I. INTRODUCTION

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Chronic injury to the pulmonary vasculature results in sustained pulmonary hypertension. Although various forms of pulmonary hypertension are a significant medical problem, mechanisms of development of this syndrome are unclear. Consequently, current options for effective prevention and therapy are limited.

One of the fastest growing areas of biomedical research during the last decade has been the biological role of endogenously produced nitric oxide (NO). Enormous evidence has accumulated showing an important role of this simple molecule in a wide variety of physiological functions, including regulation of vascular tone and mesenchymal cell growth.¹ Lots of work has also been devoted to finding out the role played by NO in the normal and hypertensive pulmonary circulation. Reviewing the findings of that work is the objective of this article. The available data support the possibility that in pulmonary hypertension, endogenous NO can act not only to suppress the increase in vascular tone, but depending on conditions not yet well characterized, also to promote the vascular wall injury. The large, promising, and rapidly expanding area of the therapeutic manipulations of NO activity in pulmonary hypertension (inhaled NO gas, gene therapy) is not covered in this review; the interested reader is referred to recent reviews published elsewhere (54, 75, 121, 145, 164, 180, 194, 214).

As reviewed extensively elsewhere (295, 389), pulmonary hypertension is defined clinically as a condition of elevated pulmonary arterial pressure and/or pulmonary vascular resistance. It is a syndrome common to a variety of lung and heart diseases, such as chronic obstructive lung disease, lung fibrosis, adult respiratory distress syndrome, mitral stenosis, or congenital heart defects. Pulmonary hypertension also exists in a primary form, which is a relatively rare but serious disease (1, 66). Pulmonary hypertension secondary to pulmonary or cardiac diseases significantly worsens the prognosis of the primary disease (389).

Pulmonary hypertension presents an increased load to the right ventricle, which consequently hypertrophies and tends to fail. In fact, right heart failure is the most common cause of death in pulmonary hypertension (389).

The elevated vascular resistance in pulmonary hypertension is a result of an increase in vascular tone and of structural remodeling of the peripheral pulmonary arteries. The remodeling affects both vascular smooth muscle, which hypertrophies and proliferates, and vascular wall connective tissue, which increases in amount and undergoes qualitative changes. The endothelium is often affected as well. All of that reduces vascular lumen and thus increases vascular resistance to blood flow. It also makes the vascular wall less compliant. A relative pathogenetic significance of the functional and structural components varies with the type and stage of the disease. In the developed pulmonary hypertension, the importance of structural remodeling prevails (298), as suggested by the relative resistance of various types of pulmonary hypertension to vasodilator therapy (see Refs. 275, 276 for review).

Although chronic pulmonary hypertension is caused by a variety of pathogenetic factors, they all lead to vasoconstriction and structural remodeling of surprising uniformity. It suggests that at least part of the pathogenetic chain is similar despite the diverse origins of the disease. Injury to the pulmonary vascular wall and resulting reparatory processes are likely to be such a common phenomenon.

Various causes of chronic lung vascular injury were studied in different models of experimental pulmonary hypertension (for review see Refs. 139, 143, 296). Three major mechanisms (Fig. 1), namely vascular wall injury, abnormal shear stress of endothelial cells due to locally increased blood flow, and increase of transmural pressure across the vascular wall, interact in most cases of pulmonary hypertension, although their relative importance may differ.

View larger version (24K): [in this window] [in a new window]   Fig. 1. Major mechanisms causing pulmonary hypertension.

As discussed in detail in recent reviews (17, 146, 290, 315, 324, 330, 353, 382, 389), a variety of intercellular and intracellular messenger molecules appear to be involved in the mechanism of pulmonary hypertension. However, their exact interplay is unclear, as is the primary stimulus to switch it on. Recent data point to alterations in the metabolism of vascular wall matrix proteins as a significant part of the mechanism of pulmonary hypertension (290). The extracellular matrix is an important denominator of mesenchymal cell migration, growth, and differentiation (331).

An important factor in the development of pulmonary hypertension is chronic or intermittent alveolar hypoxia. Lung tissue hypoxia accompanies most of the lung and heart diseases associated with pulmonary hypertension. It is not surprising, therefore, that exposure of animals to hypoxic environment has been the most often used experimental model of chronic pulmonary hypertension.

A distinction should be made between the effects of acute and chronic hypoxia. Acute hypoxia causes pulmonary vasoconstriction, which is one of the hallmarks of pulmonary vascular regulation. Systemic vessels generally respond to hypoxia with vasodilation or no change in tone. Hypoxic pulmonary vasoconstriction reduces blood flow to poorly ventilated areas of the lung in favor of the flow into the better ventilated regions. That helps to optimize lung ventilation/perfusion matching and consequently also the oxygenation of the blood. Hypoxic pulmonary vasoconstriction is a local lung regulatory mechanism. It is fast in onset (165) and is readily reversible upon reoxygenation.

Chronic hypoxia causes pulmonary hypertension, which often has a vasoconstriction component too. However, morphological remodeling of the pulmonary vascular wall appears to be more important than vasoconstriction (298).

NO is a simple molecule with one unpaired electron, i.e., it is a free radical. In the presence of oxygen, NO undergoes oxidation, which follows a second-order kinetics. Hence, when NO levels are high, it is oxidized within seconds. On the other hand, when NO levels are relatively low, as is often the case in vascular tissues (30, 167, 217), its half-life can be hours or more. Oxidation end-product of NO is nitrite (NO₂) in aqueous solutions and nitrate (NO₃) in the presence of oxyhemoglobins (e.g., in blood) (157). NO and its oxidation products are often collectively referred to as NO_x. The biologically relevant aspects of NO chemistry were recently reviewed in detail elsewhere (15, 119, 156, 178).

NO is an important endogenous vasodilator produced by endothelial cells. It also serves as a neurotransmitter. High levels of NO and products of its interaction with oxygen free radicals are toxic, a fact which is utilized by cells of the immune system to kill invading bacteria or tumor cells.

