**3. Inhaled nitric oxide**

#### **3.1 Inhaled NO as a selective pulmonary vasodilator**

In 1988, at the international conference of the American Thoracic Society, Higenbottam presented his team's paper titled "Inhaled endothelium-derived relaxing factor (EDRF) in primary pulmonary hypertension (PPH)," including the first description of NO inhalation in humans with pulmonary arterial hypertension for laboratory use [38]. In 1991, Lancet published the study [39], showing that 40 ppm NO inhalation selectively reduces PAP, with no changes in systemic pressure. Frostell et al. also showed that inhaled NO (5–80 ppm) causes selective pulmonary arterial dilatation, without changes in systemic arterial pressure in sheep [40], where PAP elevation was induced by hypoxic pulmonary vasoconstriction. Both research groups referenced the studies by Yoshida, Kasama, and Kitabatake about the metabolic fate of iNO [41, 42] because toxicity and retention in the human body should be minimal. NO has been a focus in air pollution research and thus provides

a basis for work by clinicians evaluating NO inhalation in humans. In rats with chronic hypoxia- and MCT-induced PH, iNO results in a decrease in PAP with no changes in systemic arterial pressure [43–45] (**Figure 4**).

#### **3.2 Clinical effects of iNO**

iNO dilates the pulmonary vasculature by NO combining with guanylate cyclase Fe in pulmonary vascular smooth muscle cells. Most NO diffuses into the blood at alveoli, where it reacts with the Fe of oxygenated hemoglobin (oxy-Hb, O2Hb, O2Hb(Fe2+)) in red blood cells and is converted to NO3 <sup>−</sup>. When NO reacts with oxy-Hb Fe2+ or combines with the Fe2+ of deoxygenated Hb (deoxy-Hb, deoxy-Hb(Fe2+)) in red blood cells, iNO does not have direct vasodilatory effects because it reacts or combines with Hb Fe2+ and becomes unable to combine with guanylate cyclase Fe of smooth muscle cells in systemic arteries. Thus, iNO is a selective pulmonary vasodilator, causing decreased PAP with no changes in systemic pressure (**Figure 4**).

The clinical use of NO inhalation is aimed at inducing selective pulmonary arterial dilation and treating PH and right ventricular failure. NO inhalation is also used to test pulmonary vascular reactivity in catheterization labs, which will be discussed in the last section of this chapter. Improved arterial oxygenation is also expected in patients with high intrapulmonary shunting [46]. Thus, the main target of iNO is lung and pulmonary circulation. In addition, iNO effects on remote organs, such as the kidney [47], liver [48, 49], heart [50], and muscle [51], have been investigated, with iNO shown to ameliorate inflammation and ischemia-reperfusion injury.

#### **3.3 Substances that react with NO**

NO reacts or combines with transition metal ions, such as thiols (–SH, –SS–, and HS–). Many enzymes and substances involved in regulating cell function include in their structure Fe, a transitional metal, thus suggesting its importance as an NO target. Because hemoglobin (Hb) and guanylate cyclase contain heme, which includes Fe in its structure, NO reacts or combines with Hb and guanylate cyclase. NO also combines with enzymes containing Fe–S in their structure, and combined NO (nitration) and Fe–S can prevent enzymatic activity. High concentrations of NO induce cell damage, which presumably result from this enzymatic dysfunction. Thus, NO is a double-edged sword. Although an appropriate amount is important for regulating cell function, an excess dysregulates cell function and causes damage.

RS–NO is a complex of SH– and NO. Nitrosothiol is a thionitrite including *S*-nitroso-albumin, where NO combines with cysteine, a component of albumin. –SH is a component of amino acids, peptides, and proteins. NO binds to –SH, forming *S*-nitrosothiol, 96% of which is *S*-nitrosoprotein. About 82% of *S*-nitrosoprotein is serum *S*-nitrosoalbumin. Thus, endogenous NO circulates in the form of *S*-nitrosoalbumin [52].

