Erythrocyte consumption of nitric oxide: competition experiment and model analysis.

It is generally believed that the erythrocyte membrane is highly permeable to nitric oxide (NO). To prevent NO from freely entering and being scavenged by the red blood cell (RBC), it has been suggested that NO consumption is limited by the mass transfer resistance of the diffusion layer adjacent to the erythrocyte membrane. Recently, we (Vaughn et al. (2000). J. Biol. Chem. 275, 2342) presented an experimental technique that overcomes experimental diffusional limitations and showed that RBCs also possess a mechanism to slow nitric oxide uptake. Here, we present a mathematical analysis of this technique by modeling the NO uptake of a single cell. We obtain additional data (n = 33, total) by use of the competition experiment and, through application of the model, show that either the RBC membrane permeability to NO or the intracellular reaction rate between NO and hemoglobin (Hb) is at least 2000-fold lower than previously thought. As a result, RBCs react with NO at a rate three orders of magnitude slower than free oxyHb. This phenomena may play an important role in NO bioavailability.     

Structure of nitric oxide hemoglobin

We have compared the structure of horse nitric oxide hemoglobin (HbNO) and methemoglobin in the oxy quaternary structure by difference Fourier analysis at 2.8 Å resolution. Both nitric oxide and oxygen assume bent co-ordination geometry and form low-spin complexes in binding to heme; on the basis of preferred ligand and heme stereochemistry, HbNO is the closest analog of HbO₂ (oxyhemoglobin) examined to date. To the resolution of the X-ray data, the stereochemistry of the heme-NO complex in hemoglobin and the corresponding free heme complex appears similar. In contrast, the ligand pockets in hemoglobin hinder binding of cyanide and carbon monoxide in their preferred linear axial co-ordination modes and force them to assume a strained off-axis binding stereochemistry. The structural similarity between HbNO and HbO₂ is reflected in their kinetic behavior, which is similar, and distinct from that of carboxyhemoglobin.     

Chain non-equivalence in nitric oxide binding to hemoglobin

The ratio of the apparent rates of ligand binding to the α and β subunits of human hemoglobin on mixing with non-saturating amounts of nitric oxide has been measured by two independent methods. Electron spin resonance measurements permit direct determination of the ratio of the amounts of the respective chains bound by NO. In stopped-flow kinetics measurements, use was made of the known difference in the kinetic constants of α and β chains in hemoglobin in the reaction with n-butyl isocyanide. Both methods concur in indicating that the apparent association rate constant of NO is greater for the α than for the β chain.     

Kinetics of nitric oxide binding to R-state hemoglobin

Despite earlier work indicating otherwise, some recent reports have suggested that nitric oxide (NO) binds to hemoglobin cooperatively. In particular, it has been suggested that, under physiological conditions, NO binds to the high-affinity R-state hemoglobin as much as 100 times faster than to the low-affinity T-state hemoglobin. This rapid NO binding could provide a means of preserving NO bioactivity. However, using a flash-flow photolysis technique, we have determined that the rate of NO binding to normal adult R-state hemoglobin is (2.1 +/- 0.1) x 10(7) (s(-1) M(-1)), which is essentially the same as that reported for T-state NO binding. (c)2002 Elsevier Science (USA).     

Properties of normal and glycated human hemoglobin in presence and absence of antioxidant

We present a novel approach to study properties of normal (HbA) and nonenzymatically glycated (HbA(Ic), HbA(Ia+b)) human hemoglobin using absorption spectroscopy and differential scanning calorimetry. The effect of the presence of the antioxidant fisetin on glycation of HbA is studied. Here, absorption spectroscopy has been fruitfully exploited to observe the formation of the glycated hemoglobin. With the differential scanning calorimetry, we studied the thermal unfolding of the protein hemoglobin at various conditions. The thermogram of the pure HbA showed two transition regions, with the occurrence of a partially unfolded intermediate state (the formation of which is mainly reversible) prior to complete denaturation (irreversible process). The denaturation temperature of HbA was found to be strongly dependent on the heating rate. Furthermore, there is a significant cooperativity between the two transition regions in pure HbA. The overall denaturation for the glycated hemoglobin takes place at a lower temperature, suggesting a decrease in the stability of the protein when it is glycated. In presence of fisetin, glycation is inhibited to a certain extent and the thermograms match well with that of normal HbA. Implications of the results are discussed.     

Cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease

Although the deleterious vasoconstrictive effects of cell-free, hemoglobin-based blood substitutes have been appreciated, the systemic effects of chronic hemolysis on nitric oxide bioavailability have not been considered or quantified. Central to this investigation is the understanding that nitric oxide reacts at least 1,000 times more rapidly with free hemoglobin solutions than with erythrocytes. We hypothesized that decompartmentalization of hemoglobin into plasma would divert nitric oxide from homeostatic vascular function. We demonstrate here that plasma from patients with sickle-cell disease contains cell-free ferrous hemoglobin, which stoichiometrically consumes micromolar quantities of nitric oxide and abrogates forearm blood flow responses to nitric oxide donor infusions. Therapies that inactivate plasma hemoglobin by oxidation or nitric oxide ligation restore nitric oxide bioavailability. Decompartmentalization of hemoglobin and subsequent dioxygenation of nitric oxide may explain the vascular complications shared by acute and chronic hemolytic disorders.     

Effects of nitric oxide on growth of maize seedling leaves in the presence or absence of ultraviolet-B radiation

The leaves of maize seedlings were used to measure leaf biomass including leaf length, width and weight, and to examine the relationship between nitric oxide (NO) synthase activity in microsomes and cytosol to the exo- and endo-beta-glucanase activity during growth. It was found that ultraviolet-B radiation (UV-B radiation) strongly induced nitric oxide synthase (NOS) activity but caused both a decrease of leaf biomass and exo- or endo-beta-glucanase activity. In contrast, the NOS inhibitor and NO donor largely decreased the activity of NOS in non-irradiated seedlings. The inhibitor also reduced exo- and endo-beta-glucanase activity and leaf biomass while the donor increased the enzyme activity and leaf biomass under normal conditions. Alternatively, under ultraviolet-B, the additional inhibitor of NOS and NO donor appeared to compromise the effects of ultraviolet-B on glucanase activity and leaf biomass, making the relationship between NOS activity and glucanase activity negatively correlated. This suggests that the changes of NOS activity showed a positive correlation to glucanase activity and leaf biomass in the absence of ultraviolet-B, but a negative correlation to ultraviolet-B irradiation and NO donor treatment alone. It is assumed that exo- and endogenous NO is responsible for the up-regulation of regular growth and development without ultraviolet-B. Under UV-B radiation, however, it might function as a signaling molecule of ultraviolet-B inhibiting leaf growth of maize seedlings to carry out stress-signaling transduction.     

Cooperativity in the dissociation of nitric oxide from hemoglobin.

The dissociation of nitric oxide from hemoglobin, from isolated subunits of hemoglobin, and from myoglobin has been studied using dithionite to remove free nitric oxide. The reduction of nitric oxide by dithionite has a rate of 1.4 X 10(3) M-1 S-1 at 20 degrees in 0.05 M phosphate, pH 7.0, which is small compared with the rate of recombination of hemoglobin with nitric oxide (25 X 10(6) M-1 S-1 (Cassoly, R., and Gibson, Q. H. (1975) J. Mol. Biol. 91, 301-313). The rate of NO combination with chains and myoglobin was found to be 24 X 10(6) M-1 S-1 and 17 X 10(6) M-1 S-1, respectively. Hence, the observed progress curve of the dissociation of nitric oxide is dependent upon the dithionite concentration and the total heme concentration. Addition of excess carbon monoxide to the dissociation mixture reduces the free heme yielding a single exponential process for chains and for myoglobin which is dithionite and heme concentration independent over a wide range of concentrations. The rates of dissociation of nitric oxide from alpha chains, from beta chains, and from myoglobin are 4.6 X 10(-5) S-1, 2.2 X 10(-5) S-1, and 1.2 X 10(4) S-1, respectively, both in the presence and in the absence of carbon monoxide at 20 degrees in 0.05 M phosphate, pH 7.0. Analogous heme and dithionite concentration dependence is found for the dissociation of nitric oxide from tetrameric hemoglobin. The reaction is cooperative, the intrinsic rate constants for the dissociation of the 1st and 4th molecules of NO differing about 100-fold. With hemoglobin, replacement of NO by CO at neutral pH is biphasic in phosphate buffers. The rate of the slow phase is 1 X 10(-5) S-1 and is independent of pH. The amplitude of the fast phase increases with lowering of pH. By analogy with the treatment of the HbCO + NO reaction given by Salhany et al. (Salhany, J.M., Ogawa, S., and Shulman, R.G. (1975) Biochemistry 14, 2180-2190), the fast phase is attributed to the dissociation of NO from T state molecules and the slow phase to dissociation from R state molecules. Analysis of the data gives a pH-independent value of 0.01 for the allosteric constant c (c = Kr/Kt where Kr and Kt are the dissociation constants for NO from the R and T states, respectively) and pH-dependent values of L (2.5 X 10(7) at pH 7 in 0.05 M phosphate buffer). The value of c is considerably greater than that for O2 and CO. Studies of the difference spectrum induced in the Soret region by inositol hexaphosphate are also reported. This spectrum does not arise directly from the change of conformation between R and T states. The results show that if the equilibrium binding curve for NO could be determined experimentally, it would show cooperativity with Hill's n at 50% saturation of about 1.6.     