NO is produced in mammalian cells by an oxygen-dependent, five-electron oxidation of a terminal guanidino nitrogen of L-arginine. Aside from NO, the reaction yields L-citrulline. The multistep reaction is catalyzed by a single heme-containing enzyme, NO synthase (NOS; EC 1.14.13.39), which exists in three isoforms. All isoforms are active as homodimers, are stereospecific (D-arginine is not a substrate), and require reduced nicotinamide adenine dinucleotide phosphate, 6(R)-5,6,7,8-tetrahydrobiopterin, flavin adenine dinucleotide, and flavin mononucleotide as cofactors. Isozyme I (subunit molecular mass ~160 kDa), encoded by a gene located on the human chromosome 12, is constitutively expressed in many central and peripheral neurons and is therefore often called neuronal NOS (nNOS). It can also be present in certain epithelial and vascular smooth muscle cells (including pulmonary) (340). Its activity is regulated by calcium-dependent binding of calmodulin. Type II NOS (~130 kDa; encoded by a gene on human chromosome 17) is inducible by a variety of factors related to inflammation and therefore is often referred to as inducible NOS (iNOS). It is regulated at the level of gene expression; once expressed, it produces NO at a high rate independently of the intracellular concentration of the free calcium ion ([Ca²⁺]_i). The third isoform, NOS III or endothelial NOS (eNOS; ~133 kDa), is encoded by a gene on human chromosome 7. In most endothelial cells and several other cell types, it is expressed constitutively, but the rate of the eNOS gene transcription and translation can be modulated by numerous factors, such as the shear stress of the endothelial surface (reviewed in Ref. 94). The eNOS enzyme activity is regulated by calcium-dependent binding of calmodulin and by tyrosine phosphorylation (111). Although the initial research of the physiology of NO in the vasculature focused on the eNOS, recent data suggest that at certain situations the remaining two NOS isoforms may also contribute to the vascular regulation (36, 291). More detailed discussions of NOS enzymology are available elsewhere (92, 94, 95, 150, 243, 250, 356).

The target tissue effects of NO depend on its quantity. At high concentrations, NO readily reacts with oxygen and especially with superoxide, forming highly reactive, cytotoxic substances, such as peroxynitrite. At lower concentrations, NO serves regulatory roles via activation of soluble guanylate cyclase, resulting in increased cGMP levels in target cells. In vascular smooth muscle, cGMP causes relaxation by reducing [Ca²⁺]_i and by downregulating the contractile apparatus (Fig. 2). These actions are mostly (although not exclusively; Ref. 97) mediated by type I (soluble) cGMP-dependent protein kinase (150, 398).

View larger version (28K): [in this window] [in a new window]   Fig. 2. Mechanisms of nitric oxide (NO)/cGMP-induced vasodilation. VOCC, voltage-operated calcium channels; ROCC, receptor-operated calcium channels; SERCA, sarcoplasmic/endoplasmic reticulum Ca2+-ATPase; IP3 channel, inositol 1,4,5-trisphosphate-gated calcium channel; [Ca2+]i, intracellular free calcium ion concentration.

The reduction of [Ca²⁺]_i by cGMP is accomplished in several ways. One is inhibition of calcium influx. Voltage- and receptor-operated calcium channels of the sarcolemma are directly phosphorylated and inactivated by the cGMP-dependent protein kinase (9, 28). In addition, cGMP-dependent protein kinase activates potassium channels of the sarcolemma (12, 13, 39), causing membrane hyperpolarization (13) and consequently reducing calcium influx through the voltage-operated calcium channels. In addition to the reduction of the extracellular calcium influx, cGMP also diminishes calcium release from the sarcoplasmic reticulum by blocking the inositol 1,4,5-trisphosphate-sensitive calcium release channel (186).

Enhanced calcium extrusion from the cytosol into the extracellular space also contributes to the vasorelaxant effect of cGMP. Ca²⁺-ATPase and the sodium/calcium exchanger of the sarcolemma are both stimulated by cGMP (108, 109). Calcium sequestration into the sarcoplasmic reticulum is also potentiated by cGMP via phospholamban-mediated activation of the Ca²⁺-ATPase of the sarcoplasmic reticulum (9, 374).

In vascular smooth muscle, tension depends on the phosphorylation status of the regulatory myosin light chain. Activation of the myosin light chain phosphatase is another means by which cGMP reduces vascular tone (202, 397, 398). In various tissues cGMP acts also by altering the activity of phosphodiesterases deactivating cAMP; in smooth muscle, however, this mechanism has not been documented. More details on the NO-cGMP signal transduction system can be found in extensive recent reviews (150, 246, 329).

	II. NITRIC OXIDE IN THE REGULATION OF THE BASAL TONE OF THE NORMAL PULMONARY VESSELS

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Because NO is a vasodilator continuously produced in many vascular beds, it has been suggested that a reduction of resting pulmonary vascular NO synthesis could be responsible for pulmonary hypertension (7, 71). Thus, to evaluate the role of NO in pulmonary hypertension, it is useful to first examine the basal NO production in the normal pulmonary circulation.

With respect to basal vascular tone, there is a qualitative contrast between the pulmonary circulation of a fetus and newborn on one side and that of an adult on the other. In the fetus, pulmonary vascular tone is similarly high as in the systemic circulation. In the neonatal period, pulmonary vascular tone decreases rapidly so that in older infants, children, and adults it is usually minimal. This review focuses primarily on the pulmonary circulation that has completed the perinatal transition from the fetal state. The role of NO in the fetal and neonatal pulmonary circulation, reviewed in detail elsewhere (2, 4, 89, 181, 307, 350), is summarized only briefly.

After a neonatal period, a normal, healthy pulmonary circulation is typically fully dilated. This statement is based not only on the well-known fact of a high flow and low pressure and resistance in the pulmonary circulation, but especially on a common finding that administration of vasodilators (in doses sufficient to cause profound systemic vasodilation) have very little or no effect on the pulmonary circulation of most healthy individuals (5, 69, 76, 104, 154, 317). The mechanisms responsible for this low basal tone of the pulmonary vessels are not clear. When NO was discovered as an endogenous vasodilator, an intriguing idea appeared that it could be a high continuous release of NO that keeps (or helps to keep) pulmonary vessels dilated. As discussed below, most of the data available today are not consistent with this possibility.

Approaches used to assess the role played by NO in the regulation of the normal, basal tone of the healthy pulmonary circulation include measuring pulmonary vasomotor effects of NOS inhibitors, studies of NOS mRNA and protein expression, and the use of transgenic mice.

The rationale behind using NOS inhibitors is as follows. If a continuous, basal production of NO (a vasodilator) helps to keep pulmonary vascular tone and resistance low, then inhibition of this basal production should increase pulmonary vascular resistance. Analogous reasoning led to the discovery of the continuous, "tonic" release of NO in the systemic vasculature when it was noticed that administration of NOS inhibitors caused systemic vasoconstriction (114-117, 294, 377). In the pulmonary vasculature, the reported effects of NOS inhibitors are variable. It is likely that differences in species and in NOS expression in different vascular segments contribute to the discrepancies in the literature. Therefore, in an attempt to bring some order into the plethora of findings, the following discussion of the effects of NOS inhibitors on the resting pulmonary vascular tone is sorted by species and experimental preparation.

A) RAT. I) Isolated perfused lungs. The initial attempts to elucidate the role of endothelium-derived relaxing factor (EDRF)/NO in the pulmonary circulation were started before the relatively selective NOS inhibitors, such as N-monomethyl-L-arginine (L-NMMA) or N-nitro-L-arginine methyl esther (L-NAME), became available. Brashers et al. (38) utilized the finding that EDRF activity was inhibited by the lipoxygenase antagonists eicosatetraynoic and nordihydroguaiaretic acids and by the antioxidant hydroquinone. Using isolated rat lungs, they found that the resting vascular tone was not significantly altered by these substances at doses effective in blocking the responses to endothelium-dependent vasodilators.

