Exposure of mice to chronic hypoxia attenuates pulmonary arterial contractile responses to acute hypoxia by increases in extracellular hydrogen peroxide

Abstract

Exposing mice to a chronic hypoxic treatment (10% oxygen, 21 days) that promotes pulmonary hypertension was observed to attenuate the pulmonary vasoconstriction response to acute hypoxia (HPV) both in vivo and in isolated pulmonary arteries. Since catalase restored the HPV response in isolated arteries, it appeared to be attenuated by extracellular hydrogen peroxide. Chronic hypoxia promoted the detection of elevated lung superoxide, extracellular peroxide, extracellular SOD expression, and protein kinase G (PKG) activation [based on PKG dimerization and vasodilator-stimulated phosphoprotein (VASP) phosphorylation], suggesting increased generation of extracellular peroxide and PKG activation may contribute to the suppression of HPV. Aorta from mice exposed to 21 days of hypoxia also showed evidence for extracellular hydrogen peroxide, suppressing the relaxation response to acute hypoxia. Peroxide appeared to partially suppress contractions to phenylephrine used in the study of in vitro hypoxic responses. Treatment of mice with the heme precursor δ-aminolevulinic acid (ALA; 50 mg·kg(-1)·day(-1)) during exposure to chronic hypoxia was examined as a pulmonary hypertension therapy because it could potentially activate beneficial cGMP-mediated effects through promoting a prolonged protoporphyrin IX (PpIX)-elicited activation of soluble guanylate cyclase. ALA attenuated pulmonary hypertension, increases in both superoxide and peroxide, and the suppression of in vitro and in vivo HPV responses. ALA generated prolonged detectible increases in PpIX and PKG-associated phosphorylation of VASP, suggesting PKG activation may contribute to suppression of pulmonary hypertension and prevention of alterations in extracellular peroxide that appear to be attenuating HPV responses caused by chronic hypoxia.

Keywords: aminolevulinic acid; hypoxic pulmonary vasoconstriction; protein kinase G; pulmonary hypertension.

Fig. 1.

Data from Doppler flow echocardiography detection of relative increases in pulmonary arterial pressure based on decreased acceleration time (PAT)-to-ejection time (ET) ratios showing that 21 days of exposure hypoxia (10% oxygen) causes pulmonary hypertension and an attenuation of the acute hypoxic pulmonary vasoconstriction response (2-min hypoxia) in a manner that is prevented by treating mice with δ-aminolevulinic acid (ALA) (50 mg·kg⁻¹·day⁻¹) during the exposure to chronic hypoxia. *P < 0.05 vs. control (normoxia). #P < 0.05 vs. 21-day hypoxia (N). @P < 0.05 vs. for the respective normoxia groups (n = 5 or 6).

Fig. 2.

A comparison of lung tissue from 21-day control and 21-day ALA-treated mice showing that ALA increased the levels of protoporphyrin IX (PpIX) fluorescence (A) and VASP phosphorylation (B). Summary data showing that superoxide (C) and extracellular peroxide (D) levels are significantly increased by chronic hypoxia and that ALA treatment reversed these increases in lung tissues of chronic hypoxic mice. *P < 0.05 vs. control; #P < 0.05 vs. 21-day hypoxia; n = 6.

Fig. 3.

Western blot analysis showing the effects of exposure of mice to 21 days of hypoxia compared with a 21-day exposure to normoxia control on expression of PKG-dimerization (A) (n = 8). *P < 0.05 vs. control monomer. #P < 0.05 vs. control dimer. PKG monomer and dimer were analyzed as the % of total PKG after the total PKG was normalized to actin. B: VASP-phosphorylation (n = 8); *P < 0.05 vs. control and ecSOD (C) (n = 6); *P < 0.05 vs. control in mouse lung tissue.

Fig. 4.

Effects of the absence and presence of scavenging extracellular peroxide with catalase on contractile responses of pulmonary arteries isolated from mice exposed to 21 days of normoxia or hypoxia in the absence and presence of 21-day treatment with ALA (50 mg·kg⁻¹·day⁻¹) to 10 nM phenylephrine (A) and the contractile response (B) elicited by a subsequent exposure to 20 min of acute hypoxia. *P < 0.05 vs. control; #P < 0.05 vs. 21-day hypoxia; n = 8–10. @P < 0.05 for significant effects of catalase for respective PE/acute hypoxia groups.

Fig. 5.

Effects of the absence and presence of scavenging extracellular peroxide with catalase on contractile responses of aorta isolated from mice exposed to 21 days of normoxia or hypoxia in the absence and presence of 21-day treatment of mice with ALA (50 mg·kg⁻¹·day⁻¹) to 100 nM phenylephrine (A) and the relaxation response (B) elicited by a subsequent exposure to 20 min of acute hypoxia. *P < 0.05 vs. control; @P < 0.05 for significant effects of catalase for respective PE/acute hypoxia groups; n = 6–8.

Fig. 6.

Model showing systems hypothesized to be potentially contributing to how exposing mice to 21 days of chronic hypoxia attenuates the acute pulmonary arterial hypoxic pulmonary vasoconstriction (HPV) response through increasing hydrogen peroxide. The beneficial effects of 21 days of ALA treatment functioning through attenuating pulmonary hypertension and the generation of vasodilator levels of extracellular hydrogen peroxide potentially through its attenuation of the generation of superoxide are shown. PKG, cGMP protein kinase; PpIX, protoporphyrin IX.