the heart, lung and blood.

Characterization of the Distribution of Nitrite in Human Blood.

Several groups have recently shown that blood can transport nitric oxide (NO) bioactivity so that NO can act as an endocrine signal transducer. The mechanisms of this process are uncertain. We have recently shown that nitrite ions can generate NO in the presence of deoxygenated hemoglobin (Cosby et al. Nature Medicine 2003). Thus, nitrite appears to be a major intravascular NO storage molecule and hemoglobin a nitrite reductase contributing to erythrocyte-dependent hypoxic vasodilation. Such a model requires a reaction of nitrite with intra-erythrocytic hemoglobin, however, published uptake rates of nitrite from plasma into erythrocytes are very slow and dynamic blood flow regulation requires more rapid responses. We therefore hypothesized that the red blood cell itself might be a reservoir for nitrite, with compartmentalization of nitrite and deoxyhemoglobin at the erythrocyte membrane; such a metabolon would allow for rapid nitrite reductase chemistry and NO export as hemoglobin deoxygenates. Nitrite was measured using reductive chemiluminescence in conjunction with a novel ferricyanide-based hemoglobin oxidation assay to rapidly preserve nitrite in the presence of hemoglobin following red cell lysis. We find that measurement of whole blood nitrite using ferricyanide-based hemoglobin oxidation to stabilize nitrite is a valid, sensitive, linear, and reproducible surrogate for RBC nitrite. Using the stabilization solution, nitrite in whole blood remained constant over 12 h at room temperature, whereas without it the half life of nitrite was 12 ± 4 min. Using this novel methodology, the nitrite levels measured in plasma, RBC, and calculated and measured in whole blood in normal volunteers were 121 ± 9, 620 ± 170, 245 ± 58, and 323 ± 44 nmol/L. The difference between measured and calculated blood nitrite levels reflects destruction of RBC nitrite by hemoglobin during sample processing. There was an artery-to-vein gradient for blood nitrite (A. vs V.: 169 ± 12 vs. 149 ± 11 nmol/L; p=0.03). Intraperitoneal injection of L-NAME (a potent NOS-inhibitor; 100 mg/kg) in mice caused a time dependent decrease of whole blood nitrite (0 h: 650 ± 29, 2h: 556 ± 26, 4h: 455 ± 11, 421 ± 19, 24 h: 551 ± 30 nmol/L; p=0.002). Differences of blood nitrite across the human forearm circulation correlated significantly (r2=0.94) with forearm bloodflow after infusion of acetylcholine (7.5 ug/min; n=5), consistent with nitrite formation across the vasculature with eNOS activation. We conclude that in humans, the RBC nitrite concentration is 5 times greater than plasma nitrite concentrations; A-V gradients in blood nitrite are consistent with nitrite utilization as a source of bioactive NO in the microvasculature and levels of blood nitrite are controlled by endogenous NO production. Levels of nitrite in the RBC may function as a locally available source of nitrite for deoxyhemoglobin-dependent nitrite reduction to NO, thus allowing for more efficient nitrite-deoxyhemoglobin mediated hypoxic vasodilation. Modulation of intraerythrocytic nitrite may be of therapeutic interest in diseases with a lack of bioavailable NO, such as sickle cell disease.

Contractile responses of isolated rat mesenteric arteries to acute episodes of severe hypoxia and subsequent reoxygenation

This study further investigates the mechanisms responsible for the effects of acute and severe hypoxia, and subsequent reoxygenation, on the contractility of isolated rat mesenteric arteries. In noradrenaline (NA)-contracted arteries, hypoxia caused a relaxation to near baseline levels. Reoxygenation resulted in an immediate transient contraction before tension returned more slowly to prehypoxia levels. Similar responses to hypoxia were observed in tissues precontracted by addition of KCl (60 mM) or U46619 (10 μM); however, the transient contraction upon reoxygenation was absent (KCl) or reduced (U46619). Responses to hypoxia were independent of changes in intracellular calcium ([Ca 2+] i), while those to reoxygenation were accompanied by corresponding changes in [Ca 2+] i and were completely abolished by ryanodine. In NA-contracted tissues, all responses were unaffected by endothelial removal or by inhibitors of nitric oxide synthase and cyclooxygenase. The K + channel blockers triethylamine (TEA), glibenclamide, and 4-aminopyridine (4-AP) had no effect on the responses to hypoxia. The transient contractile response to reoxygenation was, however, significantly reduced in the presence of 4-AP. The response to reoxygenation, but not that to hypoxia, was inhibited by the antioxidant dithiothreitol (DTT) and the NAD(P)H-oxidase inhibitor diphenyliodonium (DPI). These data suggest that hypoxic vasodilation occurs independently of reductions in [Ca 2+] i. Alternatively, transient contractions on reoxygenation are dependent upon the generation of reactive oxygen species and the release of stored Ca 2+.

