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Am J Physiol Heart Circ Physiol 292: H3072-H3078, 2007. First published February 16, 2007; doi:10.1152/ajpheart.01298.2006
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Nitrite-dependent vasodilation is facilitated by hypoxia and is independent of known NO-generating nitrite reductase activities

Thomas Dalsgaard,1,2 Ulf Simonsen,2 and Angela Fago1

1Department of Biological Sciences and 2Department of Pharmacology, University of Aarhus, Aarhus, Denmark

Submitted 28 November 2006 ; accepted in final form 15 February 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The reduction of circulating nitrite to nitric oxide (NO) has emerged as an important physiological reaction aimed to increase vasodilation during tissue hypoxia. Although hemoglobin, xanthine oxidase, endothelial NO synthase, and the bc1 complex of the mitochondria are known to reduce nitrite anaerobically in vitro, their relative contribution to the hypoxic vasodilatory response has remained unsolved. Using a wire myograph, we have investigated how the nitrite-dependent vasodilation in rat aortic rings is controlled by oxygen tension, norepinephrine concentration, soluble guanylate cyclase (the target for vasoactive NO), and known nitrite reductase activities under hypoxia. Vasodilation followed overall first-order dependency on nitrite concentration and, at low oxygenation and norepinephrine levels, was induced by low-nitrite concentrations, comparable to those found in vivo. The vasoactive effect of nitrite during hypoxia was abolished on inhibition of soluble guanylate cyclase and was unaffected by removal of the endothelium or by inhibition of xanthine oxidase and of the mitochondrial bc1 complex. In the presence of hemoglobin and inositol hexaphosphate (which increases the fraction of deoxygenated heme), the effect of nitrite was not different from that observed with inositol hexaphosphate alone, indicating that under the conditions investigated here deoxygenated hemoglobin did not enhance nitrite vasoactivity. Together, our results indicate that the mechanism for nitrite vasorelaxation is largely intrinsic to the vessel and that under hypoxia physiological nitrite concentrations are sufficient to induce NO-mediated vasodilation independently of the nitrite reductase activities investigated here. Possible reaction mechanisms for nitrite vasoactivity, including formation of S-nitrosothiols within the arterial smooth muscle, are discussed.

nitric oxide; soluble guanylate cyclase; hemoglobin; xanthine oxidase; endothelium

NITRITE IS THE PHYSIOLOGICAL end product of the reaction of nitric oxide (NO) produced by NO synthase with molecular oxygen in solution (25). Nitrite has long been believed to be relatively inert at the low micromolar levels normally found in vivo. In early in vitro studies, high-nitrite concentrations (~0.1–1 mM) were typically used to induce relaxation of aortic strips (14, 15, 45) or to activate purified soluble guanlylate cyclase (26), the enzyme that catalyzes the conversion of GTP into cGMP and that mediates the NO-mediated vasorelaxation. More recently, it has become clear that nitrite has a complex in vivo chemistry that is associated with NO- and S-nitrosation-dependent signaling pathways (5), particularly with regard to the control of vasodilation (18). Although some studies argue against this view (28), there is now increasing evidence that nitrite may play a crucial role in hypoxic vasodilation by acting as a recyclable source of vasoactive NO (6, 8, 19), as suggested by the arterial-to-venous differences in plasma nitrite (19). Nitrite may thus act to modulate the complex vasodilatatory response of hypoxic arterial vessels, which involves decreases in NADPH and glutathione (GSH) in the smooth muscle (20), in the voltage-dependent calcium currents, in intracellular calcium concentration (40), and in the calcium sensitivity of the contractile system (1).

Several mechanisms for the nitrite-mediated vasodilation during hypoxia have been proposed over the past years. These are essentially based on nonenzymatic (33) or enzymatic reduction of nitrite into NO. Such nitrite reductase activity has been demonstrated in vitro under anaerobic conditions for xanthine oxidase (35), the bc1 complex of the mitochondria (27), the endothelial NO synthase (17), and hemoglobin (Hb) (8, 39), where it depends on the heme redox potential and the allosteric state of the protein (24). However, the requirement of soluble guanylate cyclase and the relative contribution of these nitrite reductase activities for hypoxic vasodilation have remained unsolved.