The first study utilizing an L-arginine-derived specific blocker to investigate the effect of NOS inhibition on the basal pulmonary vascular tone was published by Archer et al. (16). They found that L-NMMA (4.7 × 10⁴ M) did not alter baseline perfusion pressure in isolated rat lungs perfused with Krebs-albumin solution at a constant flow rate. The same dose of L-NMMA completely prevented the vasodilator response of preconstricted pulmonary vasculature to bradykinin, known to be EDRF dependent (52, 107), confirming that the dose was effective in inhibiting NOS. These data indicated that, unlike in the systemic vasculature, there is no physiologically significant basal synthesis of NO in the normal rat pulmonary circulation.

The finding that L-NMMA causes minimal or no vasoconstriction is isolated rat lungs perfused with artificial solution was subsequently independently confirmed (18, 26, 44, 74, 137) and expanded by using other NOS inhibitors and variations of the technique. For example, when blood was used instead of an artificial solution as a perfusate for the isolated rat lungs, L-NMMA again caused no physiologically significant increase in vascular resistance (20, 23, 100, 210, 309, 362, 412). Using both salt solution- and blood-perfused rat lungs, numerous authors found minimal or no vasoconstrictor response to more potent NOS inhibitors, namely, L-NAME (20, 73, 83, 134, 135, 158, 319, 359, 376, 406) and N-nitro-L-arginine (L-NA) (84, 134, 265, 302, 304, 318). Using a sensitive videomicroscopy system in isolated perfused rat lungs, Suzuki, Yamaguchi, and colleagues (359, 406) found that L-NAME altered neither the resting pulmonary vascular resistance nor the resting diameters of the pulmonary precapillary arterioles (20-30 µm).

A few authors found an increased vascular resistance in isolated rat lungs after administration of L-NA (130) or L-NAME (21, 311, 394), usually using relatively high doses (>3 × 10⁴ M). We believe that this vasoconstriction may have been caused predominantly by the nonspecific, NOS-unrelated effects of higher doses (11, 40, 209, 282, 371) because lower doses, which do not elicit pulmonary vasoconstriction, are sufficient to inhibit endothelium-dependent vasodilation (16, 74, 83, 84, 135, 158, 210, 265, 304, 318). In one study, the effects of L-NAME and L-NA were directly compared between the lung and kidney isolated from the same rat (134). Doses of both NOS inhibitors, which elicited massive vasoconstriction in the kidney, had no effect in the lung (Fig. 3). This finding confirms the presence of a continuous NO production in the renal circulation and its absence in the lung.

View larger version (18K): [in this window] [in a new window]   Fig. 3. Doses of N-nitro-L-arginine (L-NA), capable of causing marked renal vasoconstriction, do not produce pulmonary vasoconstriction. Lung and kidney isolated from the same rat were perfused at constant flow rate (so that changes in perfusion pressure directly reflect changes in vascular resistance). Data are means ± SE. [Data from Hampl et al. (134). Reprinted by permission of Blackwell Science, Inc.]

Dr. Taylor's group (21, 394) found a vasoconstrictor response to a relatively high dose of L-NAME in lungs perfused at constant pressure only when using higher viscosity perfusates (blood or salt solution containing >10% albumin), but not with perfusates of low viscosity. They argued that basal NO production existed in the pulmonary circulation as a result of shear stress, which was greatly reduced in lungs perfused with low viscosity solutions (because shear stress is a function of perfusate viscosity). This idea is intriguing because shear stress is a known and potent stimulus for endothelial NO production in vitro (60, 138, 167, 189, 192, 200, 236). However, Uncles et al. (376) found that even high doses of L-NAME had no effect on resting vascular tone in isolated rat lungs perfused with artificial perfusates of viscosity equal to that of the whole blood. On the other hand, in their experiments L-NAME caused pulmonary vasoconstriction when blood (even diluted, of relatively low viscosity) was used as a perfusate (376). Thus the issue of viscosity and shear stress remains controversial. In any case, these studies do not explain the findings of many authors of a minimal or no response to lower, yet effective, doses of NOS inhibitors in lungs perfused with blood (20, 23, 83, 210, 309, 319, 362, 412).

To summarize, most studies show that low doses of NOS inhibitors, effective in inhibiting endothelium-dependent vasodilation, do not cause a significant vasoconstriction in isolated perfused rat lungs.

II) Intact rats. The studies on intact rats show no significant pulmonary vasoconstrictor response to acute administration of L-NA (265) and L-NAME (98, 155) in doses (5-15 mg/kg iv) effective in increasing systemic vascular resistance. Higher doses of L-NAME (30-300 mg/kg iv) increased pulmonary arterial pressure in catheterized rats when lung flow was held constant (153). When pulmonary blood flow was not controlled, there was no significant change in pulmonary vascular resistance in response to L-NAME (153). Chronic oral treatment with L-NAME significantly increased systemic, but not pulmonary, arterial pressure in normal rats (132). L-NMMA, on the other hand, increased pulmonary vascular resistance in conscious rats when administered acutely (50 mg/kg iv) (229). The reason for this discrepancy is obscure, but it is quite likely that differences in technique do not play a role because the same laboratory using the same technique found no response to L-NAME (98) and a significant response to L-NMMA (229). One possible explanation is that L-NMMA has more nonspecific, NOS-unrelated effects than L-NA or L-NAME, and these, rather than reduction of NO synthesis, produce pulmonary vasoconstriction.

III) Isolated pulmonary arteries. Both unchanged (61, 198, 215, 265, 322, 363, 388) and increased (16, 127, 169, 322, 349, 405, 409) tension in response to NOS inhibitors have been reported in the isolated rat pulmonary arteries. Salameh et al. (322) found a constrictor response to L-NA in pulmonary arteries of one rat strain and its absence in another. Possible reasons for the discrepancies between vasoconstrictor response to NOS inhibition in many studies with isolated pulmonary arteries and its absence in most of the studies on isolated lungs or intact rats (see above) have not been directly addressed. It is likely that three factors may play a role: vessel size/type (conduit vs. resistance), resting passive tension, and precontraction by agonists.

Most of the studies in the isolated vessels utilize large, conduit arteries, which contribute relatively little to the total pulmonary vascular resistance in the whole lung. The total resistance is controlled mostly by the small, peripheral vessels (68). The large arteries express more eNOS (see sect. IIA2) and are more reactive to exogenous NO (13) than the smaller, more peripheral arteries. Several (61, 198, 215, 363), even though not all (349), studies performed on small, distal pulmonary arteries found no significant contractile response to NOS inhibitors. On the other hand, unchanged tension after administration of NOS inhibitors has also been described on several occasions in large pulmonary arteries in vitro (265, 388).

