**3. Endogenous NO production**

vascular relaxation. In this chapter we will discuss NO and its regulation and function with special references to the development of PH as well as pulmonary vascular reactivity in PH.

NO activates soluble guanylyl cyclase (sGC) stimulating cGMP production and subsequent activation of cGMP-dependent protein kinase (PKG). This sGC-cGMP-PKG pathway plays a major role in NO-mediated regulation. In addition to this pathway, NO directly binds to proteins and induces conformational changes with subsequent functional alterations, like phosphorylation. Thus, S-nitration is also called S-nitrosylation, the term which emphasizes a biological effect of the chemical reaction of S-nitration [16]. S-nitrosylation modifies the activity of some kinases and phosphatases, thus raising the possibility that NO modifies phosphory‐

NO reacts with oxygen, transitional metal ions, thiols, and superoxides, exerting its effects via cGMP-dependent and/or -independent pathways. cGMP effector molecules include cGMPdependent protein kinases type-I and –II, cGMP-activated phosphodiesterases, and cGMPgated ion channels. Similar to phosphorylation, S-nitrosylation regulates protein function

In the vascular system, NO reacts with sGC forming cGMP, which activates cGMP-dependent protein kinase decreasing vascular smooth muscle cell cytoplasmic Ca2+ concentration by 1) activation of proteins such as Ca2+-sensitive potassium channels which decrease membrane potential thereby causing hyperpolarization and closing voltage dependent Ca2+ channels; 2) phosphorylation of voltage- and receptor-operated sarcolemmal Ca2+ channels, causing them to close; 3) inhibition of the inositol 1,3,5-trisphospate-sensitive Ca2+ release channel of the

NO mediates vasorelaxation, anticoagulation, and anti-proliferation, as well as neurotrans‐ mission. Several earlier studies demonstrated that NO inhibits smooth muscle cell growth by a cGMP-dependent mechanism [18] in addition to inhibiting growth regulating enzymes such as ribonucleotide reductase and thymidine kinase [19,20]. NO also suppresses the hypoxiainduced increase in ET-1 and platelet-derived growth factor-B, both of which have vasocon‐ striction and growth effects [21]. These effects of NO led investigators to determine whether administration of NO prevents the development of PH. Chronic NO inhalation ameliorates the development of hypertensive pulmonary vascular changes of chronic hypoxia-induced PH in rats [22], but not in monocrotaline (MCT)-induced PH [23]. In contrast, supplementation with the NO precursor, L-arginine, but not D-arginine prevented the development of PH in both models [24]. The reason for the different effects of NO inhalation is unclear, but may be a result of differing pathogenic mechanisms in the two models of PH: the increase in pulmo‐

**2.1. NO acts through the sGC pathway and S-nitrosylation of target proteins**

lation and dephosphorylation through S-nitrosylation.

allosterically or by direct modification of cysteine.

sarcoplasmic reticulum [17].

**2.2. NO prevents the development of PH**

**2. Biological effects of NO**

76 Pulmonary Hypertension

#### **3.1. Nitrate (NO3 - ) and nitrite (NO2 - ) as sources of NO (Figure 1)**

NO is produced from L-arginine by nitric oxide synthase (NOS) in the presence of oxygen, tetrahydrobiopterin (BH4), and reduced NADPH[3]. Recent studies have indicated that inorganic anions, nitrate (NO3 - ) and nitrite (NO2 - ), can be recycled to NO in vivo as alternative sources of NO in addition to the classical NOS-NO pathway. The source of nitrate includes the endogenous NOS-NO synthase pathway and the diet. Green vegetables such as lettuce and spinach provide nitrate and preservatives in cured meat and bacon include nitrite. Basically reduction of nitrate and nitrite produce NO, thus nitrate and nitrite are considered an 'endo‐ crine reservoir' of NO [28].