Other studies have proposed a predominant role of vasoactive S-nitrosothiols in the nitrite-mediated vasodilation (32), since potent S-nitrosating agents, including diffusable N2O3, may be formed from nitrite (41). The recent finding that in vivo levels of S-nitrosothiols depend linearly on nitrite concentration (5) supports this view.

Except for studies made by Gladwin and coworkers (8, 9), little is known regarding how nitrite affects aortic ring vasodilation under hypoxia. We have used the wire myograph technique to explore the origin of the nitrite effect on vasodilation, by using rat aortic rings contracted with high and low concentrations of norepinephrine (NE) under conditions of high- and low-oxygen tensions. We have determined the requirement of soluble guanylate cyclase and endothelium for the nitrite-induced hypoxic vasodilation and the extent to which S-nitrosating pathways and the nitrite reductase activities of Hb, xanthine oxidase, and the bc1 complex of the mitochondria contribute to the reaction mechanism.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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Sodium nitrite was dissolved in physiological salt solution (PSS) shortly before use and kept on ice in dark vessels until needed. PSS had the following composition (in mM): 119 NaCl, 25 NaHCO3, 4.7 KCl, 1.18 KH2PO4, 1.17 MgSO4, 1.6 CaCl2, 0.026 EDTA, and 10 glucose. The concentration of nitrite was checked by the Griess reaction (42). All reagents were from Sigma, except for the inhibitor of soluble guanylate cyclase 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ; Tocris Cookson) (16) and for S-nitrosated glutathione (GSNO; Alexis). Indomethacin was dissolved in NaHCO3 (0.5% wt/vol), and ODQ and myxothiazol were dissolved in DMSO. Diethylenetriaminepentaacetic acid (DTPA) was dissolved in 1.0 M HCl. Neither NaHCO3, DMSO, nor HCl influenced the pH of the external bath or the contractile state of the preparations in the amounts used. All other drugs were prepared in bidistilled water.

Human adult Hb was prepared from hypotonic lysis of red blood cells obtained from healthy donors and stripped of effectors by gel filtration on a Sephadex G25 column (GE Healthcare, Amersham) equilibrated with 50 mM Tris buffer, 0.5 mM EDTA, and 0.2 M NaCl, pH 8.0. Pure adult HbA0 was obtained by anion-exchange fast-protein liquid chromatography using a linear gradient of 0–0.15 M NaCl in 50 mM Tris buffer and 0.5 mM EDTA, pH 8.3, at room temperature. The column used was a Q Sepharose HP 26/10 (GE Healthcare, Amersham) at a flow rate of 5 ml/min. In vasodilation experiments, no differences were found when either unfractionated Hb after gel filtration or fast-protein liquid chromatography-purified HbA0 was used. Hb samples (<3% met, >2 mM heme) were stored as oxy derivative at –80°C and thawed on ice before use. The investigation conformed to the principles outlined in the Declaration of Helsinki and was approved by the local ethics committee of Aarhus (reference number 20040154).

Vasodilation experiments. Adult male Wistar rats (10–12 wk old) were killed in accordance with a protocol to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996). The used protocol was approved by The Danish Ministry of Justice (permission 2005/561-964). Thoracic aorta was removed, placed in cold PSS (4°C), and cut into four circular segments (~2 mm) that were mounted on a wire myograph (model 700 mo, Danish Myo Technology) for recording of isometric tension with a Myodaq 2.01.M601 software program (38). The equipment allows investigation of all four aortic segments in parallel, of which one was always a time control (i.e., without added nitrite) to follow the change in tension over time (see below). The segments were mounted in the myograph and allowed to equilibrate for 20 min at 37°C in PSS at 95% O2-5% CO2, pH 7.4. To allow for comparison between different aorta segments, a previously described normalization procedure was followed, where aortic ring segments were stretched to their optimal lumen diameter for active tension development, i.e., to an internal circumference of 90% of that achieved when the vessels were exposed to a passive tension yielding a transmural pressure of 100 mmHg (38). After normalization, NE (0.2 µM) and NE (0.05 µM) plus ACh (1 µM) were added to test smooth muscle contractility and endothelial relaxation, respectively. Preparations in which ACh induced <75% relaxation were discarded. Aortic segments were precontracted with 0.02 µM NE, unless otherwise stated.