Insulin increases glycolysis without further vasodilation in porcine coronary arteries exposed to hypoxia.

In acute ischaemia, glucose-insulin-potassium administration reduces mortality and beta-adrenoceptor antagonists have favourable effects on the outcome of ischaemic heart disease. The present study was designed to investigate whether insulin (1.4x10(-7) M) and the beta-adrenoceptor antagonist, propranolol (10(-5) M), increase hypoxic vasodilation in correspondence with changes in glycolysis. Porcine coronary arteries, precontracted with 10(-5) M prostaglandin F(2alpha), were mounted in a pressure myograph and a microdialysis catheter was inserted in the tunica media. Hypoxic vasodilation, interstitial lactate/pyruvate ratio and interstitial glucose were measured at low (2 mM) and high (20 mM) glucose concentrations. Hypoxia (60 min) caused vasodilation and doubled the lactate/pyruvate ratio. Treatment with insulin quadrupled the lactate/pyruvate ratio during hypoxia, but did not change hypoxic vasodilation. Propranolol blocked isoprenaline-evoked vasodilation, but hypoxic increases in lactate/pyruvate ratio and vasodilation did not change. The combination of insulin and propranolol did not cause further changes compared with each drug added alone, although the combination increased vasoconstriction during reoxygenation. Interstitial glucose fell during hypoxia at an organ bath glucose concentration of 2 mM, and rose at a glucose concentration of 20 mM. Addition of insulin and propranolol alone or in combination had no effect on interstitial glucose concentration. Accordingly, arteries were found to contain only minute amounts of the glucose transporter isoform GLUT4. Our findings suggest that insulin increases arterial glycolysis, but treatment with insulin, propranolol, or both, is not associated with enhanced coronary vasodilation during hypoxia.

Red blood cell nitric oxide as an endocrine vasoregulator: a potential role in congestive heart failure.

A respiratory cycle for nitric oxide (NO) would involve the formation of vasoactive metabolites between NO and hemoglobin during pulmonary oxygenation. We investigated the role of these metabolites in hypoxic tissue in vitro and in vivo in healthy subjects and patients with congestive heart failure (CHF). We investigated the capacity for red blood cells (RBCs) to dilate preconstricted aortic rings under various O2 tensions. RBCs induced cyclic guanylyl monophosphate-dependent vasorelaxation during hypoxia (35+/-4% at 1% O2, 4.7+/-1.6% at 95% O2; P<0.05). RBC-induced relaxations during hypoxia correlated with S-nitrosohemoglobin (SNO-Hb) (R2=0.88) but not iron nitrosylhemoglobin (HbFeNO) content. Relaxation responses for RBCs were compared with S-nitrosoglutathione across a range of O2 tensions. The fold increases in relaxation evoked by RBCs were significantly greater at 1% and 2% O2 compared with relaxations induced at 95% (P<0.05), consistent with an allosteric mechanism of hypoxic vasodilation. We also measured transpulmonary gradients of NO metabolites in healthy control subjects and in patients with CHF. In CHF patients but not control subjects, levels of SNO-Hb increase from 0.00293+/-0.00089 to 0.00585+/-0.00137 mol NO/mol hemoglobin tetramer (P=0.005), whereas HbFeNO decreases from 0.00361+/-0.00109 to 0.00081+/-0.00040 mol NO/mol hemoglobin tetramer (P=0.03) as hemoglobin is oxygenated in the pulmonary circulation. These metabolite gradients correlated with the hemoglobin O2 saturation gradient (P<0.05) and inversely with cardiac index (P<0.05) for both CHF patients and control subjects. We confirm that RBC-bound NO mediates hypoxic vasodilation in vitro. Transpulmonary gradients of hemoglobin-bound NO are evident in CHF patients and are inversely dependent on cardiac index. Hemoglobin may transport and release NO bioactivity to areas of tissue hypoxia or during increased peripheral oxygen extraction via an allosteric mechanism.