Nitrate in the plasma is excreted into the saliva, whereas nitrate is reduced by the oral anerobic bacteria producing nitrite. These bacteria use nitrate as an electron acceptor instead of oxygen during respiration. During its subsequent movement into the stomach, nitrite undergoes further reduction to NO, thus leading to gastric NO formation, which may play a role in gastric mucosa maintenance. This is a entro-salivary circulation of nitrate. In the systemic circulation intravascular nitrite is reduced to NO by deoxyHb, respiratory chain enzymes, xanthine oxidoreductase, deoxygenated myoglobin, and protons ( 29 ). They facilitate the transfer of protons to NO2 - , causing NO production which is intensified under acidic and hypoxic states. Artery-to-vein gradients in nitrite are observed.

Nitrite has a vasodilatory effect. Inhaled nebulized sodium nitrite reduces pulmonary artery pressure (PAP) without changes in systemic artery pressure in hypoxia- or thromboxane-

**Figure 1.** Recycling of NO from NO2- Endogenous NO includes NO produced from L-arginine by NOS and recycled NO from NO2 - . NO is converted to NO3 by the reaction with the Hb and /or to NO2 by the oxidation in the plasma with the aid of multicopper oxidase and NO oxidase ceruloplasmin. NO3 - is excreted into urine by kidney and/or into oral cavity by salivary gland. In the oral cavity anaerobic bacteria reduces NO3 converting to NO2 - , which goes down into stomach and is protonated under the gastric acidic state forming nitrous acid (HNO2) with further decomposition to NO and/or other nitrogen oxides. NO2 in the plasma is reduced and converted to NO by the reductase activity of deoxygenated hemoglobin, xanthine oxidoreductase, respiratory chain enzymes, and hydrogen ion. Hb(FeII), deoxygenated hemo‐ globin;Hb(FeII)O2, oxygenated hemoglobin; NO, nitric oxide; NOHb(FeII), nitrosylhemoglobin; NOS, nitric oxide syn‐ thase; NO2 - , nitrite, NO3 - , nitrate; XOR, xanthine oxidoreductase; Hb(FeIII), methemoglobin; REC, respiratory chain enzymes

**Figure 2. Coupled eNOS (eNOS homodimer) produces NO.** (a) eNOS homodimer produces NO, whereas eNOS monomer produces superoxide. eNOS uncoupling occurs during the conversion of eNOS homodimer to eNOS mono‐ mer. Two eNOS monomers are connected with the aid of Zn2+, making eNOS homodimer. BH4 strengthen the Zn2+ connection, maintaining the dimer form. In coupled NOS, an electron is transferred to L-arginine, producing NO and Lcitrulline. (b) electron(+) from NADPH is transferred to O2 in the uncoupled eNOS in absence of BH4(b-1) and/or Larginine(b-2), thereby producing superoxide. BH4, tetrahydrobiopterin; eNOS, endothelial nitric oxide synthase; F,

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**3.2. NOS uncoupling: NOS produces NO and superoxide depending on whether it is a**

In the process of NO formation from oxygen and L-arginine, oxygen molecules are incorpo‐ rated in both NO and L-citrulline, showing that NOS is a dioxygenase [38]. NOS containes both a reductase domain and an oxygenase domain, where electron transfer occurs from the reductase domain to the oxygenase domain. NADPH and flavin bind to the reductase domain, while oxygen, BH4 and L-arginine bind to the oxgenase domain. Electrons are transferred from NADPH through the flavin containing reductase domain to the oxygenase domain [39]. Then two cascades of further electron transfer occur depending on the presence or absence of BH4 and L-arginine. When both BH4 and L-arginine are present, NO is synthesized by oxidative deamination of arginine by NOS, where the electron is transferred to L-arginine. The initial step of L-arginine oxidation is donation of electrons to the ferrous–dioxygen complex from BH4, where trihydrobiopterin is produced and the electron is supplied through flavin regaining BH4 [40]. In contrast, in the absence of L-arginine or BH4, NOS synthesizes the superoxide, where the electron is transferred to ferrous oxygen. Intracellular deficiency of BH4 induces superoxide generation from eNOS [40]. The term "eNOS uncoupling" means func‐

flavin; NADPH, nicotinamide adenine dinucleotide phosphate

**homodimer or monomer (Figure2)**

induced PH [30]. Intravenous administration of sodium nitrite reverses PH induced by hypoxia or thromboxane analogs [31]. Furthermore, intermittent nebulization of sodium nitrite ameliorated the muscularization and hyperplasia of small pulmonary arteries, the develop‐ ment of right ventricular hypertrophy, and the rise in right ventricular pressure in chronic hypoxia- or MCT-induced PH in rats [32], which is similar to L-arginine administration[33].