Chain equivalence in reaction of nitric oxide with hemoglobin.

Mixtures of nitric oxide and hemoglobin were prepared in a rapid freeze apparatus and analyzed by EPR spectroscopy. Spectra from samples at various degrees of saturation showed that the two subunits bound NO at equal rates. Identical results were observed in 0.1 M phosphate at pH 6.5 and 0.1 M 2,2'-bis(hydroxymethyl)-2,2',2'-nitrilotriethanol, 0.1 M NaCl at pH 7.0, both in the presence and absence of inositol hexaphosphate at either buffer condition. At subsaturating levels of NO (less than 60%), or at all levels of saturation in the presence of inositol hexaphosphate, it was found that the EPR spectrum of nitrosylhemoglobin varied with the length of time before freezing. This change was characterized by the development of a hyperfine structure at g = 2.01 which appeared with a half-time of approximately 0.4 s. Maxwell and Caughey (Maxwell, J. C., and Caughey, W. S. (1976) Biochemistry 15, 388-395) have attributed this three-line EPR hyperfine structure to the formation of a pentacoordinate ferroheme-NO complex. Corresponding slow changes were observed in the visible absorption spectrum following the binding of low levels of NO to deoxyhemoglobin or inositol hexaphosphate to fully saturated nitrosylhemoglobin. Thus it appears that NO binding to the alpha and beta subunits of deoxyhemoglobin takes place at equal rates and, under conditions favoring the T quaternary state (low saturation, presence of inositol hexaphosphate), a further slow structural change takes place, resulting in the cleavage of the iron--proximal histidine bond.     

Computation of plasma hemoglobin nitric oxide scavenging in hemolytic anemias

Intravascular hemoglobin limits the amount of endothelial-derived nitric oxide (NO) available for vasodilation. Cell-free hemoglobin scavenges NO more efficiently than red blood cell-encapsulated hemoglobin. Hemolysis has recently been suggested to contribute to endothelial dysfunction based on a mechanism of NO scavenging by cell-free hemoglobin. Although experimental evidence for this phenomenon has been presented, support from a theoretical approach has, until now, been missing. Indeed, due to the low amounts of cell-free hemoglobin present in these pathological conditions, the role of cell-free hemoglobin scavenging of NO in disease has been questioned. In this study, we model the effects of cell-free hemoglobin on NO bioavailability, focusing on conditions that closely mimic those under known pathological conditions. We find that as little as 1 microM cell-free intraluminal hemoglobin (heme concentration) can significantly reduce NO bioavailability. In addition, extravasation of hemoglobin out of the lumen has an even greater effect. We also find that low hematocrit associated with anemia increases NO bioavailability but also leads to increased susceptibility to NO scavenging by cell-free hemoglobin. These results support the paradigm that cell-free hemoglobin released into plasma during intravascular hemolysis in human disease contributes to the experimentally observed reduction in NO bioavailability and endothelial dysfunction.     

Alkalin Bohr effect of nitric oxide binding by hemoglobin

The alkaline Bohr effect of nitric oxide binding by hemoglobin has been determined by differnetial titration. Binding of nitric oxide releases 2.6 protons per hemoglobin tetramer.     

The role of nitric oxide and hemoglobin in plant development and morphogenesis

Plant morphogenesis is regulated endogenously through phytohormones and other chemical signals, which may act either locally or distant from their place of synthesis. Nitric oxide (NO) is formed by a number of controlled processes in plant cells. It is a central signaling molecule with several effects on control of plant growth and development, such as shoot and root architecture. All plants are able to express non‐symbiotic hemoglobins at low concentration. Their function is generally not related to oxygen transport or storage; instead they effectively oxidize NO to NO₃^? and thereby control the local cellular NO concentration. In this review, we analyze available data on the role of NO and plant hemoglobins in morphogenetic processes in plants. The comparison of the data suggests that hemoglobin gene expression in plants modulates development and morphogenesis of organs, such as roots and shoots, through the localized control of NO, and that hemoglobin gene expression should always be considered a modulating factor in processes controlled directly or indirectly by NO in plants.     

Erythrocyte nitric oxide transport reduced by a submembrane cytoskeletal barrier

Encapsulation of hemoglobin (Hb) within red blood cells (RBCs) preserves nitric oxide (NO) bioactivity. With encapsulation, millimolar concentrations of Hb quench only a fraction of NO bioactivity, whereas mere micromolar concentrations of cell-free Hb completely quench NO bioactivity. A submembrane cytoskeletal barrier has been hypothesized to account for the lowered quenching of NO bioactivity. In order to substantiate this hypothesis, here, the underlying submembrane cytoskeletal barrier was physically reduced and the rate of NO entry into the modified RBC measured. The submembrane cytoskeletal barrier of normal and depleted RBCs was characterized using atomic force microscopy and the lipid to protein ratio measured. The reduction in the submembrane cytoskeletal barrier resulted in an increase in the rate of NO entry. We suggest that the underlying submembrane cytoskeleton may be a key component of RBC mediated regulation of NO bioavailability.     

