INVITED REVIEW
Nitric oxide production in the hypoxic lung

Timothy D. Le Cras1 and Ivan F. McMurtry2

1 Pediatric Heart Lung Center, Department of Pediatrics, and 2 Cardiovascular Pulmonary Research Laboratory, Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262


    ABSTRACT
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ABSTRACT
INTRODUCTION
OXYGEN SENSITIVITY OF NOS...
GUANOSINE 3',5'-CYCLIC...
ACUTE HYPOXIA AND NO...
ACUTE HYPOXIA AND NO...
NO PRODUCTION IN CULTURED...
NO PRODUCTION IN THE...
CONCLUSIONS AND UNANSWERED...
REFERENCES

Nitric oxide (NO) is a potent vasodilator and inhibitor of vascular remodeling. Reduced NO production has been implicated in the pathophysiology of pulmonary hypertension, with endothelial NO synthase (NOS) knockout mice showing an increased risk for pulmonary hypertension. Because molecular oxygen (O2) is an essential substrate for NO synthesis by the NOSs and biochemical studies using purified NOS isoforms have estimated the Michaelis-Menten constant values for O2 to be in the physiological range, it has been suggested that O2 substrate limitation may limit NO production in various pathophysiological conditions including hypoxia. This review summarizes numerous studies of the effects of acute and chronic hypoxia on NO production in the lungs of humans and animals as well as in cultured vascular cells. In addition, the effects of hypoxia on NOS expression and posttranslational regulation of NOS activity by other proteins are also discussed. Most studies found that hypoxia limits NO synthesis even when NOS expression is increased.

acute hypoxia; chronic hypoxia; pulmonary hypertension; nitric oxide synthase; isolated perfused lung


    INTRODUCTION
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ABSTRACT
INTRODUCTION
OXYGEN SENSITIVITY OF NOS...
GUANOSINE 3',5'-CYCLIC...
ACUTE HYPOXIA AND NO...
ACUTE HYPOXIA AND NO...
NO PRODUCTION IN CULTURED...
NO PRODUCTION IN THE...
CONCLUSIONS AND UNANSWERED...
REFERENCES

NITRIC OXIDE (NO) is a potent systemic and pulmonary vasodilator that also inhibits smooth muscle cell proliferation (10, 18, 57), migration (4, 56) and inflammation and matrix protein production (40, 68). Hence reductions in NO production in the lung may contribute to the development of pulmonary hypertension by both increasing vascular tone and promoting vascular remodeling. Studies in transgenic mice show that deletion of the endothelial NO synthase (eNOS) gene leads to development of pulmonary hypertension, which is reversed by inhalation of NO (14, 64, 65). Because chronic inhaled NO also attenuates hypoxia-induced pulmonary hypertension in rats (34, 53, 55), numerous studies have examined whether reduced NO production by the lung contributes to the pathogenesis of hypoxia-induced pulmonary hypertension.

Two main sites of NO production have been described in the lung, the vasculature, and the airways (21, 22, 33, 63). NO production in the vascular endothelium is catalyzed by eNOS (type III NOS), although some studies have suggested that the other two isoforms of NOS, inducible NOS (iNOS or type II NOS) and neuronal NOS (nNOS or type I NOS), may also be present in the vasculature and contribute to NO production (48, 49). In the airways, all three isoforms have been detected in bronchial epithelium (33, 60). Whether NO produced in the airways affects pulmonary vascular tone and the development of pulmonary hypertension is unclear.


    OXYGEN SENSITIVITY OF NOS ISOFORMS
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ABSTRACT
INTRODUCTION
OXYGEN SENSITIVITY OF NOS...
GUANOSINE 3',5'-CYCLIC...
ACUTE HYPOXIA AND NO...
ACUTE HYPOXIA AND NO...
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REFERENCES