The degree of passive stretch of the vascular ring preparations is another potentially confounding factor. In most studies, the resting, passive stretch is set to a level that results in the largest constrictor response to depolarization induced by an elevation of the extracellular potassium concentration. For pulmonary arteries, the passive stretch force found in this manner is 500 mg or more, most often around 800 mg. With the use of the Laplace equation, the wall tension can be used to calculate the corresponding transmural pressure (245, 270). A stretch force of 500 mg corresponds to a transmural pressure of ~30 mmHg in the large pulmonary arteries and of ~50 mmHg in the smaller pulmonary arteries (270). In vivo, pulmonary arterial pressure above 20 mmHg is diagnosed as pulmonary hypertension (295). In other words, vasoconstrictor reactivity of the isolated pulmonary vessels is maximal at wall stretch levels corresponding to markedly elevated pulmonary arterial pressure. When this high level of wall stretch is used as a baseline, the administration of NOS inhibitors does not accurately address the problem of NO synthesis in the normal, resting pulmonary vasculature, even though vessels isolated from normal rats are used. Instead, the finding of a vasoconstrictor response to NOS inhibitors under these conditions (16, 127, 169, 349, 405, 409) indirectly supports the idea discussed below that basal NO synthesis is elevated in situations associated with increased pulmonary arterial wall tension. To yield more direct information about the role of NO in the normal, resting pulmonary vasculature, measurements would be needed of the responses to NOS inhibitors at a range of stretch values including those corresponding to the normal physiological transmural pressures. MacLean and McCulloch (215) found a negligible response of the rat pulmonary resistance arterial rings to L-NAME when transmural tension was set to 235 mg (equivalent to intravascular pressure of ~16 mmHg).

Similarly, although it is customary to use the pulmonary arterial rings precontracted with various agonists (typically, phenylephrine or norepinephrine), the nature of the interaction between the NOS inhibitors and the preexisting active tension is poorly defined. Consequently, it is not clear which degree of precontraction in vitro models the resting, usually fully dilated pulmonary circulation (5, 69, 76, 104, 154, 317) and which precontraction is more similar to a vasoconstricted state in vivo. Thus the interpretation of the results is uncertain.

B) DOG. Most of the available evidence indicates that L-NA and L-NAME do not cause pulmonary vasoconstriction in the dog. This is true in the isolated perfused left lower lobe (129), isolated whole lung (21, 64), and intact anesthetized (205) and conscious (256) dogs. On the other hand, Perrella and co-workers (278, 279) found significant pulmonary vasoconstrictor response to L-NMMA in anesthetized dogs. Thus, as in the intact rat, pulmonary vasoconstriction in dogs appears to be produced by L-NMMA, but not by L-NA or L-NAME, supporting the possibility that L-NMMA's vasoconstrictor action could be due to its more pronounced NOS-unrelated effects.

C) CAT. Originally, L-NAME (100 mg/kg iv) was shown to cause vasoconstriction in the pulmonary circulation of the cat (70, 231). The same laboratory recently published an elegant study in cats demonstrating that a novel NOS inhibitor, L-N⁵-(1-iminoethyl)-ornithine, increased pulmonary vascular resistance only at high doses (>10 mg/kg iv). These higher doses had no more inhibitory effect on the endothelium-dependent vasodilation to acetylcholine, bradykinin, and substance P than lower doses (1-10 mg/kg iv) that were without effect on the resting pulmonary vascular resistance (70) (Fig. 4). Thus NOS-unrelated effects of L-N⁵-(1-iminoethyl)-ornithine seem to be responsible for the pulmonary vasoconstriction. The authors themselves interpret their results as showing that the "basal release of NO does not play an important role in the maintenance of baseline tone in the pulmonary vascular bed of the cat" (70).

View larger version (21K): [in this window] [in a new window]   Fig. 4. Higher doses of a nitric oxide synthase (NOS) inhibitor, L-N5-(1-iminoethyl)-ornithine (L-NIO), required to produce pulmonary vasoconstriction, are not more effective in reducing endothelium-dependent vasodilation than lower doses, which do not change pulmonary vascular resistance in intact cats. [Data from DeWitt et al. (70).]

D) RABBIT. Data in rabbits are conflicting. On one hand, Persson and co-workers (280, 281, 392) found a significant vasoconstrictor response to L-NAME (30 mg/kg iv) in open-chest rabbits. On the other hand, other groups reported an unchanged vascular resistance in isolated rabbit lungs perfused either with a buffer solution or with blood after administration of L-NMMA (128) or L-NAME (172, 208, 400). Sprague et al. (346) suggested that erythrocytes may respond to mechanical deformation during their passage through microvessels by releasing cAMP that, in turn, evokes vascular NO synthesis. They found that rabbit and human (but not dog) erythrocytes release cAMP in response to mechanical deformation. L-NAME produced vasoconstriction in isolated rabbit lungs in the presence of human, but not dog, erythrocytes (346).

E) PIG. An increased pulmonary vascular resistance in response to L-NA and L-NAME was reported in anesthetized pigs (8, 380) and in isolated pig lungs (63, 64). However, a lack of a vasoconstrictor effect of L-NAME was also found in anesthetized pigs (77).

F) SHEEP. Today, sheep are the only species where NOS inhibitors appear to consistently cause pulmonary vasoconstriction; we are unaware of studies in which NOS inhibitors would have not increased pulmonary vascular resistance in sheep. L-NA and L-NAME (20-25 mg/kg iv) were reported to cause pulmonary vasoconstriction in an intact adult sheep (185, 211, 237) and in isolated sheep lung (64).

G) HORSE. In resting horses, L-NAME (20 mg/kg iv) caused a minimal rise in pulmonary arterial pressure (218). In this study, the pulmonary arterial pressure value before L-NAME administration is not given, but it is said to be similar to that in horses in a control study (no L-NAME given), where it was 31.3 ± 1.2 mmHg. After L-NAME administration, the pulmonary arterial pressure was 31.4 ± 1.0 mmHg. At the same time, L-NAME increased systemic blood pressure by 36 mmHg. Thus the pulmonary response to L-NAME was relatively negligible in comparison with the systemic effect. This conclusion is further reinforced by the results in exercise, showing that the pulmonary vascular pressure-flow relationship was unaffected by L-NAME (218).

H) MOUSE. In mice, acute administration of L-NAME (100 mg/kg iv) caused systemic vasoconstriction, but both pulmonary arterial pressure and pulmonary vascular resistance were unchanged (354). Similarly, when L-NAME was infused in mice by minipumps for 5 days (100 mg · kg¹ · day¹), systemic arterial pressure and vascular resistance were significantly elevated, whereas pulmonary arterial pressure and vascular resistance were not (354). Endothelium-dependent vasodilation to acetylcholine was inhibited. In isolated mouse lungs perfused at constant flow rate, acute L-NA administration (10⁴ M) did not alter baseline perfusion pressure (80).

