In this study we hypothesized that if pulmonary hypertension were to be caused by reduced NO synthesis, than a long-term reduction of the endogenous NO synthesis by pharmacological means should result in pulmonary hypertension. We found that chronic administration of N^G-nitro-L-arginine methyl ester (L-NAME), an inhibitor of endogenous NO synthesis, caused - as expected - systemic hypertension (Figure). Pulmonary arterial pressure, however, was not elevated (Figure).

Chronic feeding of rats with NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of endogenous nitric oxide synthesis, causes systemic hypertension (left) but does not mimic chronic hypoxic pulmonary hypertension (right). The rats were given L-NAME in drinking water or exposed to hypoxia for 3-4 weeks before their systemic arterial pressure was measured through a cannula in the carotid artery and pulmonary arterial pressure was measured by catheterization. *P<0.05

These data show that pulmonary hypertension cannot be expained as an NO-defficiency syndrome.

In this study we focused on the question whether the basal NO production in the healthy pulmonary circulation is indeed lower than in the systemic vessels, as suggested by our previous study. We directly compared the role of NO in the pulmonary and systemic (exemplified by renal) vascular bed isolated from the same individual rat.

For this purpose I specifically designed a new experimental model by combining two well established models - isolated perfused rat lung and isolated perfused rat kidney - into one (Figure 1).We found that acute inhibition of NO synthesis by N^G-nitro-L-arginine or its methyl ester caused a marked vasoconstriction in the kidney but not in the lung (Figure 2).

Figure 1: Isolated perfused rat lung-kidney setup. A portion of the lung outflow is pumped into the kidney (the flow through the lung needs to be much higher than the flow through the kidney). The lung also serves to control the oxygenation of the perfusate (Krebs-albumin solution) for the kidney. Renal vein is not cannulated and its outflow flows freely into the heated Petri dish in which the kidney is bathed and which overflows into the main perfusate reservoir.

Figure 2: NG-nitro-L-arginine methyl ester (L-NAME) causes renal but not pulmonary vasconstriction. The organs were perfused at constant flow rate (kidney: 10.5 ml/min; lung: 28 ml/min), so that the increases in perfusion pressure reflect vasoconstriction. The results were similar for NG-nitro-L-arginine. The data are the means +/- SEM from 6 preparations. *P<0.05.

This finding confirms that NO has no substantial role in the basal tone regulation of the pulmonary vessels of a healthy rat, while being a substantial factor in the regulation of the basal tone in the renal vascular bed.

In this study we found that the basal NO production by the pulmonary vasculature is not detectable, but is increased in chronic hypoxic pulmonary hypertension (Figure 1). Acute inhibition of NO synthesis causes pulmonary vasoconstriction only in chronically hypoxic rats (confirming they did synthesize the vasodilator NO), but not in control, normoxic rats (confirming there was no basal NO synthesis to inhibit) (Figure 2). Pulmonary vasoreactivity to substance P, which acts through NO release, is not reduced in chronic hypoxia (Figure 3).

Figure 1: Basal release of nitric oxide (NO) into the perfusate of the isolated lungs is negligible in control rats and significantly elevated in rats with chronic hypoxic pulmonary hypertension. NO was measured as the total (NOx) of NO itself and its oxidation product, NO2, by a chemiluminescence assay after reduction of NO2 to NO in acidic vanadium. Accumulation of NOx in the perfusate (Krebs-albumin solution) was measured over 15 minutes. Data are the means +/- SEM. *P<0.05

Figure 2: Inhibitor of endogenous nitric oxide synthesis, NG-nitro-L-arginine methyl ester (L-NAME) causes significant pulmonary vasoconstriction in rats with chronic hypoxic pulmonary hypertension, but not in control rats. Isolated rat lungs were perfused with Krebs-albumin solution at constant flow rate (0.04 ml/min/g body weight), so that increases in perfusion pressure reflect vasoconstriction. *P<0.05.

Figure 3: Pulmonary vasodilation in response to substance P, known to act through releasing endogenous nitric oxide, is potentiated in rats with chronic hypoxic pulmonary hypertension. Isolated rat lungs were perfused with Krebs-albumin solution at constant flow rate (0.04 ml/min/g body weight), so that dexreases in perfusion pressure reflect vasodilation. Substance P was given when the pulmonary vasculature was constricted by the thromboxane analog, U46619. *P<0.05.

Taken together, these data show that basal pulmonary vascular production of NO is minimal in healthy rats and is increased in chronic pulmonary hypertension. Pulmonary hypertension is not an NO deficiency syndrome.

In this study we measured NO production by the bovine pulmonary arterial endothelial cells in culture (purchased from ATCC), as well as the recognized signal for NO synthesis initiation, increased intracellular calcium concentration([Ca²⁺]_i). Using the dual-excitation microfluorometry after loading the cultured pulmonary arteriy endothelial cells with the fluorescent calcium indicator, fura 2 (from Molecular Probes), we found that acute physiologic hypoxia (i.e. not anoxia; PO₂ = 40 mmHg) caused a transient increase in [Ca²⁺]_i (known to be a sufficient signal to a sustained NO production) (Figure). Because NO is very rapidly oxidized in the presence of oxygen, the production of NO into the culture media was measured as the total of NO itself and its oxidation products, NO₂ and NO₃, (NO_x) by the chemiluminescence assay. NO synthesis was found insignificant during normoxia and elevated during hypoxia (Figure).

Acute hypoxia increases [Ca2+]i (left) and NO production (right) in cultured pulmonary endothelial cells.

These results indicate that hypoxic pulmonary vasoconstriction is opposed by increased NO synthesis rather than being mediated by reduced NO synthesis.