NO targets are transition metal ions, oxygen, nucleophilic centers (thiols, amides, carboxyls, and hydroxyls), and free radicals. iNO targets are (**Figure 5**) also transition metal ions, namely, in the guanylate cyclase Fe, Hb Fe, iron-sulfur (Fe-S) center. Other targets include oxygen (gaseous oxygen in the airway and alveoli), dissolved oxygen in the tissue and body fluids, the nucleophilic center of organic compounds (–S–S– and –SH), and free radicals (reactive oxygen species produced in leucocytes and macrophages). Among these substances that react with iNO, The main target of an approved medical iNO gas is guanylate cyclase Fe in pulmonary vascular smooth muscle cells.

**73**

*Endogenous and Inhaled Nitric Oxide for the Treatment of Pulmonary Hypertension*

Oxidation refers to electron loss and reduction to electron gain. Reducing agents release electrons, whereas oxidizing agents receive electrons. The term redox is a combination of "reduction and oxidation reaction." Reduction and oxygenation occur simultaneously so that when a reducing agent is oxidized, an oxidizing agent

*The substances in the lung to react or combine with iNO. NO reacts with Fe in the guanylate cyclase (GC) and induces cyclic GMP and subsequent pulmonary vascular relaxation; NO reacts with Fe2+ in O2Hb and forms* 

*combine in ONOO-; NO and OH- combine in NO2-; NO and thiol (sulfhydryl group, -SH group), amine, and iron-sulfur (Fe-S) center combine in nitrosothiol, nitrosamine, and Fe-S NO, respectively. RSH is a compound including SH group. S-nitro-Hb is combination of NO and SH in the cysteine in the Hb beta subunit. Reactive* 

*<sup>−</sup>; NO and Fe2+ in deoxy-Hb combine in NOHb; NO and O2 combine in NO2; NO and O2-* 

(nitroxyl anion) [53]. Among these, NO· has a single electron, and its

(nitrosonium),

. NO· is electrically neutral, which

<sup>−</sup>) and (di)oxygen, are candidates in both

+ NO3

−

(1)

is also reduced. Nitrogen monoxides involve an array of species: NO+

*<sup>−</sup>, OH<sup>−</sup>) are produced in leucocytes and macrophages.*

O2Hb(Fe2+) [oxy‐Hb]

, whereas its addition yields NO<sup>−</sup>

contributes to its free diffusibility in aqueous medium and across cell membranes. The main NO· targets are oxygen and transition metal ions. The various redox

the gas phase and aqueous solution. Metalloproteins, such as heme-containing protein and non-heme-containing protein, and iron-sulfur clusters also react with

iNO reacts with oxy-Hb. NO oxidizes oxy-Hb to form MetHb. In other words, MetHb is oxidized oxy-Hb. Oxidized iron (MetHb) species do not catch NO, and iNO during cardiopulmonary bypass (CPB) decreases acute kidney injury [47]:

NO reacts or combines with Hb in three ways: (1) NO combines with in the heme Fe to form NOHb (nitrosyl Hb), a metal nitrosyl species; (2) NO<sup>−</sup> combines

+ NO → Hb(Fe3+)

[MetHb]

*DOI: http://dx.doi.org/10.5772/intechopen.89381*

**3.4 Metabolic fate of iNO**

NO·, and NO<sup>−</sup>

**Figure 5.**

*MetHb and NO3*

*oxygen species (O2*

NO·.

removal forms NO+

*3.4.2 NO and Hb*

*3.4.1 Oxidation and reduction of NO*

forms of oxygen, such as superoxide (O2

*Endogenous and Inhaled Nitric Oxide for the Treatment of Pulmonary Hypertension DOI: http://dx.doi.org/10.5772/intechopen.89381*