The effects of inhaled NO are not restricted to the lung. Recent studies have shown that inhaled NO improves neurological and left ventricular dysfunction after successful cardiopulmonary resuscitation [34] as well as liver function after liver transplantation [35]. Inhaled NO is converted to nitrate and nitrite when it enters the blood [36, 37]. NO can be recycled from nitrite and be used to protect organs from ischemia reperfusion injury.

**Figure 2. Coupled eNOS (eNOS homodimer) produces NO.** (a) eNOS homodimer produces NO, whereas eNOS monomer produces superoxide. eNOS uncoupling occurs during the conversion of eNOS homodimer to eNOS mono‐ mer. Two eNOS monomers are connected with the aid of Zn2+, making eNOS homodimer. BH4 strengthen the Zn2+ connection, maintaining the dimer form. In coupled NOS, an electron is transferred to L-arginine, producing NO and Lcitrulline. (b) electron(+) from NADPH is transferred to O2 in the uncoupled eNOS in absence of BH4(b-1) and/or Larginine(b-2), thereby producing superoxide. BH4, tetrahydrobiopterin; eNOS, endothelial nitric oxide synthase; F, flavin; NADPH, nicotinamide adenine dinucleotide phosphate

#### **3.2. NOS uncoupling: NOS produces NO and superoxide depending on whether it is a homodimer or monomer (Figure2)**

induced PH [30]. Intravenous administration of sodium nitrite reverses PH induced by hypoxia or thromboxane analogs [31]. Furthermore, intermittent nebulization of sodium nitrite ameliorated the muscularization and hyperplasia of small pulmonary arteries, the develop‐ ment of right ventricular hypertrophy, and the rise in right ventricular pressure in chronic hypoxia- or MCT-induced PH in rats [32], which is similar to L-arginine administration[33].

**Figure 1.** Recycling of NO from NO2- Endogenous NO includes NO produced from L-arginine by NOS and recycled NO

and is protonated under the gastric acidic state forming nitrous acid (HNO2) with further decomposition to NO and/or

hemoglobin, xanthine oxidoreductase, respiratory chain enzymes, and hydrogen ion. Hb(FeII), deoxygenated hemo‐ globin;Hb(FeII)O2, oxygenated hemoglobin; NO, nitric oxide; NOHb(FeII), nitrosylhemoglobin; NOS, nitric oxide syn‐



converting to NO2

in the plasma is reduced and converted to NO by the reductase activity of deoxygenated

, nitrate; XOR, xanthine oxidoreductase; Hb(FeIII), methemoglobin; REC, respiratory chain

by the oxidation in the plasma with the

, which goes down into stomach



by the reaction with the Hb and /or to NO2

The effects of inhaled NO are not restricted to the lung. Recent studies have shown that inhaled NO improves neurological and left ventricular dysfunction after successful cardiopulmonary resuscitation [34] as well as liver function after liver transplantation [35]. Inhaled NO is converted to nitrate and nitrite when it enters the blood [36, 37]. NO can be recycled from

nitrite and be used to protect organs from ischemia reperfusion injury.