The mouse is a species with an advantage of a technical feasibility of producing individuals with targeted gene disruption. Steudel et al. (354) were the first to study the pulmonary circulation in mice with targeted disruption of the gene encoding eNOS. As expected, the eNOS / mice had impaired endothelium-dependent vasodilator response to acetylcholine and marked systemic hypertension. They also had a somewhat higher mean pulmonary arterial pressure (19.0 ± 0.8 mmHg) than the wildtype mice (16.4 ± 0.6 mmHg) and significantly elevated total pulmonary resistance due to reduced cardiac output (354). Fagan and co-workers (79, 80) confirmed that the mice with the null mutation of the eNOS gene had increased right ventricular systolic pressure compared with the wild-type mice. Right ventricular systolic pressure was normal in nNOS / mice and elevated in mice with iNOS gene disruption, although to a lesser degree than in the eNOS / mice (80). However, it is useful to keep in mind that the studies by Fagan and co-workers (79, 80) were performed at an altitude of ~1,600 m. It has been shown previously that even the very mild hypoxia experienced at that altitude is sufficient to elicit pulmonary hypertension in a susceptible rat strain (326). It is thus possible that the elevated right ventricular systolic pressure found in eNOS / mice in mild hypoxia (79, 80) may reflect the role of eNOS in limiting the pulmonary hypertensive response to chronic hypoxia (discussed in sect. IV).

It is striking in the study of Steudel et al. (354) that the transgenic eNOS-deficient mice have moderate pulmonary hypertension whereas the wild-type mice treated with NOS inhibitor have none. An explanation of this paradox can be related to the role of NO in the neonatal pulmonary circulation (see sect. IIB). Endogenous NO production is known to be essential for a successful transition of the fetal (high pressure, low flow) pulmonary circulation into the postnatal (low pressure, high flow) one (3, 59). Thus it is likely that the postnatal transition of the pulmonary circulation of the eNOS-deficient transgenic mice was impaired in a manner similar to that shown in newborn lambs treated with NOS inhibitors (3, 59). In this respect, the moderate pulmonary hypertension seen in the transgenic eNOS-deficient mice (354) appears to be a consequence of an incomplete postnatal transition of the pulmonary circulation rather than a consequence of the absent NO production at the time of measurement. In contrast, the acute and the 5-day-long treatment of the adult wild-type mice with L-NAME did not affect the neonatal development of the pulmonary circulation and only could influence the NO synthesis at around the time of measurements. Thus, with respect to studying the role of NO in the pulmonary vascular tone regulation in adults, the results with NOS inhibitors in wild-type mice could be more relevant than the results with transgenic eNOS-deficient mice, which are invaluable in confirming the importance of NO for normal development of the pulmonary circulation. This possibility is supported by the observation that the increased pulmonary vascular resistance in the transgenic eNOS-deficient mice was not reduced by exogenous NO (354).

I) HUMAN. As in other species, data on the effects of NOS inhibition on the human pulmonary circulation are somewhat conflicting.

Stamler et al. (348) reported that L-NMMA caused pulmonary vasoconstriction in healthy human volunteers. However, their study showed a substantially lower reactivity of the pulmonary, compared with systemic, circulation to L-NMMA (Fig. 5). Pulmonary vascular resistance was increased only by the highest dose used (1 mg · kg¹ · min¹ iv), which represented the limit tolerable by the systemic circulation (31, 348). The pulmonary vascular resistance increased as a result of a drop in cardiac output; pulmonary arterial pressure was unchanged. Systemic arterial pressure and vascular resistance were significantly increased not only by this higher dose, but also by doses as much as 10 times lower, which had no effect on the pulmonary vascular resistance (Fig. 5). Thus, in humans, as in other species (see above), L-NMMA in doses sufficient to inhibit NO production in the systemic vessels had no effect on the pulmonary circulation. Perhaps the nonspecific, NOS-unrelated effects of L-NMMA could be important in the pulmonary vasoconstriction seen at high doses.

View larger version (22K): [in this window] [in a new window]   Fig. 5. N-monomethyl-L-arginine (L-NMMA) causes pulmonary vasoconstriction in human volunteers only at a high dose, whereas lower doses are sufficient for systemic vasoconstriction. Data are means ± SE. *P < 0.05. P < 0.01. [Data from Stamler et al. (348).]

Using intravascular Doppler sonography, Celermajer et al. (47) observed dose-dependent decreases in segmental pulmonary blood flow in response to locally infused L-NMMA in six children with congenital heart disease and normal pulmonary vascular resistance.

Data obtained on in vitro human preparations are also inconclusive. On one hand, a relatively high dose of L-NMMA (10⁴ M) did not increase tension of resting isolated small human intrapulmonary arteries (61). On the other hand, a vasoconstrictor response to a lower dose of L-NAME (10⁵ M) was found in isolated perfused human lungs (64). Nonetheless, this response was only partially reverted by excess L-arginine, suggesting the possibility of participation of NOS-unrelated effects of L-NAME. Samples of tissue obtained from human patients tend to be inhomogeneous, possibly contributing to discrepancies among studies.

Plasma concentration of an endogenous NOS inhibitor, N^G,N^G-dimethylarginine, is elevated in patients with chronic renal failure to a level which inhibits NOS in vitro (378). Pulmonary hypertension does not belong among the complications of chronic renal failure (22). Although very indirect, this fact is consistent with the possibility that NO is less important in the regulation of the pulmonary than systemic vascular tone in humans.

J) INTERIM SUMMARY: EFFECTS OF NOS INHIBITORS ON BASAL PULMONARY VASCULAR TONE IN ADULTS. The studies of the effects of NOS inhibitors on the resting pulmonary vascular tone in adults of different species are summarized in Table 1. In the rat, numerous studies using the isolated buffer-perfused lungs consistently show no response to NOS inhibitors. The same finding is most often reported in blood-perfused lungs, even though a significant vasoconstriction has also been repeatedly found in this preparation. Similarly, reports of no significant response appear to prevail in the intact rat, although a vasoconstriction has also been seen. The studies on the isolated rat pulmonary arterial rings in vitro, showing both an increase and no change in tone after NOS inhibition, are difficult to evaluate conclusively because of the lack of characterization and normalization of the passive and active tension.

The canine pulmonary circulation is not reactive to NOS inhibitors in most studies, although again, there are exceptions. In the cat, vasoconstriction has been shown in response to L-NAME, but not to L-N⁵-(1-iminoethyl)-ornithine at a dose sufficient to inhibit NO-dependent vasodilation. In the rabbit, a vasoconstrictor response to NOS inhibitors has been shown in open-chest studies but not in isolated lung experiments. In pig and sheep, virtually all published studies except one (77) found a pulmonary vasoconstrictor response to NOS inhibitors. In the horse and mouse, on the other hand, there is minimal or no response to L-NAME. In humans, the limited evidence available so far appears to support the existence of a vasoconstrictor response to L-NMMA and L-NAME. However, only the highest L-NMMA dose tolerated by the systemic circulation is effective in the pulmonary circulation in conscious volunteers (348), and L-NMMA does not constrict isolated human peripheral pulmonary arterial rings in vitro (61).

