Apocynin improves oxygenation and increases eNOS in persistent pulmonary hypertension of the newborn

Abstract

NADPH oxidase is a major source of superoxide anions in the pulmonary arteries (PA). We previously reported that intratracheal SOD improves oxygenation and restores endothelial nitric oxide (NO) synthase (eNOS) function in lambs with persistent pulmonary hypertension of the newborn (PPHN). In this study, we determined the effects of the NADPH oxidase inhibitor apocynin on oxygenation, reactive oxygen species (ROS) levels, and NO signaling in PPHN lambs. PPHN was induced in lambs by antenatal ligation of the ductus arteriosus 9 days prior to delivery. Lambs were treated with vehicle or apocynin (3 mg/kg intratracheally) at birth and then ventilated with 100% O(2) for 24 h. A significant improvement in oxygenation was observed in apocynin-treated lambs after 24 h of ventilation. Contractility of isolated fifth-generation PA to norepinephrine was attenuated in apocynin-treated lambs. PA constrictions to NO synthase (NOS) inhibition with N-nitro-l-arginine were blunted in PPHN lambs; apocynin restored contractility to N-nitro-l-arginine, suggesting increased NOS activity. Intratracheal apocynin also enhanced PA relaxations to the eNOS activator A-23187 and to the NO donor S-nitrosyl-N-acetyl-penicillamine. Apocynin decreased the interaction between NADPH oxidase subunits p22(phox) and p47(phox) and decreased the expression of Nox2 and p22(phox) in ventilated PPHN lungs. These findings were associated with decreased superoxide and 3-nitrotyrosine levels in the PA of apocynin-treated PPHN lambs. eNOS protein expression, endothelial NO levels, and tetrahydrobiopterin-to-dihydrobiopterin ratios were significantly increased in PA from apocynin-treated lambs, although cGMP levels did not significantly increase and phosphodiesterase-5 activity did not significantly decrease. NADPH oxidase inhibition with apocynin may improve oxygenation, in part, by attenuating ROS-mediated vasoconstriction and by increasing NOS activity.

Fig. 1.

Apocynin improves oxygenation in ventilated lambs with persistent pulmonary hypertension of the newborn (PPHN). Arterial-to-alveolar Po₂ ratio (a/A ratio) was measured in 1 group of lambs with PPHN ventilated with 100% O₂ (PPHN-100 O₂) and 1 group of lambs with PPHN ventilated with 100% O₂ and treated with apocynin (3 mg/kg) administered intratracheally at birth (PPHN-Apocynin). PPHN lambs ventilated with 100% O₂ were critically ill, and 3 died: 1 at ∼8 h, 1 at 11 h, and 1 at 12 h (arrows). Dashed line beyond this point represents mean ± SE for surviving lambs. *P < 0.05 vs. PPHN-100% O₂.

Fig. 2.

Intratracheal apocynin reduces pulmonary artery (PA) contractility and enhances relaxation in ventilated PPHN lambs. Cumulative concentration-response curves to increasing doses of norepinephrine (NE) in 5th-generation PA isolated from 1-day spontaneously breathing (Control-1DSB) lambs, PPHN lambs ventilated for 24 h with 100% O₂ (Oxygen), and PPHN lambs ventilated for 24 h with 100% O₂ and treated with intratracheal apocynin. *P < 0.05 vs. Control-1DSB. †P < 0.05 vs. Oxygen.

Fig. 3.

A: apocynin restores endogenous nitric oxide synthase (NOS) activity in ventilated PPHN lamb PA. Contraction response of PA to 10⁻⁶ M NE was measured with or without pretreatment with the NOS inhibitor N-nitro-l-arginine (l-NA, 1 mM) in 1DSB-Control lambs (n = 6), PPHN lambs ventilated for 24 h with 100% O₂ (PPHN-Oxygen, n = 5), and PPHN lambs ventilated for 24 h with 100% O₂ and treated with intratracheal apocynin (PPHN-Apocynin, n = 5). *P < 0.05 vs. Control-1DSB. †P < 0.05 vs. PPHN-Oxygen. ‡P < 0.05 vs. corresponding PA without l-NA. B: apocynin reduces PA contractility and enhances relaxation to the NOS stimulant A-23187. Response was measured in 5th-generation PA that were constricted to half-maximal capacity with NE and relaxed with A-23187. *P < 0.05 vs. PPHN-Oxygen at 10⁻⁶ M only. C: apocynin reduces PA contractility and enhances relaxation to the NO donor S-nitrosyl-N-acetyl-penicillamine (SNAP). Response was measured in 5th-generation PA constricted to half-maximal capacity with NE and relaxed with SNAP. *P < 0.05, PPHN-Apocynin vs. PPHN-Oxygen. †P < 0.05, Control-1DSB vs. PPHN-Oxygen.