from NO2 -

78 Pulmonary Hypertension

thase; NO2 -

enzymes

. NO is converted to NO3

other nitrogen oxides. NO2

, nitrite, NO3


aid of multicopper oxidase and NO oxidase ceruloplasmin. NO3



by salivary gland. In the oral cavity anaerobic bacteria reduces NO3

In the process of NO formation from oxygen and L-arginine, oxygen molecules are incorpo‐ rated in both NO and L-citrulline, showing that NOS is a dioxygenase [38]. NOS containes both a reductase domain and an oxygenase domain, where electron transfer occurs from the reductase domain to the oxygenase domain. NADPH and flavin bind to the reductase domain, while oxygen, BH4 and L-arginine bind to the oxgenase domain. Electrons are transferred from NADPH through the flavin containing reductase domain to the oxygenase domain [39]. Then two cascades of further electron transfer occur depending on the presence or absence of BH4 and L-arginine. When both BH4 and L-arginine are present, NO is synthesized by oxidative deamination of arginine by NOS, where the electron is transferred to L-arginine. The initial step of L-arginine oxidation is donation of electrons to the ferrous–dioxygen complex from BH4, where trihydrobiopterin is produced and the electron is supplied through flavin regaining BH4 [40]. In contrast, in the absence of L-arginine or BH4, NOS synthesizes the superoxide, where the electron is transferred to ferrous oxygen. Intracellular deficiency of BH4 induces superoxide generation from eNOS [40]. The term "eNOS uncoupling" means func‐ tionally that electron transfer to L-arginine is uncoupled, when the electron is transferred to ferrous-dioxygen instead of L-arginine, producing superoxide. NOS homodimer produces NO from L-arginine and oxygen, whereas NOS monomer produces superoxide [41]. Thus, the molecular basis of eNOS uncoupling is conversion of the NOS dimer to the NOS monomer. To maintain the NOS dimer, BH4 is essential and dihydrobiopterin (BH2) is the oxidized form of BH4. Peroxinitrite oxidizes BH4 to BH2, reducing the BH4 amount and/or the BH2/BH4 ratio, both of which induce eNOS uncoupling [42]. The effects of BH4 are mediated through the regulation of NO compared with superoxide synthesis by endothelial NOS. Since BH4 might both augment NO synthesis and decrease superoxide production, BH4 deficiency may play a role in the pathogenesis of PH.

is uncoupled eNOS, producing superoxide [49]. Genetic deletion of caveolin in mice causes PH and treatment with a superoxide scavenger and/or a NOS inhibitor prevents PH associated vascular remodeling [49]. Although caveolin expression in total lung determined by Western blotting is not altered in severe PH, its immunohistological expression in plexiform lesions is

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A 90-kDa heat shock protein (HSP90) is a molecular chaperone of proteins that modu‐ lates protein functions. Along with many other proteins, eNOS and sGC are targets for HSP90. HSP90 interacts with eNOS and HSP90 facilitates the displacement of eNOS from caveolin 1, activating eNOS. HSP90 activity is dependent on adenosine triphosphate (ATP). Asymmetric dimethylarginine (ADMA) inhibits HSP90 activity in pulmonary endothelial cells through mitochondrial dysfunction, caused by ADMA induced eNOS uncoupling with subsequent superoxide production and nitration of mitochondrial protein, which reduce

To examine whether the change in eNOS expression and its activity is associated with vascular endothelial dysfunction in PH, many studies have been performed in several species of animals and humans, using isolated lung, isolated pulmonary artery, and in vivo. eNOS is expressed in not only vascular endothelial cells, but lung epithelial cells. In addition, eNOS expression

mRNA and protein expression of eNOS in rat lung and eNOS expression localized in pulmo‐ nary vascular endothelial cells and epithelial cells is upregulated in acute hypoxia [52]. In that study, nitrate/nitrite in rat lung homogenate also increased, suggesting augmented eNOS activity. The enhancement of eNOS activity in hypoxic pulmonary vasoconstriction (HPV) in normal rat lung also has been shown in other studies using NOS inhibitors [53, 54] (see sect. 3.1). eNOS protein expression was time-dependently increased in rats in chronic hypoxiainduced PH [55,56], while phosphorylated eNOS (peNOS), active form, was impaired [55]. MCT-induced PH rats showed decreased expression of both eNOS [57,58,59] and peNOS [59].

Many studies of eNOS expression and its activity have been performed in adult human PAH. However, the results are not consistent: eNOS expression is reduced in pulmonary vessels from adults with primary and secondary PH, but is increased in plexiform lesions [60]. Western blot analysis showed that eNOS expression is not changed in the lung tissue of idiopathic PAH (IPAH) patients [61]. However, several studies reported lower exhaled nitrate/nitrite (NOx) in PAH patients [62,63]. Overall, these results suggest that eNOS activity might be depressed in

and/or activity might be different between conduit PAs and resistance PAs.

absent or decreased [50].

ATP production [51].

**Animal models**

**Human**

adult human PAH.