Haldane, hot dogs, halitosis, and hypoxic vasodilation: the emerging biology of the nitrite anion

While it has long been known that the reduction of nitrite to nitric oxide (NO) forms iron-nitrosyl-myoglobin and is the basis of meat curing, a greater biological activity of the nitrite anion has only recently been appreciated. In the stomach, NO is formed from acidic reduction of nitrite and increases mucous barrier thickness and gastric blood flow (see the related study beginning on page 106). Nitrite levels in blood reflect NO production from endothelial NO synthase enzymes, and recent data suggest that nitrite contributes to blood flow regulation by reaction with deoxygenated hemoglobin and tissue heme proteins to form NO.

Locally released norepinephrine in the oxygen-dependent regulation of vascular tone of human umbilical vein.

In a previous study, human umbilical vein preparations constricted at PO(2) values above the physiologic intrauterine PO(2) range and dilated at hypoxia. Denudation of the endothelium reversed the hypoxic vasodilatation only, suggesting the release of a nonendothelial vasoconstrictor. We therefore hypothesized that norepinephrine from adrenergic nerve terminals could be responsible for the observed constricting effects. We measured intracellular membrane potential and isometric tension of human umbilical vein strips with and without functional adrenergic denervation by 6-OH-dopamine during variation of PO(2). With PO(2) increasing from 5 to 104 mm Hg, intact preparations depolarized of from -58.6 +/- 1.1 mV (SEM) to -53.3 +/- 1.0 mV (p < 0.001) and isometric tension increased from 0.673 +/- 0.037 g to 0.825 +/- 0.044 g (p < 0.02). Intact preparations pretreated with 6-OH-dopamine depolarized from -58.0 +/- 0.5 mV to -55.8 +/- 0.6 mV (p < 0.01), and isometric tension increased from 0.598 +/- 0.040 g to 0.661 +/- 0.018 g (p < 0.02). At Po(2) values above the physiologic intrauterine umbilical venous Po(2) range, membrane potential and isometric tension were significantly lower in preparations with 6-OH-dopamine pretreatment compared with matched controls (p < 0.05). Denudation of the endothelium reversed the hypoxic hyperpolarization and vasodilatation observed in intact preparations. However, membrane potential and isometric tension were not different between endothelium-denuded preparations with and without 6-OH-dopamine pretreatment. We conclude that locally released norepinephrine contributes to the depolarization and vasoconstriction of the human umbilical vein at hyperoxia but does not antagonize the endothelium-dependent vasodilation at hypoxia.

Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation.

Nitrite anions comprise the largest vascular storage pool of nitric oxide (NO), provided that physiological mechanisms exist to reduce nitrite to NO. We evaluated the vasodilator properties and mechanisms for bioactivation of nitrite in the human forearm. Nitrite infusions of 36 and 0.36 micromol/min into the forearm brachial artery resulted in supra- and near-physiologic intravascular nitrite concentrations, respectively, and increased forearm blood flow before and during exercise, with or without NO synthase inhibition. Nitrite infusions were associated with rapid formation of erythrocyte iron-nitrosylated hemoglobin and, to a lesser extent, S-nitroso-hemoglobin. NO-modified hemoglobin formation was inversely proportional to oxyhemoglobin saturation. Vasodilation of rat aortic rings and formation of both NO gas and NO-modified hemoglobin resulted from the nitrite reductase activity of deoxyhemoglobin and deoxygenated erythrocytes. This finding links tissue hypoxia, hemoglobin allostery and nitrite bioactivation. These results suggest that nitrite represents a major bioavailable pool of NO, and describe a new physiological function for hemoglobin as a nitrite reductase, potentially contributing to hypoxic vasodilation.

Prostaglandins.

Prostaglandins.

Hypoxic regulation of blood flow in humans. Skin blood flow and temperature regulation.