Molecular oxygen (O2) is an essential substrate for NO synthesis by NOS, and biochemical studies using purified NOS isoforms have estimated the Michaelis-Menten constant (Km) values for O2 to be in the 5-20 µM range (50, 51) and for nNOS to be as high as 400 µM (2). Several studies (1-3, 27) have shown that when NO is formed, it can bind to the heme iron of NOS, which inhibits NOS activity and increases the apparent Km for O2. Because O2 and NO compete for the heme iron of nNOS, the overall O2 dependence of this isoform depends on the rate of decay of the heme iron-NO complex, which is itself dependent on O2 concentration (2). Because the apparent O2 sensitivities of the NOS isoforms are within the range of tissue O2 concentrations, Rengasamy and Johns (51) have suggested that O2 substrate limitation may regulate NO production in pathophysiological conditions including hypoxia. However, whether these in vitro data could be extrapolated to NO production in the hypoxic lung was uncertain, and numerous investigators have examined the effects of hypoxia on NO production in human subjects, intact animals, perfused lungs, and cultured cells. This review compares these various studies of the acute and chronic effects of hypoxia on NO production in the lung as well as in cultured cells.


    GUANOSINE 3',5'-CYCLIC MONOPHOSPHATE AS AN INDEX OF NO SYNTHESIS
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ABSTRACT
INTRODUCTION
OXYGEN SENSITIVITY OF NOS...
GUANOSINE 3',5'-CYCLIC...
ACUTE HYPOXIA AND NO...
ACUTE HYPOXIA AND NO...
NO PRODUCTION IN CULTURED...
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REFERENCES

Because NO stimulates soluble guanylyl cyclase (sGC) and hence cGMP formation, a number of studies (30, 54, 61, 62) have measured cGMP levels in isolated vessels and cultured cells exposed to hypoxia to assess the effects of hypoxia on NO production. For the most part, these studies showed reduced cGMP levels with exposure to hypoxia, suggesting that NO production is decreased by low O2 tension. However, cGMP levels may not necessarily directly reflect changes in NO production. Steady-state cGMP levels are determined by the rate of cGMP breakdown by phosphodiesterases and the rate of cGMP formation by sGC, and although in many of the studies, breakdown of cGMP was blocked with phosphodiesterase inhibitors, NO-independent changes in sGC activity with hypoxia were generally not accounted for. It is now clear that other vascular products can regulate cGMP formation; for example, superoxide anion-derived hydrogen peroxide is an important O2-sensitive regulator of vascular cGMP formation (for a review, see Ref. 71). In addition, recent studies in rats suggested that chronic hypoxia increases lung sGC expression and activity (36) and that increased cGMP production by hypoxia-induced hypertensive lungs is due to atrial natriuretic peptide rather than NO (41). Many of the studies examining the effects of hypoxia on NO-stimulated cGMP formation were performed before the development of sensitive instruments for directly measuring NO and its immediate products (nitrite, peroxynitrite, and nitrate; NO<UP><SUB>x</SUB><SUP>−</SUP></UP>), and although they do not directly prove that NO production is reduced by hypoxia, they are certainly suggestive. Because numerous studies have now measured NO and NO<UP><SUB>x</SUB><SUP>−</SUP></UP>, we will direct most of this review to these studies.


    ACUTE HYPOXIA AND NO PRODUCTION IN ANIMAL MODELS
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ABSTRACT
INTRODUCTION
OXYGEN SENSITIVITY OF NOS...
GUANOSINE 3',5'-CYCLIC...
ACUTE HYPOXIA AND NO...
ACUTE HYPOXIA AND NO...
NO PRODUCTION IN CULTURED...
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REFERENCES

Several studies have addressed the effects of acute ventilation with hypoxic gas on NO production in isolated perfused lungs (Table 1). Perfusate NO<UP><SUB>x</SUB><SUP>−</SUP></UP> or nitrite levels and/or exhaled NO were measured to assess NO production. Nelin et al. (43) measured both perfusate NO<UP><SUB>x</SUB><SUP>−</SUP></UP> and exhaled NO production in piglet lungs and found that acute hypoxia reduced perfusate NO<UP><SUB>x</SUB><SUP>−</SUP></UP> by 60% and exhaled NO by 30%. The authors concluded that hypoxia inhibited a continuous basal production of NO in the piglet lungs. Another study in older piglets by Cremona et al. (11) also found that exhaled NO production was reduced during hypoxic ventilation (perfusate NO<UP><SUB>x</SUB><SUP>−</SUP></UP> was not measured). In addition, because exhaled NO production was reduced when N-nitro-L-arginine methyl ester was added to the vascular perfusate, Cremona et al. suggested that NO produced by the vascular endothelium contributes to exhaled NO. Pearl et al. (46) performed in vivo studies on anesthetized piglets and measured exhaled NO and plasma nitrite levels after 90 min of hypoxia, 1 h of reoxygenation on cardiopulmonary bypass, and 2 h of recovery. Exhaled NO levels decreased to 36% of baseline by the end of the hypoxic period and then even further by the end of the reoxygenation and/or recovery period (20% of baseline). Aortic plasma nitrite levels decreased to 48% of baseline and pulmonary arterial nitrite levels decreased to 73% of baseline during hypoxia. Both aortic and pulmonary arterial plasma nitrite levels returned to baseline after the recovery period. Because reductions in exhaled NO persisted after hypoxia-reoxygenation, whereas plasma nitrite levels returned to baseline, the authors (46) suggested that alterations in exhaled NO might be unrelated to plasma nitrite levels and could be due to a dysfunctional bronchial epithelium resulting from acute lung injury.