In all experiments, aortic segments were incubated for 30 min with indomethacin (3 µM) and asymmetric dimethylarginine (300 µM) to inhibit cyclooxygenase and endothelial NO synthase, respectively (44).

The gas mixture equilibrating the organ bath was delivered and controlled by precision gas-mixing pumps (Wösthoff, Bochum, Germany). In hypoxia experiments, aortic segments were allowed to equilibrate to 1% O2-5% CO2-94% N2 for 5 min before addition of NE. Oxygen tension, measured in the myograph chamber with an oxygen microelectrode (OX500, Unisense), was 13 Torr.

The effect of cumulative additions of nitrite (0.01–300 µM) on the degree of vasodilation was studied in the absence and presence of GSH (500 µM), inositol hexaphosphate (IHP, 100 µM), and Hb (25 µM heme) in the myograph chamber. ODQ (3 µM) (16), allopurinol (300 µM) (21), and myxothiazol (10 µM) (27) were added to selectively inhibit soluble guanylate cyclase, xanthine oxidase, and the bc1 complex of the mitochondria, respectively. The endothelium was removed by gently rubbing with a human hair. Removal of the endothelium was verified by lack of response to ACh, as described above.

Data analysis. The mechanical responses of aortic segments were measured as changes in force ({Delta}F) and expressed as wall tension, {Delta}T = {Delta}F/2l, where l is the segment length (38). For each nitrite addition, relaxation was expressed as relative to the tension measured in the time-control vessel (without added nitrite). A two-way ANOVA was used to calculate the differences between dose-response curves obtained with different conditions; in cases of significant difference, t-tests were applied with Bonferroni correction for number of comparison. Statistical significance was taken at P < 0.05. To calculate EC50 values, dose-response curves obtained under identical conditions were fitted according to the Hill equation, logT/(1 – T) vs. log[NaNO2], where T is the relative tension. In addition, the slope of the Hill plots gives the overall order number of the vasodilation response on nitrite concentration.

Measurement of S-nitrosothiols. Concentration of total S-nitrosothiols formed in the myograph solution 3 min after the addition of 10 µM nitrite in the absence and presence of GSH (500 µM) and of Hb (25 µM heme) and IHP (100 µM) was measured by the Saville reaction after degradation of unreacted nitrite by ammonium sulfamate, as previously described (22).


   RESULTS
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 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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Aortic segments mounted in the wire myograph contracted on addition of NE and relaxed in a concentration-dependent manner with cumulative addition of nitrite. However, the degree of relaxation of aortic segments measured after addition of nitrite depended considerably on the oxygen tension and on the level of precontraction (Fig. 1A). Thus vasodilation curves were left shifted when measured at a lower oxygen tension (1% O2 rather than 95% O2) and when using a lower concentration of NE (0.02 µM rather than 1 µM) to precontract the vessel. The increase in force development was clearly dependent on the concentration of NE and on oxygen tension (Table 1). The relative decrease in tension due to hypoxia was not significantly different in vessels precontracted with low and high NE (39 ± 9% and 50 ± 2%, respectively). The degree of vasodilation induced by a given concentration of nitrite was the same at 1% O2 and 95% O2 when precontraction and/or force was adjusted to the same level by addition of different concentrations of NE (data not shown). Figure 1B shows the unitary slope of the Hill plots of the vasodilation curves measured at high- and low-oxygen levels, indicating that in both cases the vasodilation response followed an overall first-order dependency on the nitrite concentration.