**3.4. eNOS expression and activity in PH**

eNOS uncoupling is evaluated by the eNOS dimer/monomer ratio in cold SDS-PAGE Western blot analysis. While oxidative stress reduces the eNOS dimer/monomer ratio in a cardiac hypertrophic model suggesting eNOS uncoupling, exogenous BH4 restored the eNOS dimer/ monomer ratio [43]. Administration of exogenous BH4 might be used for eNOS uncoupling diseases. BH4 deficiency might cause PH in mice and BH4 augmentation might ameliorate the development of PH. Mice with low BH4 tissue levels develop PH which is reversed by increasing BH4 with targeted transgenic overexpression of the rate-limiting enzyme in BH4 synthesis, guanosine triphosphate(GTP) cyclohydrolase [44]. Lung BH4 availability is con‐ trolled by pulmonary vascular tone, right ventricular hypertrophy, and vascular structural remodeling. BH4 is a cofactor of NOS in the production of NO. BH4 deficiency causes decreased NO production with concomitant production of superoxide by NOS. Chronic administration of BH4 analogues improves NO-mediated pulmonary artery dilatation in rats with chronic hypoxic pulmonary hypertension [45]. Copresence of increased levels of NOS and reduced NO bioactivity might be explained by the deficiency of BH4 and/or L-arginine.

Long-term increases in NO might increase eNOS expression and eNOS uncoupling, thereby producing superoxide. Long-term administration of nitroglycerin (TNG) increased eNOS mRNA and protein expression and vascular superoxide (O2 •- ) in intact vessels monitored using ESR spectroscopy [46]. An earlier study showed that endothelial denudation improves vascular relaxation induced by TNG in isolated vessels from nitrate-tolerant animals [47].

#### **3.3. Caveolin and NOS (Figure 3)**

Caveolae are flask-shaped invaginations on the cell surface, which contain structural proteins called caveolin and other signaling proteins. In endothelial cells, eNOS is inactivated when it is conjugated to caveolin-1, a structural protein of endothelial caveolae; eNOS is activated when it dissociates from caveolae. Stimulation of β2 adrenergic receptors cause this dissocia‐ tion through phosphorylation of Tyr in caveolin-1. The mouse pulmonary endothelial β2 adrenergic receptor coupled to Gi/o proteins causes phosphorylation of caveolon-1 by Src kinase and eNOS phosphorylation at ser1177 by the Src kinase - phosphatidylinositol 3 kinase (PI3kinase) - Akt kinase pathway [48]. Thus, stimulation of the β2 adrenergic receptor causes endothelial NO synthase-dependent relaxation.

Loss of caveolin-1 induces chronic activation of eNOS and subsequent tyrosine nitration of PKG in lungs from patients with idiopathic pulmonary hypertension, where activated eNOS is uncoupled eNOS, producing superoxide [49]. Genetic deletion of caveolin in mice causes PH and treatment with a superoxide scavenger and/or a NOS inhibitor prevents PH associated vascular remodeling [49]. Although caveolin expression in total lung determined by Western blotting is not altered in severe PH, its immunohistological expression in plexiform lesions is absent or decreased [50].

A 90-kDa heat shock protein (HSP90) is a molecular chaperone of proteins that modu‐ lates protein functions. Along with many other proteins, eNOS and sGC are targets for HSP90. HSP90 interacts with eNOS and HSP90 facilitates the displacement of eNOS from caveolin 1, activating eNOS. HSP90 activity is dependent on adenosine triphosphate (ATP). Asymmetric dimethylarginine (ADMA) inhibits HSP90 activity in pulmonary endothelial cells through mitochondrial dysfunction, caused by ADMA induced eNOS uncoupling with subsequent superoxide production and nitration of mitochondrial protein, which reduce ATP production [51].

#### **3.4. eNOS expression and activity in PH**

To examine whether the change in eNOS expression and its activity is associated with vascular endothelial dysfunction in PH, many studies have been performed in several species of animals and humans, using isolated lung, isolated pulmonary artery, and in vivo. eNOS is expressed in not only vascular endothelial cells, but lung epithelial cells. In addition, eNOS expression and/or activity might be different between conduit PAs and resistance PAs.