Regulation of cutaneous vascular tone in humans is complex due to the different types of skin in various regions of the body and the vast array of nerves involved in regulation of blood flow. Due to these complexities, it is unclear how the cutaneous vasculature responds to hypoxia. There are reports of exaggerated vasoconstriction and vasodilation, while others suggest the skin is unresponsive to a hypoxic stimulus. Preliminary work in our laboratory suggests hypoxic vasodilation may be unmasked with alpha-receptor blockade. In contrast to skeletal muscle, hypoxic cutaneous vasodilation is not blunted by beta-blockade, but may be abolished with NO-synthase inhibition. Furthermore, effects of hypoxia on skin blood flow may be more pronounced during combined hypoxic and thermoregulatory challenges. Along these lines, overall thermoregulation may be impacted by hypoxic effects on the cutaneous vasculature and hypobaric effects on sweating and evaporation. During supine heat stress, for example, skin blood flow can increase to 8 Liters per minute. This dramatic rise in skin blood flow is accomplished by an increase in cardiac output and redistribution of blood flow from the splanchnic and renal vascular beds. During hypoxia, splanchnic blood flow has been shown to increase. Thus, during a hypoxic challenge in the heat, a competition for blood flow between the compliant skin and splanchnic regions must exist, but is not well understood. In this review, the effects of hypoxia on the regulation of cutaneous vascular tone and the impact on temperature regulation will be discussed.

Does nitric oxide allow endothelial cells to sense hypoxia and mediate hypoxic vasodilatation? In vivo and in vitro studies.

Hypoxia-evoked vasodilatation is a fundamental regulatory mechanism that is often attributed to adenosine. The identity of the O(2) sensor is unknown. Nitric oxide (NO) inhibits endothelial mitochondrial respiration and ATP generation by competing with O(2) for its binding site on cytochrome oxidase. We proposed that in vivo this interaction allows endothelial cells to release adenosine when O(2) tension falls or NO concentration increases. Using anaesthetised rats, we confirmed that the increase in femoral vascular conductance (FVC, hindlimb vasodilatation) evoked by systemic hypoxia is attenuated by NO synthesis blockade with L-NAME, but restored when baseline FVC is restored by infusion of NO donor. This "restored" hypoxic response, like the control hypoxic response, is inhibited by the adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. Similarly, the FVC increase evoked by adenosine infusion was attenuated by L-NAME but restored by infusion of NO donor. However, when baseline FVC was restored after L-NAME with 8-bromo-cGMP, the FVC increase evoked by adenosine infusion was restored, but not in response to systemic hypoxia, suggesting that adenosine was no longer released by hypoxia. Infusion of NO donor at a given rate after treatment with L-NAME evoked a greater FVC increase during systemic hypoxia than during normoxia, both responses being reduced by 8-cyclopentyl-1,3-dipropylxanthine. Finally, both bradykinin and NO donor released adenosine from superfused endothelial cells in vitro; L-NAME attenuated only the former response. We propose that in vivo, shear-released NO increases the apparent K(m) of endothelial cytochrome oxidase for O(2), allowing the endothelium to act as an O(2) sensor, releasing adenosine in response to moderate falls in O(2).

Nitric oxide in the human respiratory cycle.

Interactions of nitric oxide (NO) with hemoglobin (Hb) could regulate the uptake and delivery of oxygen (O(2)) 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 HbO(2) saturation (FeO(2)). Moreover, we show that red blood cell (RBC)/SNO-mediated vasodilator activity is inversely proportional to FeO(2) over a wide range, whereas RBC-induced vasoconstriction correlates directly with FeO(2). Thus, native RBCs respond to changes in oxygen tension (pO2) with graded vasodilator and vasoconstrictor activity, which emulates the human physiological response subserving O(2) uptake and delivery. The ability to monitor and manipulate blood levels of NO, in conjunction with O(2) 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.

Cerebrovascular effects of carbon monoxide.