Hence, in most species NOS inhibitors do not consistently cause significant pulmonary vasoconstriction in doses that minimize the risk of nonspecific effects yet are effective in NOS inhibition. However, the presence of a significant pulmonary vasoconstrictor response to NOS inhibitors has been reported more often in certain species, namely, the pig, sheep, and human. Thus the crucial question whether the response in humans is substantially similar to that in experimental animals remains without a definitive answer.

In rats, NADPH diaphorase staining (a classical method for constitutive NOS localization), immunohistochemical studies using eNOS antibodies, and in situ hybridization with eNOS mRNA probe found essentially no NOS in the endothelium of the small, peripheral pulmonary arteries (those most responsible for pulmonary vascular resistance) (173, 201, 375, 402, 403). In contrast, eNOS expression was considerable in large pulmonary vessels, while <25% of medium-sized vessels showed eNOS staining (Fig. 6). No NOS immunostaining was detected in the smooth muscle of the pulmonary vessels of all sizes (403). Inducible NOS mRNA was not detectable by reverse-transcriptase polymerase chain reaction in the pulmonary arterial wall of normal rats (44).

View larger version (12K): [in this window] [in a new window]   Fig. 6. Endothelial NOS (eNOS) expression is marked in the large pulmonary vessels of the rat and negligible in the peripheral ones. [Data from Xue et al. (403).]

Data on NOS expression in humans are contradictory. Kobzik et al. (184) found variable NADPH diaphorase staining in the endothelium of large pulmonary arteries and no staining in the pulmonary microvasculature. That resembles the results in the rat (173, 201, 402, 403). On the other hand, Giaid and Saleh (125) reported dense eNOS immunostaining in pulmonary arteries of all sizes. Several details of their technique were challenged by Xue and Johns (401), who themselves observed only a weak eNOS immunostaining in normal human pulmonary vessels.

An interesting possible source of NO in the pulmonary circulation might be NO produced in the paranasal sinuses (213) and inhaled with each breath. When autoinhalation of nasal NO was prevented in patients recovering from open heart surgery by having them breathe through their mouth, their pulmonary vascular resistance was slightly but significantly higher than when they breathed through their nose (336). However, pulmonary vascular resistance was slightly reduced in only 4 of 12 intubated, ventilated patients (who cannot inhale NO from their nose) when the air derived from the patient's own nose was aspirated and led into the inhalation limb of the ventilator (212). Hence, this interesting idea and its relevance to the normal, healthy pulmonary circulation needs more experimental clarification.

Pulmonary circulation in the fetus differs substantially from that in the adult. In the adult, where blood is oxygenated in the lung, the whole cardiac output flows through the lung at a low pressure. Vascular resistance is very low. In the fetus, the oxygenation of the blood does not take place in the lung, and the lung receives only a fraction of cardiac output at high pressure. Vascular resistance is high. In these aspects the normal fetal pulmonary circulation bears more similarities to the systemic vascular beds or to the hypertensive pulmonary circulation of the adult than to the normal adult pulmonary vascular bed. Correspondingly, the role of NO may differ between normal fetal and adult pulmonary circulation. A detailed discussion of the role of NO in the fetal and neonatal pulmonary circulation, available in relevant reviews (2, 4, 89, 181, 307, 350), is beyond the scope of this review; we briefly summarize only the aspects important for a comparison with the situation in the adult.

eNOS immunostaining is dense and eNOS mRNA level is high in the fetal pulmonary circulation; they both decrease postnatally (131, 148, 173, 258, 404). Lung eNOS mRNA levels and immunoreactivity are high in the fetal rat, highest around the first postnatal day, and minimal in adult rats (148, 173, 404) (Fig. 7). There is even evidence for iNOS expression and hemodynamically relevant activity of iNOS in the fetal pulmonary circulation (291, 404), reminiscent of the iNOS expression in pulmonary hypertension in adults (see sect. IVC). NOS inhibitors cause pulmonary vasoconstriction in fetal (3, 182, 241) and newborn (67, 88, 251, 252, 277) lambs, piglets, and guinea pigs. The magnitude of this response decreases with postnatal age (277).

View larger version (51K): [in this window] [in a new window]   Fig. 7. Pulmonary eNOS gene expression culminates at the perinatal period and decreases with postnatal age. RNA extracted from lungs of individual fetal (days 18.5, 19.5, 20.5, and 21.5 of 22-day gestation), postnatal (1, 4, and 8 h and 1, 2, 8, 16, and 26 days after birth), and adult (Ad) rats were hybridized with radiolabeled NOS cDNA and 18S oligonucleotide probes (the later to confirm integrity of the extracted RNA). [From Kawai et al. (173).]

Endogenous NO synthesis plays an important role in the transition of the pulmonary circulation from the high-resistance fetal state to the low-resistance postnatal one at birth. The postnatal decline of pulmonary vascular resistance in lambs is attenuated by acute (3, 59, 182, 241) and chronic (90) L-NA treatment. The pulmonary vasodilation at birth is caused to a great extent by the increase in lung PO₂. The oxygen-induced decline in the pulmonary vascular resistance in late fetal lambs is attenuated by L-NA (49, 59, 233, 241, 364).

Thus the available evidence is consistent with the view that basal NO synthesis in the pulmonary circulation is high in the fetus, culminates at the time of birth, declines postnatally, and is relatively small during adulthood.

	III. ROLE OF NITRIC OXIDE IN PULMONARY VASOCONSTRICTION

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Vasoconstriction is among the factors contributing to the development of most forms of chronic pulmonary hypertension. It is therefore useful to briefly explore the NO activity during pulmonary vasoconstriction.

One of the most physiologically important pulmonary vasoconstrictor stimuli is alveolar hypoxia, and much of our knowledge of the pulmonary vasoreactivity comes from experiments studying hypoxic pulmonary vasoconstriction. Because oxygen is needed for NO synthesis, hypoxia has been hypothesized to cause pulmonary vasoconstriction by inhibiting basal NO synthesis in the pulmonary vasculature. Indeed, the apparent Michaelis constant (K_m) of the eNOS for oxygen, 7.7 µM (299), predicts that NO output can be significantly reduced when PO₂ drops below ~30 mmHg. However, as discussed below, there is a solid evidence available today that physiologically relevant degrees of acute hypoxia actually potentiate NO synthesis in the pulmonary circulation, as do other vasoconstrictor stimuli. This increased NO production limits the vasoconstrictor-induced increase in intravascular pressure. Apparently, the effect of the reduced substrate (O₂) availability on the eNOS activity can be overridden during pulmonary vasoconstriction by other regulatory mechanisms, such as the elevated [Ca²⁺]_i (133), at least if hypoxia is not too severe. In this context it is useful to keep in mind that in vivo, PO₂ in the adult pulmonary circulation does not drop below ~30 mmHg even in hypoxia as extreme as that experienced during exercise on the summit of Mt. Everest (358).