Figure 1
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  		Fig. 1. A: effect of sodium nitrite concentration on the relative tension of rat aortic segments at 37°C and pH 7.4, in the presence of 300 µM asymmetric dimethylarginine (ADMA) and 3 µM indomethacin, in aortic segments contracted with 0.02 or 1.0 µM norepinephrine (NE) at low-oxygen (1% O2-5% CO2-94% N2) or high-oxygen (95% O2-5% CO2) tensions, as indicated. Data are means ± SE of 4 preparations (1 from each animal). B: Hill plots of the mean values of the relative tension (T) measured at 0.02 µM NE at 95% O2 (from A) and at 1% O2 (from Figs. 2 and 3). The lines represent the linear regression of the data. C: representative traces showing the time course of the relative tension achieved in aortic segments in the presence of 300 µM ADMA and 3 µM indomethacin after addition of 0.02 µM NE (black line) and after addition of 0.02 µM NE and sodium nitrite (gray line) at the indicated (µM) final concentrations. Conditions: 1% O2-5% CO2-94% N2, 37°C, pH 7.4.

 

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  		Table 1. {Delta}T induced by NE, EC50 values for nitrite, and the maximum relaxation under the conditions shown in Fig. 1

 
At 95% O2, complete vasodilation of the aortic segments was not achieved at the highest concentration of nitrite used (Fig. 1A, Table 1). Conversely, at 1% O2 and after precontraction with 0.02 µM NE, nitrite induced a marked (up to 50%) vasodilation at concentrations equivalent to those normally found in vivo (≤10 µM) (Fig. 1A). Accordingly, the effect of physiological levels of nitrite on vasodilation was studied under these experimental conditions.

As shown in Fig. 1C, in experiments performed under hypoxia, it was critical to follow over time the tension of control segments kept in the absence of nitrite, since the tension changed with time in a characteristic multiphasic manner after the addition of NE, as also observed in a previous study (31). This slow increase in tension in hypoxic aortic segments was thus taken into account to calculate the relative tension in segments exposed to cumulative additions of nitrite (Fig. 1C).

To identify the target and the origin of the nitrite vasoactivity under hypoxia, we used selective inhibitors of key enzymatic activities or removed the endothelium. The vasoactive effect of nitrite disappeared after incubation of aortic segments with 3 µM ODQ (Fig. 2), which specifically inhibits soluble guanylate cyclase, indicating that the classical soluble guanylate cyclase-cGMP pathway is essential for the vasodilatory response induced by nitrite. The response remained unchanged after removal of the endothelium (Fig. 2) or after addition of 300 µM allopurinol in the presence and absence of 10 µM myxothiazol (Table 2), which specifically inhibit xanthine oxidase and the bc1 complex of the mitochondria, respectively. Together, these data indicate that nitrite must be converted into NO to be vasoactive and that xanthine oxidase, the bc1 complex, or enzymes located in the endothelium do not play a significant role in the conversion of nitrite to NO under the conditions investigated here. No significant differences were observed when experiments were performed in the presence of the metal chelator DTPA (Table 2), indicating that the nitrite reduction observed in this study was not catalyzed by contaminant-free metals in the preparation. Addition of 10 µM FeSO4 increased nitrite-induced relaxation, an effect that was reversed by DTPA (not shown).


Figure 2
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  		Fig. 2. Effect of increasing concentration of sodium nitrite on the relative tension of rat aortic segments in the presence and absence of 3 µM 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase, and without endothelium, as indicated, at 1% O2-5% CO2-94% N2, 37°C, pH 7.4, in the presence of 300 µM ADMA and 3 µM indomethacin. Data are means ± SE of 6 preparations (1 from each animal). Differences were evaluated by 2-way ANOVA. *P < 0.05, significantly different from control treatment with nitrite alone.