Fig. 4.

Apocynin decreases reactive oxygen species levels in ventilated PPHN PA. A: frozen lung sections were probed with dihydroethidium (DHE), which is oxidized by superoxide to a fluorescent red product. Sequential sections were additionally treated with polyethylene glycol (PEG)-SOD to determine SOD-inhibited DHE fluorescence. Images show PA and airways (AW). B: average SOD-inhibited fluorescence intensities from PA were quantified by MetaMorph and are expressed relative to Control-1DSB. C: lung sections were fixed and probed with an antibody against 3-nitrotyrosine (3-NT). Fluorescence from an appropriate secondary antibody was evident within PA and airways. D: 3-NT fluorescence intensities within the PA were quantified by MetaMorph and are expressed relative to Control-1DSB. Values are means ± SE; n = 6 Control-1DSB, n = 6 PPHN-Oxygen, n = 5 PPHN-Apocynin. *P < 0.05 vs. Control-1DSB. †P < 0.05 vs. PPHN-Oxygen.

Fig. 5.

Apocynin decreases Nox2 and p22^phox expression and p22^phox/p47^phox association in ventilated PPHN lungs. A: representative Western blots for Nox2, p22^phox, and p47^phox. Bands separated by a black line were from the same Western blot membrane and exposure, and images were processed identically. B: band intensities were normalized to β-actin and are expressed relative to Control-1DSB. Values are means ± SE; n = 6 Control-1DSB, n = 6 PPHN-Oxygen, n = 5 PPHN-Apocynin. *P < 0.05 vs. Control-1DSB. †P < 0.05 vs. PPHN-Oxygen. C: lung protein was immunoprecipitated using an antibody against p47^phox, and Western blot was probed with an anti-p22^phox antibody. Also depicted is precipitating IgG protein from the immunoprecipitation reaction. D: p22 band intensities were normalized to the IgG band and are expressed relative to Control-1DSB. Values are means ± SE; n = 5 Control-1DSB, n = 5 PPHN-Oxygen, n = 5 PPHN-Apocynin. *P < 0.05 vs. Control-1DSB. †P < 0.05 vs. PPHN-Oxygen.

Fig. 6.

Apocynin increases endothelial NOS (eNOS) expression and nitric oxide levels in ventilated PPHN lungs. A: representative Western blot. Bands separated by a black line were from the same Western blot membrane and exposure, and images were processed identically. B: band intensities were normalized to β-actin and are expressed relative to Control-1DSB. C: frozen lung sections were probed with 4,5-diaminofluorescein diacetate (DAF2-DA), which fluoresces when it interacts with nitric oxide. Sections show PA and airways. D: average DAF2-DA fluorescence intensities from endothelium of PA were quantified by MetaMorph and are expressed relative to Control-1DSB. Values are means ± SE; n = 6 Control-1DSB, n = 6 PPHN-Oxygen, n = 5 PPHN-Apocynin. *P < 0.05 vs. Control-1DSB. †P < 0.05 vs. PPHN-Oxygen.

Fig. 7.

Apocynin increases the ratio of tetrahydrobiopterin (BH₄) to dihydrobiopterin (BH₂) in ventilated PPHN PA. A: total biopterin, BH₄, and BH₂ levels were determined by HPLC. B: BH₄-to-BH₂ ratios were calculated for each group. Values are means ± SE; n = 6 Control-1DSB, n = 4 PPHN-Oxygen, n = 4 PPHN-Apocynin. *P < 0.05 vs. PPHN-Oxygen.

Fig. 8.

A: apocynin increases cGMP levels in ventilated PPHN PA. cGMP levels were determined by enzyme immunoassay and normalized to protein content. Values are means ± SE; n = 6 Control-1DSB, n = 5 PPHN-Oxygen, n = 4 PPHN-Apocynin. *P < 0.05 vs. Control-1DSB. B–D: apocynin increases phosphodiesterase 5 (PDE5) protein expression but decreases activity in ventilated PPHN PA. B: representative Western blot. C: band intensities normalized to β-actin and expressed relative to Control-1DSB. Values are means ± SE; n = 5 Control-1DSB, n = 8 PPHN-Oxygen, n = 5 PPHN-Apocynin. *P < 0.05 vs. Control-1DSB. †P < 0.05 vs. PPHN-Oxygen. D: specific activity of PDE5 in resistance PA. Values are means ± SE; n = 4 Control-1DSB, n = 6 PPHN-Oxygen, n = 5 PPHN-Apocynin. *P < 0.05 vs. Control-1DSB.