                              
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Table 1.   Acute hypoxia and NO production in normotensive isolated perfused lungs

Several studies of perfused rabbit lungs have found somewhat different effects of acute hypoxia on perfusate NO<UP><SUB>x</SUB><SUP>−</SUP></UP> production. Despite ventilation of rabbit lungs with 3% O2, Grimminger et al. (20) did not detect any change in intravascular release of NO but did observe a small reduction in exhaled NO that was very rapid and preceded an increase in pulmonary arterial pressure (hypoxic pulmonary vasoconstriction). Similarly, Deem et al. (12) found that although there was a small decrease in exhaled NO levels in rabbit lungs ventilated with hypoxic gas, perfusate NO<UP><SUB>x</SUB><SUP>−</SUP></UP> levels did not change. These authors also observed that exhaled NO levels rapidly returned to baseline on return to normoxia and were reduced when red blood cells were added to the perfusate. The addition of red blood cells also potentiated hypoxic pulmonary vasoconstriction, which they attributed to scavenging of NO by red blood cells because NOS inhibition with N-nitro-L-arginine eliminated the red blood cell dependence of hypoxic pulmonary vasoconstriction (12). Kantrow et al. (32) reported no effect of ventilation of rabbit lungs with hypoxic gas on perfusate nitrite accumulation but did find a 90% reduction in perfusate nitrite when anoxic gas was used (exhaled NO was not measured). Pulmonary vasoconstriction was observed during severe but not during moderate hypoxia. They suggested that decreased NO production contributed to the pulmonary vasoconstrictor response to severe hypoxia. Carlin et al. (7) studied the effects of graded hypoxia on exhaled NO production in blood-perfused rabbit lungs and found that although ventilation with 6% O2 did not reduce NO levels, both 3 and 0% O2 did.

Only one study has examined the relative importance of vascular versus airway hypoxia. Ide et al. (28) examined the effects of perfusate hypoxia alone, alveolar hypoxia alone, and combined perfusate and alveolar hypoxia using a membrane oxygenator-deoxygenator on the inlet limb to the pulmonary artery in perfused rabbit lungs to control perfusate PO2 separate from alveolar PO2. The authors found that perfusate hypoxia alone did not affect either perfusate NO<UP><SUB>x</SUB><SUP>−</SUP></UP> or exhaled NO production and did not elicit pulmonary vasoconstriction. In contrast, alveolar hypoxia produced by ventilation with 1% O2 reduced perfusate NO<UP><SUB>x</SUB><SUP>−</SUP></UP> by 56% and exhaled NO by 34% and increased pulmonary arterial pressure. The combination of perfusate and alveolar hypoxia caused a similar reduction in NO production as alveolar hypoxia alone. From the reduction in exhaled NO production with alveolar hypoxia, the authors (28) calculated a Km for O2 of 23.2 µM for buffer-perfused rabbit lungs. They suggested that exhaled NO production is dependent on the level of inspired O2 and speculated that epithelial NOS is O2 sensitive over the physiological range of alveolar PO2 and controls the pulmonary circulation.