 

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  		Table 2. {Delta}T induced by 0.02 µM NE, nitrite EC50 values, and the maximum relaxation achieved (at 300 µM nitrite) in the absence and presence of allopurinol (300 µM), myxothiazol (10 µM), and DTPA (100 µM)

 
To assess the role of Hb in generating vasoactive NO from nitrite, 25 µM Hb (heme basis), 100 µM IHP (a potent allosteric effector of Hb), or both were added to the myograph chamber. These experimental conditions, which were equivalent to those of a previous study (8), were used here to verify whether an increase in the fraction of deoxy Hb, i.e., with IHP added, could increase the degree of vasodilation induced by nitrite alone. As shown in Fig. 3A, Hb in the absence of IHP significantly reduced vasodilation at nitrite concentrations >10 µM. Conversely, in the presence of Hb and IHP, a significant increase in vasodilation was observed at 3 µM nitrite (Fig. 3A). However, this increase was identical to that observed with IHP alone at the same concentration of nitrite (Fig. 3A). In these experiments, addition of IHP alone induced a significant increase in vasodilation in the range 1–10 µM nitrite (Fig. 3A).


Figure 3
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  		Fig. 3. Effect of increasing concentration of sodium nitrite on the relative tension of rat aortic segments in the presence of 300 µM ADMA and 3 µM indomethacin under the following conditions: control, 100 µM inositol hexaphosphate (IHP), 25 µM hemoglobin (Hb), and 100 µM IHP + 25 µM Hb (A) and control, 500 µM GSH, 500 µM + 25 µM Hb, and 500 µM GSH + 25 µM Hb + 100 µM IHP (B) at 1% O2-5% CO2-94% N2, 37°C, pH 7.4. C: effect of increasing concentration of sodium nitrite or S-nitrosated glutathione (GSNO), as indicated, on the relative tension of rat aortic segments in the presence of 300 µM ADMA and 3 µM indomethacin, at 1% O2-5% CO2-94% N2, 37°C, pH 7.4. Data are means ± SE of 6 preparations (1 from each animal). Differences were evaluated by 2-way ANOVA. *P < 0.05, significantly different from control treatment with nitrite alone.

 
Similar results were obtained in experiments performed in the presence of 500 µM GSH (Fig. 3B) to evaluate the contribution of S-nitrosative reactions in the nitrite-mediated vasodilation. As also observed in the absence of GSH, the effect of nitrite on vasodilation measured with Hb alone was significantly reduced, whereas it remained unchanged when Hb and IHP were present together with GSH. Addition of GSH alone had no effect on the vasodilation induced by nitrite (Fig. 3B). The total concentration of S-nitrosothiols formed in the myograph chamber after addition of 10 µM nitrite was found to be below the detection limit of the Saville reaction (<0.2 µM) under all conditions examined, i.e., in the presence or in the absence of Hb and IHP with or without GSH added. As a control, the potent effect of externally added GSNO on vasodilation compared with that of nitrite measured under identical conditions is reported in Fig. 3C.


   DISCUSSION
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we report several novel observations that demonstrate that the nitrite-dependent vasodilation is facilitated by hypoxia, is intrinsic to the vessel, and follows a reaction mechanism that is overall first order in nitrite, regardless of the oxygen level. When attempting to assess the relative importance of potential reaction mechanisms, we found that the effect of nitrite on aorta relaxation under hypoxic conditions relies on the activity of soluble guanylate cyclase, as has been shown to be the case at high-oxygen levels (36), but not on NO-generating nitrite reductases, including xanthine oxidase, Hb, and the bc1 complex of the mitochondria or on endothelium components. As discussed below, these findings give novel insight into the origin of nitrite vasoactivity.