It should also be noted that in contrast to the pulmonary vessels, the production of NO in the distal airways is reduced during ventilatory hypoxia (128). It is possible that at least a portion of this NO is produced by the neuronal isoform of NOS. Its K_m for oxygen (23.2 µM) is higher than that of the endothelial isoform (299). Even less severe degrees of hypoxia therefore may suppress its activity. However, pharmacological inhibition of the airway NO production does not mimic hypoxic pulmonary vasoconstriction (128), suggesting that the hypoxic decrease in airway NO synthesis is not responsible for the increase in the pulmonary vascular tone.

The studies of the effects of NOS inhibitors on pulmonary vasoconstriction are summarized in Table 2. As already mentioned, the initial attempts to determine the role of EDRF in the pulmonary circulation started before the selective NOS inhibitors became available. It was shown that several putative blockers of the EDRF-cGMP pathway, including eicosatetraynoic and nordihydroguaiaretic acids (lipoxygenase antagonists), hydroquinone (antioxidant), and methylene blue (gyanlylate cyclase inhibitor), potentiated the hypoxic vasoconstriction in the isolated rat lungs (38, 228). The baseline tone was unaffected.

After the more specific, L-arginine-based NOS inibitors became available, Archer et al. (16) were the first to show that L-NMMA, while not changing the baseline vascular tone, significantly potentiates the vasoconstrictor responses of isolated rat lungs to hypoxia and angiotensin II (Fig. 8). A logical interpretation is that vasoconstriction increases the normally low NO synthesis in the pulmonary circulation. This observation was subsequently confirmed many times in isolated rat lungs using L-NMMA, L-NAME, and L-NA for NOS inhibition and hypoxia, angiotensin II, endothelin-1, dexfenfluramine, or a thromboxane analog U46619 as vasoconstrictor stimuli (18, 20, 23, 73, 83, 100, 137, 158, 210, 265, 302, 309, 359, 362, 390, 395, 406, 412). Vasoconstrictor responses to acute hypoxia and angiotensin II were also potentiated in lungs isolated from rats after treating them with L-NAME for 3 wk (132). Acute administration of L-NAME or L-NMMA potentiated hypoxic pulmonary vasoconstriction in awake (98, 229) and anesthetized (155) rats. Pulmonary vasoconstrictor response to U46619 was potentiated by L-NAME in anesthetized rats (153). NOS inhibitors added to the bath of isolated rat pulmonary arterial rings in vitro contracted by various stimuli cause further increase in tension (11, 16, 127, 169), although no change (198) or even a decrease (169, 322) in hypoxic contraction has also been observed in this preparation. Using intravital videomicroscopy, Suzuki et al. (359) observed no potentiation of the hypoxic contraction of very small pulmonary arterioles (~25 µm) by L-NAME, although the total pressor response of the whole lung was markedly increased. This suggests that NO production is stimulated only in a portion of vessels affected by vasoconstriction.

View larger version (16K): [in this window] [in a new window]   Fig. 8. Inhibition of NOS by N-nitro-L-arginine methyl ester (L-NAME) potentiates pulmonary vasoconstrictor reactivity to angiotensin II and hypoxia. A representative tracing is shown of an experiment in isolated rat lungs perfused with Krebs-albumin solution at constant flow rate (so that increases in pressure directly reflect vasoconstriction). Thin line, control run without L-NAME; thick line, a subsequent run in the presence of 5 × 105 M L-NAME. Two subsequent control runs did not differ from one another in a separate group of lungs.

A potentiation of the pulmonary vasoconstrictor responses to hypoxia and U46619 by L-NA, L-NAME, or L-NMMA was observed in conscious (256) or anesthetized (41, 204, 205, 278) dogs and in isolated left lower lobe of the dog lung (129). Increased reactivity to hypoxia, U46619, or angiotensin II was also found after NOS inhibitors administration in intact (347) and open-chest rabbits (280, 281, 392) and in isolated rabbit lungs (128). Hypoxic pulmonary vasoconstriction was increased by L-NAME in intact pigs (77, 102) and by L-NMMA in isolated pig intrapulmonary arteries (263). Hypoxic pulmonary vasoconstriction potentiated by L-NMMA was also reported in human patients (31). Phenylephrine-increased tension was further elevated by L-NMMA in human small pulmonary arteries in vitro (61).

Hypoxic vasoconstriction is potentiated by L-NA in isolated perfused mouse lungs (80). Lungs isolated from transgenic mice with targeted disruption of the eNOS gene show hypoxic vasoconstriction that is about twice as large as that seen in lungs of wild-type mice, but cannot be increased further by L-NA (80). This suggests that eNOS is the main source of NO produced in the pulmonary circulation during acute hypoxia. Hypoxic vasoconstriction is normal in lungs isolated from both nNOS / mice and iNOS / mice (80).

The potentiation of pulmonary vasoconstriction by NOS inhibitors suggests that vasoconstriction increases NO synthesis in the pulmonary circulation. A more indirect proof for this conclusion was added by Cohen et al. (58). They found that hypoxic vasoconstriction was reduced in isolated rat lungs by an inhibition of cGMP phosphodiesterase, which did not change the baseline tone. Because cGMP phosphodiesterase inactivates NO's second messenger, cGMP, this finding is consistent with increased NO levels during hypoxic pulmonary vasoconstriction, although other factors than NO can elevate cGMP.

Direct measurements of NO and its oxidation products (NO_x) in the lung effluent during acute hypoxia are infrequent. Grimminger et al. (128) found unchanged NO_x in perfusate of isolated rabbit lungs during ventilation with a hypoxic gas, although hypoxic pulmonary vasoconstriction in that study was markedly potentiated by L-NMMA. Naoki et al. (249), on the other hand, reported a significant increase in perfusate NO_x during acute hypoxia in isolated rat lung. The reason for the discrepancy is unknown, although it might be related to gradual improvements in the sensitivity of NO detection.

The mechanism whereby vasoconstriction increases NO synthesis in the pulmonary circulation has not been much studied. Wilson et al. (395) found that L-NMMA potentiated the U46619-induced vasoconstriction only in lungs perfused at constant flow, but not at constant pressure. One of the key differences between constant flow and constant pressure perfusion is that vasoconstriction increases shear stress only in the former. Therefore, these data suggest that the stimulus for increased NO synthesis during pulmonary vasoconstriction is the increase in shear stress. Shear stress is known to be a potent stimulus for endothelial NO synthesis (60, 138, 167, 189, 192, 200, 236). However, additional factors must be at play because NOS inhibitors potentiate vasoconstriction in isolated pulmonary arterial rings in vitro (11, 16, 127, 169, 265), where there are no changes in shear stress. Hypoxia itself, in the absence of changes of shear stress or vascular smooth muscle tone, increases NO synthesis in cultured pulmonary artery endothelial cells (133). Further supporting a direct, shear stress-independent influence of hypoxia on the eNOS in the pulmonary vasculature is a recent report by Le Cras et al. (200). Using rats, they found that eNOS is upregulated by chronic hypoxia equally in the normal right lung and in the left lung, in which blood flow (and therefore shear stress) had been severely reduced by creating a left pulmonary artery stenosis (see also sect. IVC).

