**5. Superoxide scavenges NO producing peroxynitrite (Figure 4)**

#### **5.1. Oxidative stress**

In pulmonary hypertension, endothelial NOS expression is increased, which may not neces‐ sarily indicate an increase in NO production [87]. NOS might produce superoxide, which is due to uncoupling of NOS [88]. Increased levels of NOS and reduced NO bioactivity might be explained by the deficiency of BH4 and/or L-arginine. Oxidative stress induces the changes of BH4 to BH2. Oxidative stress also induces S-glutathionylation and subsequent eNOS uncou‐ pling [39], in which S-glutathionylation of eNOS reversibly decreases NOS activity with an increase in O2 •- generation primarily from the reductase and endothelium-dependent relaxa‐ tion is impaired. Oxidative stress upregulates nuclear factor (NF)-kappaB, a key transcription factor that is involved in vascular tissue remodeling. NF-kappaB nuclear localization and vascular cell adhesion molecule 1(VCAM-1) expression is temporally and spatially associated with the development of MCT-induced PH in rats, which is ameliorated by administering a NF-kappaB inhibitor, pyrrolidine dithiocarbamate(PDTC)[89].

Deficiency of superoxide dismutase (SOD) may play a role in the development of PH. Expression and activity of mitochondrial SOD2 in patients and animal models of PH is decreased [92,93] in pulmonary arteries and plexiform lesions. SOD produces H2O2 from mitochondrial superoxide. H2O2 is less potent than superoxide and acts as a signaling molecule to inhibit transcriptional factors such as hypoxia-inducible factor-1α. Epigenetic suppression of SOD with selective hypermethylation of CpG islands in SOD2 gene induces excessive proliferation and decreases apoptosis in pulmonary artery smooth muscle cells [92], suggest‐

tion of protein kinases, thereby activating or suppressing their activities. PKG phosphorylates Rho kinase, Akt, and ion channels. Phosphorylation of ion channels makes Ca2+ ion channels closed and potassium channel open. Peroxynitrite further oxidize BH4 to BH2, inducing eNOS uncoupling with subsequent superoxide production. BH4, tetrahydrobiop‐ terin; BH2, dihydrobiopterin; eNOS, endothelial nitric oxide synthase; ERK, extracellular signal-regulated kinase; IP3,

.- producing peroxynitrite(ONOO-

Nitric Oxide in Pathophysiology and Treatment of Pulmonary Hypertension

NO reacts with superoxide more rapidly than SOD producing peroxinitrite. Peroxynitrite is a

from peroxynitrous acid (HOONO) and/or its reactive activated isomer (HOONO\*) attacks biological targets [94] including cyclic GMP-dependent protein kinase (PKG). In the setting of eNOS uncoupling, eNOS synthesizes superoxide which reacts with NO to create peroxynitrite.

depresses the function of PKG ( 42 ).

and NO2 produced

.-) is produced by uncoupled eNOS,

http://dx.doi.org/10.5772/55680

85

) with subsequent nitrosyla‐

, peroxynitrite; PKC, pro‐

more potent and versatile oxidant than NO or superoxide, in which HO+

**Figure 4. Peroxynitrite production from NO and superoxide.** Superoxide (O2

inositol triphosphate receptor; MAPK, mitogen-activated protein kinase; OX, oxidase; ONOO-

tein kinase C; PKG, cyclic-GMP dependent protein kinase( protein kinase G); XOX, xanthine oxidase;

ing a causative role of SOD deficiency in PH.

NADPH oxidase, and xanthine oxidase. NO reacts with O2

Nitrosylation of PKG by ONOO-

#### **5.2. Production of superoxide in PH: role of NADPH oxidase and SOD**

NAD(P)H oxidase enzyme complex catalyzes one electron reduction of oxygen using NADPH or NADH as an electron donor, which produces superoxide : NAD(P)H + 2O2 → NAD(P) <sup>+</sup> + H+ +2O2 -' NADPH oxidase expression is increased in pulmonary arteries from a lamb model of persistent pulmonary hypertension of the newborn (PPHN) [90]. The expression was determined by the Western blotting of the levels of p67phox a subunit of the NADPH oxidase complex and immunostaining of the pulmonary vessels in lung sections. Another study demonstrated that expression and activity of the NADPH oxidase complex are upregulated in PH with increased pulmonary blood flow [91].

expression of COX2, otherwise known as PGH synthase, was increased in rat lungs in chronic hypoxia, and a PGH2/TXA2 receptor antagonist attenuated the rise in PAP induced by chronic