As shown in Fig. 1, a decrease from 95% to 1% O2 shifts the vasodilation curves of aortic segments to lower nitrite concentrations, similar to those normally found in the mammalian circulation (0.1–10 µM) (4, 11). This finding shows that the mechanism for nitrite vasoactivity is intrinsic to the vessel and has important biological implications, as it illustrates that the contribution of endogenous nitrite to vasodilation is inversely related to local oxygen tensions and that nitrite bioavailability within aortic tissue increases when oxygen levels decrease. Recent studies have shown that, in human subjects, blood flow increases after nitrite infusion, particularly during metabolic activity and regional hypoxia (8, 18, 19). In aortic bioassay experiments comparable to those shown here, Crawford et al. (9) noticed changes in the nitrite vasodilatatory response only in the presence of red blood cells and not with nitrite alone when oxygen tensions were in the range of 15–60 Torr. The difference between their findings and ours may be due to different experimental protocols possibly in the precontraction procedure. As shown here, the initial degree of vessel contraction is an important factor that modulates the extent to which nitrite affects vasodilation during hypoxia, so that that the magnitude of the nitrite-induced vasodilation is regulated by oxygen tension and by the degree of NE contraction. Although in vivo sympathetic activation occurs without measurable increases in plasma NE (29), our in vitro findings agree with the observations that regional {alpha}-adrenoceptor blockade enhances hypoxic vasodilation in the forearm of healthy men (46), hence suggesting that sympathetic vasoconstriction counteracts hypoxic vasodilation. Together, these observations indicate that the effect of NE on the nitrite-dependent vasodilation becomes more pronounced during hypoxia.

The sensitivity of nitrite vasoactivity to changes in oxygen levels and precontraction is likely to be a major source for the variability in the EC50 values for nitrite reported in the literature for aortic bioassays experiments, as illustrated in Table 3. Although it was measured at 95% O2, the often-cited high-EC50 value for nitrite of ~100 µM (Table 3) has long precluded nitrite from being considered physiologically relevant in the control of vasodilation. However, as shown in this study, when aortic segments are exposed to low NE and oxygen levels, they relax considerably (up to 50%) at physiological concentrations of nitrite (0.1–10 µM) (Fig. 1A), thus indicating that nitrite is a more potent vasodilator than previously appreciated.


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  		Table 3. Order of magnitude for EC50 values for nitrite under different precontraction and oxygenation conditions obtained in aortic ring or strip bioassays

 
The molecular mechanism of the nitrite-mediated vasodilation depends on active soluble guanylate cyclase, as seen here at low-oxygen levels and by others at high-oxygen levels (36) and is the same at high- and at low-oxygen tensions, where the vasodilation response follows identical apparent first-order dependency on the nitrite concentration. NO generation from protonation of nitrite according to the reactions 2NO2– + 2H+ {leftrightarrow} N2O3 + H2O and N2O3 {leftrightarrow} NO + NO2 is second order in nitrite and thus does not seem to contribute significantly to the vasoactive effects observed here. It has been suggested that sufficient NO would be generated via the nonenzymatic acidic reduction of nitrite to mediate vasodilation, particularly under hypoxic conditions (33, 36). However, as discussed in detail by Butler and Ridd (7), the dissociation of N2O3 into NO and NO2 is extremely slow in solution but very rapid and irreversible in the gas phase, where NO was originally detected (36). Moreover, the intracellular pH measured within small arteries remains remarkably constant even under severe hypoxia (1), whereby a nonenzymatic acidic conversion of nitrite into NO cannot occur to a significant extent.

When searching for possible first-order mechanisms of nitrite vasoactivity, it is notable that the in vitro reaction between thiols and nitrite to form S-nitrosothiols is first order in nitrite and involves H2NO2+ rather than N2O3 as the S-nitrosating agent (37). Such a reaction mechanism may well occur in the aortic rings of the present study and in vivo, since the levels of S-nitrosothiols found within rat tissues also follow a first-order dependency on the administered nitrite, with aorta containing among the highest basal levels of nitrite (~10 µM) and S-nitrosothiols (~0.2 µM) observed among tissues (5). The absence of detectable S-nitrosothiols (Hb-SNO and GSNO) formed from 10 µM nitrite in the myograph chamber solution and the lack of a GSH effect on the nitrite-mediated vasodilation suggest that nitrite is rapidly taken up in tissues (5), possibly as nitrous acid (34), and that a yet unknown enzymatic activity located within the aorta tissue may be required to generate sufficient H2NO2+ (or NO+) from nitrous acid (pKa 3.3) at physiological pH. Further studies that use other approaches, e.g., highly sensitive chemiluminescence methods, could verify whether S-nitrosothiols formed within vessel smooth muscle are responsible for the vasoactive effects of nitrite observed here.

