Upregulation of nitric oxide synthase in mice with severe hypoxia-induced pulmonary hypertension

Abstract

Background: The importance of nitric oxide (NO) in hypoxic pulmonary hypertension has been demonstrated using nitric oxide synthase (NOS) knockout mice. In that model NO from endothelial NOS (eNOS) plays a central role in modulating pulmonary vascular tone and attenuating hypoxic pulmonary hypertension. However, the normal regulation of NOS expression in mice following hypoxia is uncertain. Because genetically engineered mice are often utilized in studies of NO, we conducted the present study to determine how hypoxia alters NOS expression in wild-type mice.

Method: Mice were exposed to sea level, ambient conditions (5280 feet) or severe altitude (17,000 feet) for 6 weeks from birth, and hemodynamics and lung NOS expression were assessed.

Results: Hypoxic mice developed severe pulmonary hypertension (right ventricular systolic pressure [RVsP] 60 mmHg) as compared with normoxic mice (27 mmHg). Using quantitative reverse-transcription PCR, it was found that expressions of eNOS and inducible NOS (iNOS) increased 1.5-fold and 3.5-fold, respectively, in the lung. In addition, the level of lung eNOS protein was increased, neuronal NOS (nNOS) protein was unchanged, and iNOS was below the limit of detection. Immunohistochemistry demonstrated no change in lung iNOS or nNOS staining in either central or peripheral areas, but suggested increased eNOS in the periphery following hypoxia.

Conclusion: In mice, hypoxia is associated with increases in lung eNOS, possibly in iNOS, but not in nNOS; this suggests that the pattern of lung NOS expression following hypoxia must be considered in studies using genetically engineered mice.

Figure 1

Right ventricular systolic pressure (RVsP) in mice exposed to sea level conditions (SL, n = 5), ambient conditions (DEN, n = 9), or hypobaric hypoxia (HA, n = 6) for 6 weeks after birth.^*P < 0.005 versus SL or DEN.

Figure 2

Lung nitric oxide synthase (NOS) protein. (a) Western blots of homogenized whole lung from mice exposed to sea level conditions (S, n = 5), ambient conditions (D, n = 4), or hypobaric hypoxia (H, n = 5) for endothelial NOS (eNOS; 140 kDa), inducible NOS (iNOS; 130 kDa), neuronal NOS (nNOS; 164 and 160 kDa), and β-actin (42 kDa). Positive control is present for iNOS (lipopolysaccharide-treated mouse spleen protein) and nNOS (mouse brain protein). nNOS protein on Western analysis has previously been reported to appear as a doublet [45]. MW, molecular weight marker. (b) Densitometry evaluation for eNOS protein comparing sea level (SL, n = 5) with ambient conditions (DEN, n = 3) and hypobaric hypoxia (HA, n = 5). ^*P < 0.05 SL versus HA.

Figure 3

Quantitative reverse-transcription PCR for endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS) in homogenized whole lungs from mice exposed to sea level conditions (SL, n = 5), ambient conditions (DEN, n = 4), or hypobaric hypoxia (HA, n = 5) for 6 weeks after birth.^*P < 0.05 versus SL, ^#P < 0.05 versus DEN.

Figure 4

Lung nitric oxide synthase (NOS) immunolocalization. Lung endothelial NOS (eNOS) immunostaining in (a) normoxic versus (b) hypoxic mice, demonstrating an increase in peripheral distribution of eNOS (arrow, red staining). Lung inducible NOS (iNOS) immunostaining in (c) normoxic versus (d) hypobaric mice, demonstrating no difference in the airway epithelial localization of iNOS (arrow, brown staining). Lung neuronal NOS (nNOS) immunostaining in (e) normoxic versus (f) hypoxic mice, demonstrating no change in airway epithelial distribution of nNOS (arrow, brown staining). Magnification 40×.

Figure 5

Plasma nitric oxide metabolites (NOX) from mice exposed to sea level conditions (SL, n = 5), ambient conditions (DEN, n = 9), or hypobaric hypoxia (HA, n = 6) for 6 weeks after birth. ^*P < 0.05 versus DEN.