#### **Figure 5.**

*The substances in the lung to react or combine with iNO. NO reacts with Fe in the guanylate cyclase (GC) and induces cyclic GMP and subsequent pulmonary vascular relaxation; NO reacts with Fe2+ in O2Hb and forms MetHb and NO3 <sup>−</sup>; NO and Fe2+ in deoxy-Hb combine in NOHb; NO and O2 combine in NO2; NO and O2 combine in ONOO-; NO and OH- combine in NO2-; NO and thiol (sulfhydryl group, -SH group), amine, and iron-sulfur (Fe-S) center combine in nitrosothiol, nitrosamine, and Fe-S NO, respectively. RSH is a compound including SH group. S-nitro-Hb is combination of NO and SH in the cysteine in the Hb beta subunit. Reactive oxygen species (O2 <sup>−</sup>, OH<sup>−</sup>) are produced in leucocytes and macrophages.*

#### **3.4 Metabolic fate of iNO**

#### *3.4.1 Oxidation and reduction of NO*

Oxidation refers to electron loss and reduction to electron gain. Reducing agents release electrons, whereas oxidizing agents receive electrons. The term redox is a combination of "reduction and oxidation reaction." Reduction and oxygenation occur simultaneously so that when a reducing agent is oxidized, an oxidizing agent is also reduced. Nitrogen monoxides involve an array of species: NO+ (nitrosonium), NO·, and NO<sup>−</sup> (nitroxyl anion) [53]. Among these, NO· has a single electron, and its removal forms NO+ , whereas its addition yields NO<sup>−</sup> . NO· is electrically neutral, which contributes to its free diffusibility in aqueous medium and across cell membranes.

The main NO· targets are oxygen and transition metal ions. The various redox forms of oxygen, such as superoxide (O2 <sup>−</sup>) and (di)oxygen, are candidates in both the gas phase and aqueous solution. Metalloproteins, such as heme-containing protein and non-heme-containing protein, and iron-sulfur clusters also react with NO·.

iNO reacts with oxy-Hb. NO oxidizes oxy-Hb to form MetHb. In other words, MetHb is oxidized oxy-Hb. Oxidized iron (MetHb) species do not catch NO, and iNO during cardiopulmonary bypass (CPB) decreases acute kidney injury [47]:

$$\text{O}\_2\text{Hb}\text{(Fe}^{2+}\text{)} + \text{NO} \rightarrow \underset{\text{[oxy-Hb]}}{\text{Hb}\text{(Fe}^{3+}\text{)}} + \text{NO}\_3^{-}\tag{1}$$

*3.4.2 NO and Hb*

NO reacts or combines with Hb in three ways: (1) NO combines with in the heme Fe to form NOHb (nitrosyl Hb), a metal nitrosyl species; (2) NO<sup>−</sup> combines with amines in Hb to form *S*-nitroso-Hb, a nitrosamine, where NO combines with cysteine in the beta subunit of Hb; and (3) O<sup>−</sup> or NO+ combines with the sulfhydryl center (-SH) in Hb. NO reacts with oxy-Hb and combines with deoxy-Hb. If NO reacts with oxy-Hb (Fe2+), MetHb (Fe3+) and nitrate (NO3 <sup>−</sup>) are formed. If NO combines with deoxy-Hb (Fe2+), NOHb (Fe2+) is formed, after which NOHb (Fe2+) reacts with O2 to form MetHb (Fe3+) and NO3 <sup>−</sup>. MetHb (Fe3+) is reduced to deoxy-Hb (Fe2+) by MetHb reductase. The depletion of MetHb reductase or high production of MetHb causes methemoglobinemia. NO3 <sup>−</sup> is excreted in the urine (**Figures 6**, **7**).

In an in vitro experiment, Wennmalm [54] incubated NO with arterial and venous blood and measured MetHb, NOHb, NO3 − , and NO2 − . The reaction of NO with O2Hb was rapid in the arterial blood (oxygen saturation 94–99%). NOHb was low in arterial blood and high in venous blood (oxygen saturation 36–86%). These results suggest that O2Hb (oxy-Hb) gives O2 to NO making NO3 <sup>−</sup>. In contrast, deoxy-Hb directly combines with NO making NOHb in the absence of oxy-Hb (i.e., in the presence of deoxy-Hb). The NO and oxy-Hb reaction is completed in 100 ms [55].