From these studies, it seems that although acute hypoxia reduces NO production in both piglet and rabbit lungs, the severity of hypoxia required to do so differs. Although <= 7.5% O2 reduced exhaled NO production in piglet lungs (43), 6% O2 did not reduce exhaled NO in rabbit lungs, but <= 3% O2 did (7, 20, 28). Similarly, moderate hypoxia (7.5% O2) reduced perfusate NO<UP><SUB>x</SUB><SUP>−</SUP></UP> production in piglet lungs (43), but in rabbit lungs, no change in perfusate NO<UP><SUB>x</SUB><SUP>−</SUP></UP> production was seen until the inspired O2 fraction was <= 1% O2 (7, 28, 32). Whether the more severe level of hypoxia that is apparently required to reduce NO production in rabbit lungs compared with piglet lungs is due to a species or age difference is unclear.


    ACUTE HYPOXIA AND NO PRODUCTION IN HUMANS
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ABSTRACT
INTRODUCTION
OXYGEN SENSITIVITY OF NOS...
GUANOSINE 3',5'-CYCLIC...
ACUTE HYPOXIA AND NO...
ACUTE HYPOXIA AND NO...
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Several studies in normal humans (Table 2) have examined the effects of acute hypoxia on exhaled NO production. Although measurements of exhaled NO in subjects wearing a noseclip can include NO produced by both the lung and the nasopharynx, measurements made during endotracheal intubation should exclude the nasopharynx and just represent NO produced by the lung. Tsujino et al. (69) measured exhaled NO using both approaches. The concentration of exhaled NO was ~55-60% lower with intubation than with the noseclip, suggesting that in normal humans only ~40-45% of NO in exhaled air originates from the lungs, with the remainder originating from the nasopharynx. In the subjects wearing a noseclip, inhalation of hypoxic gas (10% O2) for 3 min did not affect exhaled NO (69). Schmetterer et al. (59) examined the effects of breathing 10-100% O2 on exhaled NO measured from end-expiratory single-breath exhalation with nasal occlusion. They observed concentration-dependent changes in exhaled NO during graded O2 breathing; exhaled NO levels were 31 ± 3 parts/billion while breathing room air and increased by 25% with 100% O2. Although exhaled NO levels while breathing 10% O2 were 26 ± 3 parts/billion, this was not significantly less than baseline (59). Dweik et al. (13) measured real-time bronchiolar NO levels in normal individuals by sampling gas through a bronchoscope and found that exhaled NO levels were reduced with hypoxia (5-15% O2). Small decreases in NO levels were seen between 21 and 10%, but exhaled NO decreased by 60% in subjects breathing 5% O2. Dweik et al. calculated the apparent Km for O2 to be 190 µM, which is well within the physiological range (0-250 µM). None of these studies measured the effects of hypoxia on plasma NO<UP><SUB>x</SUB><SUP>−</SUP></UP>.

                              
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Table 2.   Effects of hypoxia and hyperoxia on exhaled NO production in healthy humans


    NO PRODUCTION IN CULTURED CELLS EXPOSED TO HYPOXIA
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ABSTRACT
INTRODUCTION
OXYGEN SENSITIVITY OF NOS...
GUANOSINE 3',5'-CYCLIC...
ACUTE HYPOXIA AND NO...
ACUTE HYPOXIA AND NO...
NO PRODUCTION IN CULTURED...
NO PRODUCTION IN THE...
CONCLUSIONS AND UNANSWERED...
REFERENCES

Several studies have examined the regulation of NOS activity by O2 tension in cultured cells (Table 3). Hong et al. (26) measured NO production in cultured smooth muscle cells under basal conditions and after induction of iNOS (type II NOS) with bacterial lipopolysaccharide and interferon-gamma . Although 0.2% O2 did not suppress NO production under basal conditions, it did reduce NO production when iNOS expression was induced. Cormick et al. (9) found that although hypoxia increased iNOS gene expression in macrophages activated with lipopolysaccharide and/or interferon-gamma , NO synthesis was markedly reduced. By exposing cells to a range of O2 tensions, Cormick et al. estimated the Km for iNOS to be around 11% O2, which is considerably higher than the O2 tension in tissues and similar to that reported for purified recombinant iNOS (9). Hence, although hypoxia may increase iNOS expression in cultured cells, it also limits NO production by the enzyme.