#### **Animal models**

tionally that electron transfer to L-arginine is uncoupled, when the electron is transferred to ferrous-dioxygen instead of L-arginine, producing superoxide. NOS homodimer produces NO from L-arginine and oxygen, whereas NOS monomer produces superoxide [41]. Thus, the molecular basis of eNOS uncoupling is conversion of the NOS dimer to the NOS monomer. To maintain the NOS dimer, BH4 is essential and dihydrobiopterin (BH2) is the oxidized form of BH4. Peroxinitrite oxidizes BH4 to BH2, reducing the BH4 amount and/or the BH2/BH4 ratio, both of which induce eNOS uncoupling [42]. The effects of BH4 are mediated through the regulation of NO compared with superoxide synthesis by endothelial NOS. Since BH4 might both augment NO synthesis and decrease superoxide production, BH4 deficiency may

eNOS uncoupling is evaluated by the eNOS dimer/monomer ratio in cold SDS-PAGE Western blot analysis. While oxidative stress reduces the eNOS dimer/monomer ratio in a cardiac hypertrophic model suggesting eNOS uncoupling, exogenous BH4 restored the eNOS dimer/ monomer ratio [43]. Administration of exogenous BH4 might be used for eNOS uncoupling diseases. BH4 deficiency might cause PH in mice and BH4 augmentation might ameliorate the development of PH. Mice with low BH4 tissue levels develop PH which is reversed by increasing BH4 with targeted transgenic overexpression of the rate-limiting enzyme in BH4 synthesis, guanosine triphosphate(GTP) cyclohydrolase [44]. Lung BH4 availability is con‐ trolled by pulmonary vascular tone, right ventricular hypertrophy, and vascular structural remodeling. BH4 is a cofactor of NOS in the production of NO. BH4 deficiency causes decreased NO production with concomitant production of superoxide by NOS. Chronic administration of BH4 analogues improves NO-mediated pulmonary artery dilatation in rats with chronic hypoxic pulmonary hypertension [45]. Copresence of increased levels of NOS and reduced NO bioactivity might be explained by the deficiency of BH4 and/or L-arginine. Long-term increases in NO might increase eNOS expression and eNOS uncoupling, thereby producing superoxide. Long-term administration of nitroglycerin (TNG) increased eNOS

using ESR spectroscopy [46]. An earlier study showed that endothelial denudation improves vascular relaxation induced by TNG in isolated vessels from nitrate-tolerant animals [47].

Caveolae are flask-shaped invaginations on the cell surface, which contain structural proteins called caveolin and other signaling proteins. In endothelial cells, eNOS is inactivated when it is conjugated to caveolin-1, a structural protein of endothelial caveolae; eNOS is activated when it dissociates from caveolae. Stimulation of β2 adrenergic receptors cause this dissocia‐ tion through phosphorylation of Tyr in caveolin-1. The mouse pulmonary endothelial β2 adrenergic receptor coupled to Gi/o proteins causes phosphorylation of caveolon-1 by Src kinase and eNOS phosphorylation at ser1177 by the Src kinase - phosphatidylinositol 3 kinase (PI3kinase) - Akt kinase pathway [48]. Thus, stimulation of the β2 adrenergic receptor causes

Loss of caveolin-1 induces chronic activation of eNOS and subsequent tyrosine nitration of PKG in lungs from patients with idiopathic pulmonary hypertension, where activated eNOS

•- ) in intact vessels monitored

play a role in the pathogenesis of PH.

80 Pulmonary Hypertension

**3.3. Caveolin and NOS (Figure 3)**

endothelial NO synthase-dependent relaxation.

mRNA and protein expression and vascular superoxide (O2

mRNA and protein expression of eNOS in rat lung and eNOS expression localized in pulmo‐ nary vascular endothelial cells and epithelial cells is upregulated in acute hypoxia [52]. In that study, nitrate/nitrite in rat lung homogenate also increased, suggesting augmented eNOS activity. The enhancement of eNOS activity in hypoxic pulmonary vasoconstriction (HPV) in normal rat lung also has been shown in other studies using NOS inhibitors [53, 54] (see sect. 3.1). eNOS protein expression was time-dependently increased in rats in chronic hypoxiainduced PH [55,56], while phosphorylated eNOS (peNOS), active form, was impaired [55]. MCT-induced PH rats showed decreased expression of both eNOS [57,58,59] and peNOS [59].