The involvement of nitric oxide in formation of hemoglobin oxygen-binding properties

The analysis of literature and results of our investigations indicate the possible involvement of L-arginine-nitric oxide (NO) system in formation of blood oxygen-carrying capacity. In reaction with hemoglobin NO forms methemoglobin, nitrosyl-hemoglobin (HbFe2+NO) and S-nitrosohemoglobin (SNO-Hb). The NO-hemoglobin derivatives have the various biological functions (NO transport, storage, elimination etc.) and are involved in the genesis of different pathologic conditions. The presence of different NO-hemoglobin derivatives can differently influence on the whole blood hemoglobin-oxygen affinity (HOA): methemoglobin and SNO-Hb increases, and HbFe2+NO decreases it. Their effect on the blood oxygen-binding properties may be important for the gas exchange processes. At the level of lung capillaries such effect may be the additional mechanism promoting a blood oxygenation, and in the systemic microcirculation it may optimize blood desaturation and hence the tissue oxygen delivery. Blood oxygen-binding properties affect the state of L-arginine-NO system, however this system also may determine HOA through the intraerythrocytic regulatory mechanisms, oxygen-dependent nature of NO generation, regulation of vascular tone and effect of peroxynitrite.     

Reductive nitrosylation and S-nitrosation of hemoglobin in inhomogeneous nitric oxide solutions.

Elucidating the reaction of nitric oxide (NO) with oxyhemoglobin [HbFe(II)O2] is critical to understanding the metabolic fate of NO in the vasculature. At low concentrations of NO, methemoglobin [HbFe(III)] is the only detectable product from this reaction; however, locally high concentrations of NO have been demonstrated to result in some iron-nitrosylhemoglobin [HbFe(II)NO] and S-nitrosohemoglobin (SNO-Hb) formation. Reductive nitrosylation through a HbFe(III) intermediate was proposed as a viable pathway under such conditions. Here, we explore another potential mechanism based on mixed valenced Hb tetramers. The oxidation of one or two heme Fe(II) in the R-state HbFe(II)O2 has been observed to lower the oxygen affinity of the remaining heme groups, thus creating the possibility of oxygen release and NO binding at the heme Fe(II) sites. This mixed valenced hypothesis requires an allosteric transition of the Hb tetramer. Hence, this hypothesis can account for HbFe(II)NO formation, but not SNO-Hb formation. Here, we demonstrate that cyanide attenuated the formation of SNO-Hb by 30-40% when a saturated NO bolus was added to 0.1-1.0 mM HbFe(II)O2 solutions. In addition, HbFe(II)NO formation under such inhomogeneous conditions does not require allostericity. Therefore, we concluded that the mixed valenced theory does not play a major role under these conditions, and reductive nitrosylation accounts for a significant fraction of the HbFe(II)NO formed and approximately 30-40% of SNO-Hb. The remaining SNO-Hb is likely formed from NO oxidation products.     

Effect of dietary nitric oxide on gastric mucosal mast cells in absence or presence of an experimental gastritis in rats

Synthetic nitric oxide donors are known to protect the gastric mucosa from damage and dietary nitrate is known to release NO in the stomach. Mast cells have been found to be involved in gastric mucosal damage in humans or in rodents, and recent studies have pointed out the possibility of nitric oxide from endogenous or exogenous origin to modulate mast cell reactivity. This study aimed to determine whether the protective effect afforded by dietary nitrate against gastric mucosal damage was linked to mast cell stabilization. Mast cell involvement in iodoacetamide-induced gastritis was investigated in rats receiving oral administration of iodoacetamide together with the mast cell stabilizer doxantrazole (ip) or its solvent. The effects of dietary nitrate on mast cells during gastritis were investigated in rats receiving iodoacetamide orally, associated or not with KNO3. Control groups were given water instead of iodoacetamide either with or without KNO3, doxantrazole or its solvent. After sacrifice, blood samples were taken to determine RMCP II serum level and the stomach was resected in order to determine myeloperoxidase (MPO) activity and mucosal mast cell (MMC) number. Iodoacetamide significantly increased gastric MPO activity but did not modify RMCP II serum level or MMC number. Doxantrazole and KNO3 significantly reduced iodoacetamide-induced increase in gastric MPO activity, increased MMC number, and decreased RMCP II serum level in basal conditions. Only doxantrazole was able to modify all parameters under inflammatory conditions. These results suggest that nitric oxide released by dietary nitrate in the stomach stabilizes mast cells in basal conditions but exerts its protective effect against experimental gastritis through other pathways.