                              
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Table 3.   Hypoxia and NO production in cultured cells

Whorton et al. (70) examined the effects of graded hypoxia on thapsigargin-stimulated NO production by eNOS (type III NOS) in bovine aortic endothelial cells (BAECs). Exposure to hypoxia caused a concentration-dependent reduction in NO production as the severity of hypoxia was increased. Thus Whorton et al. suggested that O2 may be an important determinant of eNOS activity and NO production in hypoxic tissues and vascular beds such as the pulmonary arterial and fetal circulations where PO2 can be low. Xu et al. (72) also exposed cultured BAECs to hypoxia and found that NO production by eNOS stimulated with the calcium ionophore A-23187 was reduced compared with NO production by normoxic cells stimulated with the same agent. These authors also reported that cyclooxygenase inhibition prevented the hypoxia-induced decrease in NO production and that iloprost, a prostacyclin analog, directly suppressed NO production. They suggested that endothelium-derived prostanoids produced in response to hypoxia rather than reduced O2 itself regulated NO production via an autocrine negative-feedback mechanism.

In addition to the effects of hypoxia on the availability of O2 substrate for NO synthesis, a recent study by Su and Block (66) showed that hypoxia may also affect NOS activity by reducing 90-kDa heat shock protein (HSP90) levels. HSP90 has been shown to bind to eNOS and to increase its activity in response to agonists that stimulate production of NO (17). Su and Block found that hypoxia reduced eNOS activity but not eNOS protein levels in pulmonary arterial endothelial cells (PAECs) and showed that the reductions in eNOS activity were due to a decrease in HSP90 levels caused by calpain. Whether HSP90 and calpain regulate eNOS activity in the hypoxic lung in vivo remains to be determined.

A number of studies have shown that O2 tension affects eNOS gene expression in cultured cells. Liao et al. (35) found that exposure to 24 h of 3% O2 reduced eNOS mRNA and protein levels in bovine PAECs compared with cells cultured in 20% O2. Similarly, hypoxic exposure reduced both eNOS gene transcription and mRNA stability after 24-48 h in human umbilical vein endothelial cells (HUVECs) (39). In cocultures with smooth muscle cells, the hypoxic HUVECs also stimulated less cGMP formation than corresponding normoxic cells. North et al. (44) reported that as O2 tension was increased from 50 to 150 mmHg, eNOS mRNA, protein, and activity increased in ovine fetal PAECs. Hence these studies indicated that NO production by eNOS may be reduced in hypoxia through transcriptional and posttranscriptional regulation of eNOS expression as well as through reductions in eNOS activity due to reduced O2 substrate.

In contrast to these reports of hypoxic inhibition of NO synthesis and NOS expression, Hampl et al. (23) found that acute hypoxia increased basal and bradykinin-stimulated NO production in cultured bovine PAECs. The increase in NO production was attributed to hypoxia-induced increases in cytosolic calcium. Arnet et al. (6) reported that although hypoxic exposure increased eNOS mRNA and protein expression in BAECs, eNOS activity was unaffected. Whether HSP90 limited eNOS activity in the hypoxic BAECs was not determined. Justice et al. (31) compared cultured endothelial cells from resistance and nonresistance epicardial arteries and found that although hypoxia increased NO production and eNOS protein in both cell populations, eNOS mRNA increased only in the nonresistance epicardial endothelial cells, suggesting that increased NO production in the microvascular (resistance) endothelial cells may be due to translational or posttranslational regulation.


    NO PRODUCTION IN THE CHRONICALLY HYPOXIC LUNG
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ABSTRACT
INTRODUCTION
OXYGEN SENSITIVITY OF NOS...
GUANOSINE 3',5'-CYCLIC...
ACUTE HYPOXIA AND NO...
ACUTE HYPOXIA AND NO...
NO PRODUCTION IN CULTURED...
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REFERENCES

