**2. Biological effects of NO**

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

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‐ lation and dephosphorylation through S-nitrosylation.

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 allosterically or by direct modification of cysteine.

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 sarcoplasmic reticulum [17].

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

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‐ nary pressures precedes the vascular structural changes in chronic hypoxia-induced PH, whereas the reverse sequence of events occurs in MCT-induced PH. Endogenous NO from Larginine could prevent the development of new muscularization of peripheral pulmonary arteries in both models, whereas exogenous inhaled NO would be effective only in hypoxiainduced PH because of the reduction in pulmonary vascular pressures caused by NO mediated vasodilation.

Inhaled NO likely attenuates the hypertensive vascular structural changes through pulmonary vasodilation by a cGMP-mediated mechanism. Endogenous NO from L-arginine might also prevent the development of structural changes through a cGMP-mediated mechanism. This hypothesis is supported by another study that showed that pulmonary gene transfection of atrial natriuretic peptide (ANP), another inducer of cGMP, attenuates the development of chronic hypoxia-induced pulmonary vascular changes [25]. Treatment to increase NO production in the pulmonary vascular bed by eNOS gene transfection ameliorates the development of PH. Studies have demonstrated that eNOS transfected smooth muscle cell administration prevented the development of MCT-induced PH [26] and that eNOS trans‐ fected bone marrow-derived endothelial-like progenitor cell venous administration reversed established MCT-induced PH [27].