PA vascular functional changes in rats with MCT-induced PH have been compared with PAs from animals with chronic hypoxia-induced PH. Many vasodilation studies have reported a depressed relaxation response to Ach in MCT-induced rat conduit PA rings [76,81,82,83,84]. Many of these studies described impaired SNP relaxation, [76,82] with the exception of one study [84]. While Ach-induced relaxation was impaired in the pulmonary circulation in MCTinduced PH, the SNP relaxation response has been reported to be impaired [85] or not impaired [86]. Taken together, in MCT-induced PH, vascular endothelial dysfunction is observed from proximal to distal PAs; however, smooth muscle functional alteration is not apparent in

**5. Superoxide scavenges NO producing peroxynitrite (Figure 4)**

In pulmonary hypertension, endothelial NOS expression is increased, which may not neces‐ sarily indicate an increase in NO production [87]. NOS might produce superoxide, which is due to uncoupling of NOS [88]. Increased levels of NOS and reduced NO bioactivity might be explained by the deficiency of BH4 and/or L-arginine. Oxidative stress induces the changes of BH4 to BH2. Oxidative stress also induces S-glutathionylation and subsequent eNOS uncou‐ pling [39], in which S-glutathionylation of eNOS reversibly decreases NOS activity with an

tion is impaired. Oxidative stress upregulates nuclear factor (NF)-kappaB, a key transcription factor that is involved in vascular tissue remodeling. NF-kappaB nuclear localization and vascular cell adhesion molecule 1(VCAM-1) expression is temporally and spatially associated with the development of MCT-induced PH in rats, which is ameliorated by administering a

NAD(P)H oxidase enzyme complex catalyzes one electron reduction of oxygen using NADPH or NADH as an electron donor, which produces superoxide : NAD(P)H + 2O2 → NAD(P) <sup>+</sup>

of persistent pulmonary hypertension of the newborn (PPHN) [90]. The expression was determined by the Western blotting of the levels of p67phox a subunit of the NADPH oxidase complex and immunostaining of the pulmonary vessels in lung sections. Another study demonstrated that expression and activity of the NADPH oxidase complex are upregulated


NF-kappaB inhibitor, pyrrolidine dithiocarbamate(PDTC)[89].

in PH with increased pulmonary blood flow [91].

**5.2. Production of superoxide in PH: role of NADPH oxidase and SOD**

•- generation primarily from the reductase and endothelium-dependent relaxa‐

+

hypoxia [80].

84 Pulmonary Hypertension

peripheral PAs.

**5.1. Oxidative stress**

increase in O2

H+ +2O2

**MCT-induced PH in rats**

**Figure 4. Peroxynitrite production from NO and superoxide.** Superoxide (O2 .-) is produced by uncoupled eNOS, NADPH oxidase, and xanthine oxidase. NO reacts with O2 .- producing peroxynitrite(ONOO- ) with subsequent nitrosyla‐ tion of protein kinases, thereby activating or suppressing their activities. PKG phosphorylates Rho kinase, Akt, and ion channels. Phosphorylation of ion channels makes Ca2+ ion channels closed and potassium channel open. Peroxynitrite further oxidize BH4 to BH2, inducing eNOS uncoupling with subsequent superoxide production. BH4, tetrahydrobiop‐ terin; BH2, dihydrobiopterin; eNOS, endothelial nitric oxide synthase; ERK, extracellular signal-regulated kinase; IP3, inositol triphosphate receptor; MAPK, mitogen-activated protein kinase; OX, oxidase; ONOO- , peroxynitrite; PKC, pro‐ tein kinase C; PKG, cyclic-GMP dependent protein kinase( protein kinase G); XOX, xanthine oxidase;

Deficiency of superoxide dismutase (SOD) may play a role in the development of PH. Expression and activity of mitochondrial SOD2 in patients and animal models of PH is decreased [92,93] in pulmonary arteries and plexiform lesions. SOD produces H2O2 from mitochondrial superoxide. H2O2 is less potent than superoxide and acts as a signaling molecule to inhibit transcriptional factors such as hypoxia-inducible factor-1α. Epigenetic suppression of SOD with selective hypermethylation of CpG islands in SOD2 gene induces excessive proliferation and decreases apoptosis in pulmonary artery smooth muscle cells [92], suggest‐ ing a causative role of SOD deficiency in PH.

NO reacts with superoxide more rapidly than SOD producing peroxinitrite. Peroxynitrite is a more potent and versatile oxidant than NO or superoxide, in which HO+ and NO2 produced from peroxynitrous acid (HOONO) and/or its reactive activated isomer (HOONO\*) attacks biological targets [94] including cyclic GMP-dependent protein kinase (PKG). In the setting of eNOS uncoupling, eNOS synthesizes superoxide which reacts with NO to create peroxynitrite. Nitrosylation of PKG by ONOO depresses the function of PKG ( 42 ).