Numerous studies have measured lung NO production in animal models of chronic hypoxia-induced pulmonary hypertension (Table 4). Fike et al. (16) exposed neonatal pigs to hypoxia (10% O2) for 10-12 days. Plasma NO<UP><SUB>x</SUB><SUP>−</SUP></UP> levels were measured when the piglets were breathing room air, and perfusate NO<UP><SUB>x</SUB><SUP>−</SUP></UP> levels were measured in lungs ventilated with 17% O2. Both plasma NO<UP><SUB>x</SUB><SUP>−</SUP></UP> and lung perfusate NO<UP><SUB>x</SUB><SUP>−</SUP></UP> levels were lower in the chronic hypoxic group. Exhaled NO levels were also lower in the chronic hypoxic piglets compared with those in the normoxic control piglets (16). Because eNOS protein levels in whole lung homogenates were lower in the chronic hypoxic group compared with those in control animals and because NO<UP><SUB>x</SUB><SUP>−</SUP></UP> measurements were made in room air or with normoxic ventilation, lower NO levels in plasma, perfusate, and exhaled air may have been due to reduced amounts of eNOS rather than to decreased eNOS activity. However, a more recent study by Fike et al. (15) showed that perfusate NO<UP><SUB>x</SUB><SUP>−</SUP></UP> production by the chronically hypoxic piglet lungs was markedly increased after the addition of L-arginine, which suggests that impaired L-arginine uptake contributes to the reduced NO production.

                              
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Table 4.   NO production in the chronically hypoxic lung

Three studies (29, 42, 58) have examined NO production in perfused lungs from chronically hypoxic rats. All three studies found perfusate NO<UP><SUB>x</SUB><SUP>−</SUP></UP> levels to be increased when the chronically hypoxic lungs were ventilated with 21% O2, consistent with reported increases in expression of all three NOS isoforms in the lungs of chronically hypoxic rats (37, 52, 60). Sato et al. (58) examined perfusate NO<UP><SUB>x</SUB><SUP>−</SUP></UP> production when chronically hypoxic rat lungs were ventilated with hypoxic gas and found that although 3% O2 tended to reduce basal NO<UP><SUB>x</SUB><SUP>−</SUP></UP>, 0% O2 markedly reduced both basal and thapsigargin-stimulated NO<UP><SUB>x</SUB><SUP>−</SUP></UP> accumulation. The hypoxic inhibition of lung perfusate NO<UP><SUB>x</SUB><SUP>−</SUP></UP> accumulation was not reversed by supplementary L-arginine or tetrahydrobiopterin and was not mimicked by the inhibition of Ca2+ influx, suggesting that none of these factors were limiting NO production. In contrast to perfusate NO<UP><SUB>x</SUB><SUP>−</SUP></UP> levels, exhaled NO levels in the chronically hypoxic lungs were not increased and were unaffected by ventilation with 0% O2 (58). In addition to perfused lung studies, Sato et al. (58) also measured plasma NO<UP><SUB>x</SUB><SUP>−</SUP></UP> levels in chronically hypoxic rats breathing either 21 or 10% O2. Although plasma NO<UP><SUB>x</SUB><SUP>−</SUP></UP> levels were higher in chronically hypoxic rats breathing 21% O2 compared with control normoxic rats, plasma NO<UP><SUB>x</SUB><SUP>−</SUP></UP> levels were not increased when the chronically hypoxic rats were breathing 10% O2. These findings suggest that hypoxia limits NO production in the lungs of chronically hypoxic rats despite increased expression of NOS.

Exhaled NO production has been shown to be reduced in humans with chronic obstructive pulmonary disease, and a recent report by Clini et al. (8) reported that NO production from the airways was lower and inversely related to the development of cor pulmonale in patients with severe chronic obstructive pulmonary disease. It is unknown whether these reductions in exhaled NO in patients with cor pulmonale are due to hypoxia or other mechanisms.


    CONCLUSIONS AND UNANSWERED QUESTIONS
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ABSTRACT
INTRODUCTION
OXYGEN SENSITIVITY OF NOS...
GUANOSINE 3',5'-CYCLIC...
ACUTE HYPOXIA AND NO...
ACUTE HYPOXIA AND NO...
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Although perfused lung studies show that acute moderate to severe hypoxia can reduce exhaled NO and perfusate NO<UP><SUB>x</SUB><SUP>−</SUP></UP> production, the severity of the hypoxia required varies with animal species and age. Studies in normal humans also suggest that exhaled NO is sensitive to inspired O2 levels such that exhaled NO levels increase with hyperoxia and decrease with hypoxia. Studies in cultured cells are conflicting, with examples of both reduced and increased NOS expression and NO production. Exposure of animals to chronic hypoxia alters lung NOS levels, which are reduced in piglets and increased in adult rats. Thus alterations in NO production with chronic hypoxia may be due to changes in both NOS level and activity. Although depending on whether chronically hypoxic rats were breathing room air or hypoxic gas, NO production was either increased or unchanged, indicating that despite the fact that lung NOS expression was increased in chronic hypoxic rats, NO production was not when the rats were breathing hypoxic gas.

