**4. Endothelium-dependent and -independent NO-mediated relaxation in pulmonary circulation**

#### **4.1. Role of endothelium-derived NO in basal tone**

L-NMMA (N omega-monomethyl–L-arginine), L-NNA (N omega-nitro-L-arginine), L-NAME (N omega-nitro-L-arginine methyl ester), L-NA(N omega-nitro-L-arginine) and other NOS inhibitors have been used to examine the physiological role of NO in pulmonary vascular tone. The increase in vascular tone in the presence of NOS inhibitors may indirectly represent NO production and/or release in the pulmonary circulation.

#### **Animals**

L-NMMA [53] and L-NNA [64,65] did not change pulmonary basal tone in normal rat PA rings. Normal isolated perfused lungs were not affected by NOS inhibitors such as L-NMMA [53], L-NNA [64], and L-NA [66] except for a few studies showing a moderate increase with L-NAME [67]. In chronic hypoxia, many studies showed markedly enhanced vascular tone by L-NNA [64] or L-NAME [67]. Although these NOS inhibitors caused different results, the findings suggested that 1) NO might not be involved in vascular basal tone in normal pulmo‐ nary circulation, and 2) basal NO production might be increased in hypoxia-induced chronic 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 in HPV is increased in both normal and hypoxic PH rats.

#### **Humans**

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 inconclusive.

#### **4.2. Vasoreactivity to endothelium-dependent and independent NO-related relaxing substances in rat lung**

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

#### **Rats with hypoxic PH**

**4. Endothelium-dependent and -independent NO-mediated relaxation in**

**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‐

L-NMMA (N omega-monomethyl–L-arginine), L-NNA (N omega-nitro-L-arginine), L-NAME (N omega-nitro-L-arginine methyl ester), L-NA(N omega-nitro-L-arginine) and other NOS inhibitors have been used to examine the physiological role of NO in pulmonary vascular tone. The increase in vascular tone in the presence of NOS inhibitors may indirectly represent NO

L-NMMA [53] and L-NNA [64,65] did not change pulmonary basal tone in normal rat PA rings. Normal isolated perfused lungs were not affected by NOS inhibitors such as L-NMMA [53], L-NNA [64], and L-NA [66] except for a few studies showing a moderate increase with L-NAME [67]. In chronic hypoxia, many studies showed markedly enhanced vascular tone by L-NNA [64] or L-NAME [67]. Although these NOS inhibitors caused different results, the findings suggested that 1) NO might not be involved in vascular basal tone in normal pulmo‐ nary circulation, and 2) basal NO production might be increased in hypoxia-induced chronic

**pulmonary circulation**

82 Pulmonary Hypertension

**Animals**

**4.1. Role of endothelium-derived NO in basal tone**

phogenetic ptotein II receptor; PKA, cyclic AMP-dependent protein kinase

production and/or release in the pulmonary circulation.

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 occurred independently.

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

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 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 hypoxia [80].

#### **MCT-induced PH in rats**

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 peripheral PAs.