This review examines the influence of endogenous and exogenous carbon monoxide (CO) on the cerebral circulation. Although CO generated from neuronal heme oxygenase can modulate neurotransmission, evidence supporting its role in cerebral vasodilation is limited. In newborn piglets, heme oxygenase is enriched in microvessels and contributes to hypoxic vasodilation. Low CO concentrations dilate piglet arterioles by opening calcium-activated potassium channels. With inhalation of CO and formation of carboxyhemoglobin, cerebral vasodilation can be greater than that occurring with hypoxic hypoxia at equivalent reductions of arterial oxygen content. This additional vasodilation is probably attributable to additional release of hypoxic vasodilators secondary to increased oxyhemoglobin affinity, although direct effects of CO on cerebral arterioles may also occur. When CO exposure is prolonged, cerebral endothelium undergoes oxidant stress as evident by nitrotyrosine formation. As CO levels increase, modest decreases in oxygen consumption are detectable, which may reflect CO or nitric oxide interactions with cytochrome oxidase in regions with very low oxygen availability. If subsequent CO concentration increases sufficiently to depress cardiac function and limit cerebral perfusion, cerebral oxygen consumption becomes further reduced, and oxidant stress becomes amplified by leukocyte sequestration and xanthine oxidase activity with consequent lipid peroxidation. Specific regions of the brain, such as central white matter, globus pallidus, and hippocampus, are selectively vulnerable to CO toxicity, but whether the mechanisms involved in selective injury differ from other forms of hypoxia-ischemia needs to be clarified.

Effects of regional phentolamine on hypoxic vasodilatation in healthy humans.

1. Limb vascular beds exhibit a graded dilatation in response to hypoxia despite increased sympathetic vasoconstrictor nerve activity. We investigated the extent to which sympathetic vasoconstriction can mask hypoxic vasodilatation and assessed the relative contributions of beta-adrenergic and nitric oxide (NO) pathways to hypoxic vasodilatation. 2. We measured forearm blood flow responses (plethysmography) to isocapnic hypoxia (arterial saturation approximately 85%) in eight healthy men and women (18-26 years) after selective alpha-adrenergic blockade (phentolamine) of one forearm. Subsequently, we measured hypoxic responses after combined alpha- and beta-adrenergic blockade (phentolamine and propranolol) and after combined alpha- and beta-adrenergic blockade coupled with NO synthase inhibition (N(G)-monomethyl-L-arginine, L-NMMA). 3. Hypoxia increased forearm vascular conductance by 49.0 +/- 13.5% after phentolamine (compared to +16.8 +/- 7.0% in the control arm without phentolamine, P < 0.05). After addition of propranolol, the forearm vascular conductance response to hypoxia was reduced by approximately 50%, but dilatation was still present (+24.7 +/- 7.0%, P < 0.05 vs. normoxia). When L-NMMA was added, there was no further reduction in the forearm vascular conductance response to hypoxia (+28.2 +/- 4.0%, P < 0.05 vs. normoxia). 4. Thus, selective regional alpha-adrenergic blockade unmasked a greater hypoxic vasodilatation than occurs in the presence of functional sympathetic nervous system responses to hypoxia. Furthermore, approximately half of the hypoxic vasodilatation in the forearm appears to be mediated by beta-adrenergic receptor-mediated pathways. Finally, since considerable dilatation persists in the presence of both beta-adrenergic blockade and NO synthase inhibition, it is likely that an additional vasodilator mechanism is activated by hypoxia in humans.

Anaemia in end-stage renal disease: pathophysiological considerations

Anaemia is a frequent complication of many diseases but the mechanisms that link reduced blood oxygen content to the long-term consequences of anaemia are incompletely understood. The maintenance of oxygen supply to the tissues during anaemia involves complex cardiovascular adaptations, including an increase in cardiac output, reduced peripheral resistance and increased oxygen extraction from haemoglobin (Hb). In addition, hypoxia-inducible factors are associated with the transcriptional activation of genes involved in adaptive mechanisms that increase oxygen delivery and provide alternative metabolic pathways. The complex pathophysiology of chronic kidney disease alters the adaptations to anaemia in uraemic patients. The increased cardiac output induced by anaemia is associated with left ventricular hypertrophy and cardiac disease in renal patients. Alterations in endothelial cell function, common in renal disease, may diminish endothelium-induced vasodilatation, increase the risk of atherosclerosis and impair angiogenesis. Many potential reasons for erythropoietin-induced hypertension in uraemic patients have been postulated, including increased blood viscosity as haematocrit rises, a reversal of hypoxic vasodilatation, increased blood volume that is not compensated by haemodialysis, ultrafiltration and impaired nitric oxide synthesis, preventing vascular relaxation in response to increased blood viscosity. In view of this impaired vascular reactivity, rapid increases in haematocrit should be avoided during epoetin treatment. As the interaction between anaemia and uraemia is very complex, it is not possible to derive the optimal Hb concentration for individual patients by using simple physiological or pathophysiological models and there is a need for good randomized controlled clinical trials to address this issue.