Whether reduced NO production contributes to hypoxic pulmonary vasoconstriction is controversial. Some studies using pulmonary artery rings have reported that blockade of NOS activity inhibits hypoxic vasoconstriction (19, 67), whereas other ring studies and all perfused lungs studies have shown that inhibition of NO production potentiates hypoxic vasoconstriction (5, 25, 38). Interestingly, Hasunuma et al. (25) also reported that acute inhibition of NO synthesis had little effect on baseline pulmonary vascular resistance in either perfused rat lungs or intact rats, suggesting that basal NO production is not solely responsible for the low vascular tone of the normoxic rat lung. Although the role of NO in hypoxic pulmonary vasoconstriction is unclear, studies have shown that endogenous NO production actively opposes vasoconstrictor stimuli in the lungs of rats with chronic hypoxia-induced pulmonary hypertension (29, 45). So although inhibition of NO production may not be the sole factor responsible for the development of hypoxia-induced pulmonary hypertension, NO synthesis in the hypoxic lung may play an important role in attenuating the severity of the disease. The vast majority of studies reviewed above suggest that reduced O2 tension may limit NO production by NOS despite increases in lung NOS expression observed in some species. Most studies implicate reduced O2 substrate as the limiting factor, but the role of other factors such as HSP90 in the regulation of NO production in the hypoxic lung remains to be defined.

Recently, the role of NO in attenuating hypertensive pulmonary vascular remodeling has been questioned in a study by Quinlan et al. (47). These investigators found that in contrast to other studies (14, 64), their eNOS-deficient mice showed decreased muscularization and wall thickening of small pulmonary arteries in response to chronic hypoxia. The authors suggested that differences in the genetic background of the eNOS-deficient and control mice might have accounted for the opposite findings in their study versus those of Fagan et al. (14) and Steudel et al. (64). The role of NO in the pathogenesis of chronic pulmonary hypertension is still controversial and has recently been reviewed by Hampl and Herget (24). There is evidence for both increased and reduced NO production in chronic pulmonary hypertension, and Hampl and Herget present evidence for both beneficial and potentially adverse effects of increased NO in the development of this disease. They suggest that the protective and adverse effects of NO in pulmonary hypertension are determined by the relative amounts of NO and reactive oxygen species and that the balance may be shifted toward injury during exacerbations of chronic diseases associated with pulmonary hypertension (24).


    NOTE ADDED IN PROOF

Since acceptance, two additional papers that are relevant to this review have been published. Otto and Baumgardner have described hypoxic inhibition of NO production by cultured macrophages. (Otto CM and Baumgardner JE. Effect of culture PO2 on macrophage (RAW 264.7) nitric oxide production. Am J Physiol Cell Physiol 280: C280-C287, 2001). Budts et al. showed that iNOS gene transfer reduced hypoxia-induced pulmonary hypertension and vascular remodeling in mice (Budts W, Pokreisz P, Nong Z, Van Pelt N, Gillijns H, Gerard R, Lyons R, Collen D, Bloch KD, and Janssens S. Aerosol gene transfer with inducible nitric oxide synthase reduces hypoxic pulmonary hypertension and pulmonary vascular remodeling in rats. Circulation 102: 2880-2885, 2000).


    ACKNOWLEDGEMENTS

We thank Drs. B. Fouty and J. Weil for critical review of the manuscript.


    FOOTNOTES

This work was supported by an American Heart Association Scientist Development Award (to T. D. Le Cras) and National Heart, Lung, and Blood Institute Grant HL-14985 (to I. F. McMurtry).

Address for reprint requests and other correspondence: T. D. Le Cras, Dept. of Pediatrics, Box C218, Univ. of Colorado Health Sciences Center, 4200 E. Ninth Ave., Denver, CO 80262 (E-mail: Timothy.Lecras{at}uchsc.edu).


    REFERENCES
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5.   Archer, SL, Tolins JP, Raij L, and Weir EK. Hypoxic pulmonary vasoconstriction is enhanced by inhibition of the synthesis of an endothelium derived relaxing factor. Biochem Biophys Res Commun 164: 1198-1205, 1989[ISI][Medline].

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