Another possible mechanism for the nitrite-mediated vasodilation is that of a nitrite reductase activity, which would catalyze the one-electron reduction of nitrite into NO. We have probed specifically the role of enzymes that may potentially contribute to such activity, including xanthine oxidase (30, 35), the endothelial NO synthase(17), the mitochondrial bc1 complex (27), and Hb (8).

The vasoactive effect of nitrite under the hypoxic conditions investigated here (in the presence of an inhibitor of NO synthase) was independent of the activities of xanthine oxidase and of the bc1 complex and of the presence of the endothelium. Although these reductase activities are able to generate NO from high (mM) nitrite levels under anoxic conditions in vitro, they appear to have a marginal role in stimulating hypoxic vasodilation at the low-micromolar levels of nitrite close to those normally found in vivo. Interestingly, in agreement with these conclusions, a recent study (10) shows that hypoxic pulmonary vasoconstriction can partly be reversed by nitrite and that neither physiological changes in pH nor xanthine oxidase takes part in the nitrite vasoactivity.

An additional factor that may potentiate the intrinsic vasoactivity of nitrite is the nitrite reductase activity of circulating deoxygenated Hb, which has been the focus of several recent studies. The role of purified Hb in the nitrite-mediated hypoxic vasodilation was studied using the conditions reported by Cosby et al. (8). With half-saturation oxygen tensions of 9 and 45 Torr in the absence and presence of IHP, respectively (8), oxygen saturation levels for Hb at the oxygen partial pressure of 13 Torr were 74 and 3% in the absence and presence of IHP, respectively. We found that, in the absence of IHP, Hb significantly reduced the effect of nitrite on vasodilation, as also observed by Cosby et al. This reduction in vasodilation may reflect the reaction of nitrite with oxy-Hb that oxidizes nitrite into nitrate. This reaction is considerably faster than that between nitrite and deoxy-Hb, which reduces nitrite into NO (Fe2+ + NO2– + 2H+ -> Fe3+ + NO + H2O), as shown by the overall rate constants for the two reactions, 29 and ~0.2 M–1·s–1, respectively (12, 23). The overall vasodilating effect of nitrite found here did not increase significantly when Hb and IHP were present, either in the absence or in the presence of GSH, suggesting that any NO generated from nitrite and deoxy-Hb reacted stoichiometrically with the Hb in the myograph chamber before reaching the aorta smooth muscle (2, 13). Although the nitrite reductase activity of Hb is not essential to the nitrite vasoactivity seen in the present study, it cannot be excluded that Hb may contribute to increased NO production from nitrite at higher (~50%) oxygen saturation than those studied here (24) or that other yet unidentified processes occurring at low but not high hematocrit levels (10) take place in intact red blood cells and favor nitrite reduction (9).

Interestingly, we found that IHP alone enhanced the vasodilating effect of nitrite over a broad range of nitrite concentrations. This relaxing effect of IHP has previously been noticed (32) and may relate to calcium-chelating properties of IHP (3, 43) that would decrease the activity of extracellular calcium and reduce muscle contraction.

In conclusion, the results presented here support the view that arterial vasodilation is linked to physiological levels of nitrite, particularly during hypoxic conditions, and suggest that S-nitrosothiols formed from nitrite within the smooth muscle cells may well be at the origin of the nitrite-mediated vasodilation during hypoxia. Such an intrinsic mechanism of vascular tissue response effectively links the nitrite with the hypoxia response and does not appear to depend on previously identified nitrite reductase activities that may generate NO from nitrite.


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This work was supported by the Danish Natural Science Research Council, the Novo Nordisk Foundation, and the Lundbeck Foundation.


   ACKNOWLEDGMENTS
 
We thank Dr. Roy Weber for reading the manuscript and Anna Sofia Fägersten for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Fago, Dept. of Biological Sciences, Universitetsparken 1131, Univ. of Aarhus, DK-8000 Aarhus C, Denmark (e-mail: angela.fago{at}biology.au.dk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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