#### **Human**

Many studies of eNOS expression and its activity have been performed in adult human PAH. However, the results are not consistent: eNOS expression is reduced in pulmonary vessels from adults with primary and secondary PH, but is increased in plexiform lesions [60]. Western blot analysis showed that eNOS expression is not changed in the lung tissue of idiopathic PAH (IPAH) patients [61]. However, several studies reported lower exhaled nitrate/nitrite (NOx) in PAH patients [62,63]. Overall, these results suggest that eNOS activity might be depressed in adult human PAH.

PH. On exposure to acute hypoxia, NOS inhibitors augmented vascular contraction in normal [53,67,68] and hypoxia-induced PH rat models [67]. This finding suggests that NO production

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Inhibition of NO production by L-NMMA caused the reduction of pulmonary flow in conscious healthy adults [69,70], suggesting the possible role of continuous production of NO in maintaining basal vascular tone. In PAH patients, several studies reported decreased expression of NOS. Although several studies reported decreased exhaled nitrogen oxide (NOx) levels in PAH patients, others have reported higher levels. The results therefore remain

**4.2. Vasoreactivity to endothelium-dependent and independent NO-related relaxing**

Many studies have been performed using acetylcholine (Ach) and sodium nitroprusside (SNP), endothelium-dependent and -independent NO-related vasorelaxants, to examine functional changes in vascular endothelial and smooth muscle cells in PH. As Ach-induced relaxation was abolished by NOS inhibitors [64] and restored with L-arginine [71,72], reactivity

The relaxation response to Ach is impaired in rat isolated conduit pulmonary arteries (PAs) [65,73,74,75,76]. Many of these studies also described an impaired relaxation response to SNP in conduit PAs [65,74,76]. These results suggested 1) decreased production and release of NO in endothelial cells or 2) decreased responsiveness to NO in smooth muscle cells, or both. Impaired relaxation in Ach and SNP was partially restored after exposure to chronic hypoxia. As the recovery process was different between the responses of Ach and SNP [65], it was speculated that NO-related functional abnormalities in endothelial and smooth muscle cells

In contrast, in hypoxic vasoconstriction resistant rat PA rings, the relaxation response to Ach was not changed [74,75] or augmented [77] in chronic hypoxia. It is likely that Ach-reactive NO production and/or release varies in a vascular site-specific manner. Conduit arteries produce and release more eNOS than peripheral arteries. The vascular functional change in response to stimuli such as abnormal shear stress, circumferential wall stretch and hypoxia itself may occur in conduit PAs more than in peripheral resistant arteries. Although conduit arteries do not directly relate to pulmonary vascular resistance, the pathophysiological change

Impaired response to Ach was partly restored in the presence of a non-selective inhibitor of cyclooxygenase (COX) [65] or prostaglandin (PG) H2 / thromboxane (TX) A2 receptor antago‐ nist [79], suggesting the possibility of 1) imbalance between the production of vasocontracting and vasorelaxing prostanoid in vascular endothelial cells, and 2) simultaneously release of vasocontracting prostanoids such as PGH2 and/or TXA2. Pidgeon et al. showed that the basal

in conduit arteries may play a key role in pulmonary vascular remodeling [78].

in HPV is increased in both normal and hypoxic PH rats.

may partly reflect changes in NOS expression and/or activity.

**Humans**

inconclusive.

**substances in rat lung**

**Rats with hypoxic PH**

occurred independently.

**Figure 3. Inactive form of eNOS associated with caveola.** eNOS is associated with caveola, which is the inactive form of eNOS. The active form of eNOS is dissociated from caveola. Stimulation of BMPIIR induces dissociation of eNOS from caveola as well as phosphorylation of eNOS through PKA and/or Akt activation. eNOS, endothelial nitric oxide synthase; B2-AR, beta 2-adrenergic receptor; SrcK, src kinase; peNOS, phosphorylated eNOS; BMPIIR, bone mor‐ phogenetic ptotein II receptor; PKA, cyclic AMP-dependent protein kinase