	IV. NITRIC OXIDE SYNTHESIS IN CHRONIC PULMONARY HYPERTENSION

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The methods used to study the role of NO in chronic pulmonary hypertension include measurements of the effects of NOS inhibitors (or eNOS gene deficiency) on pulmonary vascular tone, measurements of NOS activity, studies of NOS expression, and evaluation of reactivity to endothelium-dependent vasodilators.

Several laboratories have shown that acute administration of L-NMMA, L-NAME, or L-NA to isolated adult rat lungs, which is usually without much effect in normal, control rats (see sect. IIA1), causes a marked vasoconstriction in lungs of rats with chronic hypoxic pulmonary hypertension (20, 158, 265, 319, 375, 376) (Fig. 9). Similar presence of a marked vasoconstrictor response to NOS inhibitors (minimal in controls) was reported in intact rats with chronic hypoxic pulmonary hypertension (155, 265) and in pulmonary arterial rings isolated from such rats (215, 265, 388). In contrast, one study found that constriction in response to L-NA was reduced by chronic hypoxia in rat conduit pulmonary artery rings in vitro (169).

View larger version (18K): [in this window] [in a new window]   Fig. 9. Vasoconstrictor response to L-NA in isolated rat lungs increases with progression of chronic hypoxic pulmonary hypertension. Baseline perfusion pressure in the isolated rat lungs and its increase upon acute addition of L-NA are shown (means ± SE). [Data from Oka et al. (265).]

An increase in vascular resistance in response to L-NMMA or L-NA was also found in isolated lungs of rats with pulmonary hypertension induced by an injection of an alkaloid, monocrotaline (100, 265, 375), and of a rat strain with spontaneous pulmonary hypertension (375). These findings suggest that the increased vasoconstrictor reactivity to NOS inhibitors is related to the presence of pulmonary hypertension rather than to chronic hypoxia itself. This conclusion is further supported by the observation that the hyperreactivity to NOS inhibitors persists after 2 days of recovery from chronic hypoxia (when some degree of pulmonary hypertension is still present) (265). Pulmonary resistance arterial rings isolated from chronically hypoxic rats constrict significantly in response to L-NAME only when they are passively stretched to a tension corresponding to pulmonary hypertension (215). On the other hand, vasoconstrictor response to L-NA was not found in control lungs even after increasing flow rate to such a degree that the perfusion pressure reached the level found in chronically hypoxic lungs (20, 265). That suggests that the high intravascular pressure per se is not responsible. The vasoconstrictor response to NOS blockers in isolated lungs can be prevented by endothelin receptor blockers, as has been shown in rats with pulmonary hypertension induced by chronic hypoxia (247) or monocrotaline injection (100). The authors of these studies interpreted the data as showing that endogenous NO synthesis is elevated in the hypertensive lung and opposes vasoconstriction produced by the potent endogenous pulmonary vasoconstrictor endothelin-1 (50, 199, 287), the levels of which are increased in numerous forms of pulmonary hypertension (126, 206, 352). Endothelin is a known stimulus for NO synthesis (86, 287, 410).

Aside from studies showing an increased pulmonary vasoconstrictor response to NOS inhibitors in rats with chronic hypoxic pulmonary hypertension, there are also publications reporting a lack of such a potentiation (302, 304, 318, 412). The doses of L-NMMA and L-NA in these studies were sufficient to inhibit endothelium-dependent vasodilation (304) or to potentiate reactivity to vasoconstrictor stimuli (302, 318, 412). Cremona et al. (64) found no significant difference in vasoconstrictor response to L-NAME between isolated lungs from healthy human donors and lungs of patients with pulmonary hypertension. The reason for this discrepancy from other studies is unclear. Nevertheless, it is safe to conclude that experiments studying the effects of NOS inhibitors on the resting pulmonary vascular tone in adults do not support NO deficiency in pulmonary hypertension (Table 3). In the neonates, where the role of NO in the basal pulmonary vascular tone regulation seems different than in adults (see sect. IIB), there is support both for (85) and against (29) reduced lung NO production in pulmonary hypertension.

Recently, mice with a targeted disruption of the eNOS gene began to be utilized to study the role of eNOS in pulmonary hypertension (79, 355). Steudel et al. (355) found that after 3-6 wk of hypoxia, several indices of pulmonary hypertension were greater in the eNOS-deficient than in the wild-type mice, including right ventricular systolic pressure, pulmonary arterial pressure, incremental total pulmonary vascular resistance, pulmonary vascular remodeling, and right ventricle free wall weight and thickness. The difference between the eNOS-deficient and wild-type mice was especially marked in the proportion of muscularized small pulmonary vessels. This proportion is often considered an essential factor determining the severity of pulmonary hypertension (297). Fagan et al. (79) reported that eNOS-deficient mice were especially sensitive to a relatively mild degree of chronic hypoxia, whereas with severe hypoxia these authors did not find right ventricular systolic pressure to be significantly different between the wild-type and genetically manipulated mice. As in the study of Steudel et al. (355), the proportion of muscularized small pulmonary vessels was increased by chronic hypoxia considerably more in the eNOS-deficient than in the wild-type mice (79).

Only a few studies measured NOS activity in adult pulmonary hypertension. Isaacson et al. (158) found negligible accumulation of NO_x in the recirculating artificial perfusate of lungs isolated from control rats. In lungs isolated from rats with chronic hypoxic pulmonary hypertension, however, NO_x accumulation in the perfusate was significantly elevated (158, 325, 375) (Fig. 10). The faster NO_x accumulation in the perfusate of lungs of chronically hypoxic rats can be prevented by endothelin type B receptor antagonism (325). This suggests that endothelin-1, known to be upregulated in pulmonary hypertension (126, 206, 352), activates NOS via its action on type B receptors. Sato et al. (325) also found that perfusate NO_x accumulation is not accelerated in lungs of chronically hypoxic rats if they are ventilated with anoxic gas. The relevance of this finding to less severe hypoxia, compatible with long-term survival, remains to be elucidated. Although acute hypoxia decreases otherwise elevated plasma NO_x in intact rats with chronic hypoxic pulmonary hypertension (325), the contribution to this finding of NO produced in the systemic circulation is likely but unknown. The tendency for an increased release of NO into the perfusate of the isolated rat lungs did not reach significance in monocrotaline-induced pulmonary hypertension (375).

View larger version (11K): [in this window] [in a new window]   Fig. 10. Accumulation of NO and its oxidation product, nitrite, in the perfusate of isolated rat lungs is increased in chronic hypoxic pulmonary hypertension. Nitrite plus NO (NOx) was measured by chemiluminescence. Nitrite was first reduced to NO by potassium iodide at low pH. Data are means ± SE. *P < 0.05. [Data from Isaacson et al. (158).]

NOS activity in a whole