Inhaled nitric oxide decreases pulmonary endothelial nitric oxide synthase expression and activity in normal newborn rat lungs

Animal models

All experimental protocols and animal housing procedures were approved by the National Institute of Health and Biomedical Research (INSERM, UMR1141, Paris, France), and complied with the institutional instructions for animal care and use. Timed-pregnant Sprague-Dawley rats were maintained at 20–24°C in a room with a 12-h light/dark cycle and free access to standard food. Controls for rat behaviours, food and drink sources, and gas concentrations (NO, NO₂ and O₂) were performed twice a day on weekdays and once a day on weekends.

Experimental protocols

All pregnant Sprague-Dawley rats were kept in normoxic conditions and randomly divided into three groups at the last day of their gestation: control (room air), low-dose iNO (iNO-5 ppm) and high-dose iNO (iNO-20 ppm) groups [14]. Rats from the iNO groups were placed in a transparent 50×50×70-cm Plexiglas chamber that was connected to the NO source (BioSpherix, Redfield, IL, USA) so that rat pups would receive NO from their first inspiration. The concentration of NO₂ was continuously monitored and kept at <1 ppm so that potential harmful effects of NO₂, i.e. generation of methaemoglobin, could be prevented. The iNO period lasted for 7 days. Rat pups were sacrificed at post-natal day (P) 7 to study early lung development and at P14 to study late lung development. For the P14 group, all rats (control, iNO-5 ppm and iNO-20 ppm groups) were maintained under the same conditions with room air from P7 to P14.

Tissue preparations

Litter sizes were between 10 and 16; at least six littermates per group were killed by decapitation. The thoracic cavity was opened, and the lungs were removed and weighed. The left lung was washed in 0.9% NaCl (pH 7.4), fixed with paraformaldehyde buffer (4% in PBS, pH 7.4) until total submersion (2–5 days) and waxed in paraffin using an automated procedure (Shandon Citadel 2000 Tissue Processor; Rankin Biomedical, Holly, MI, USA). Lung blocks were cut into 5-µm sections for immunohistochemical study. The right lung was snap-frozen in liquid nitrogen and stored at −80°C until use for quantitative reverse transcriptase PCR (RT-PCR) or Western blot assays.

Chemical reagents and antibodies

All chemical reagents were purchased from Sigma-Aldrich (St Louis, MO, USA) except VECTASTAIN Elite ABC-Peroxidase kits (PK-7100) and VectaMount Permanent Mounting Medium (H-5000) for immunohistochemical staining, which were from Vector Laboratories (Burlingame, CA, USA). Primary antibodies included a rabbit anti-endothelial NOS (eNOS) antibody from BD Transduction Laboratories (San Jose, CA, USA; 610297) and a mouse anti-β-actin antibody from Santa Cruz Biotechnology (Dallas, TX, USA; sc-47778). Secondary antibodies were horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology; sc-2004) and goat anti-mouse IgG (Santa Cruz Biotechnology; sc-2005) antibodies for Western blotting, and biotinylated goat anti-rabbit IgG antibody (Vector Laboratories) for immunohistochemistry.

Immunohistochemistry for eNOS

Immunohistochemical staining for eNOS on rat lung sections was performed as previously described [15] with a minor modification. Sections of rat lung tissues were incubated with primary anti-eNOS antibody diluted to 1:50 (overnight, 4°C). For semiquantitative analyses, eNOS expression was separately evaluated in bronchial and arterial sections, using arbitrary intensity scores, assigned as no staining (0), focal staining (1), diffuse weak staining (2), diffuse moderate staining (3) and diffuse strong staining (4). For each animal, at least 10 images (×400 magnification) were randomly taken and “blindly” scored by two observers. Mean values of eNOS expression for each animal were used for comparison among groups of rat pups (control, iNO-5 ppm and iNO-20 ppm groups).

Western blotting analysis for eNOS

Protein extraction from frozen lung samples was performed as previously described [2]. For Western blots, equal amounts of pulmonary homogenates (50 µg per animal) were subjected to 7.5% SDS-PAGE and then transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Molsheim, France). Membranes were blocked in 5% nonfat milk for 1 h at room temperature and then hybridised with primary anti-eNOS antibody (1:500, overnight, 4°C). Blots were revealed after incubation with appropriate secondary HRP-conjugated antibody using enhanced chemiluminescence reagent (ECL Plus; Amersham, Courtaboeuf, France). Protein densities were quantified using the Genius2 image software system (Syngene, Cambridge, UK) and normalised to β-actin densities.

Quantitative RT-PCR experiments

Transcript levels of eNOS in lung tissues were determined by quantitative RT-PCR using a LightCycler® 480 System (Roche Applied Biosystems, Foster City, CA, USA) and normalised to hypoxanthine-guanine phosphoribosyltransferase (HPRT) as previously described [16].

Oligonucleotide primers were designed with the Primer Express software package (Applied Biosystems, Saint Aubin, France) and purchased from Eurogentec (Liege, Belgium): eNOS forward: 5′-GGAGAAGATGCCA- AGGCTGCTG-3′/eNOS reverse: 5′-CTTCCAGTGTCCAGACGCACCA-3′ (224 bp) and HPRT forward: 5′-GGTGAAAAGGACCTCTCGAA-3′/HPRT reverse: 5′-CAAGGGCATATCCAACAACA-3′ (260 bp).

Total RNA was extracted from frozen lung tissues using a Qiagen RNeasy Mini kit (Courtaboeuf, France). RNA quality and concentrations were assessed using a NanoDrop 1000 spectrophotometer (NanoDrop, Wilmington, DE, USA), based on relative absorbance at 260 versus 280 nm. Total RNA (1 µg per sample) was subjected to reverse transcription using an iScript cDNA Synthesis kit from Bio-Rad (Hercules, CA, USA).

For quantitative RT-PCR, samples were denatured at 95°C for 3 min, followed by 40 cycles of 60 s (96°C for 20 s, 60°C for 20 s and 72°C for 20 s), fusion at 68°C for 1 min and cooling at 45°C for 30 s. Relative quantification values of the target genes were standardised according to the value of HPRT used as housekeeping gene.

Determination of nitrate concentrations in rat lung homogenates

A NOS activity assay kit was used to measure newly synthesised NO from l-arginine by the action of NOS in the presence of essential cofactors, according to the manufacturer's instructions (Cayman Chemical, Ann Arbor, MI, USA). The final products of the reaction were nitrates, measured by a colorimetric method (540 nm), which indirectly represented the activity of NOS. Nitrate concentrations were determined by extrapolating from a standard curve.

Analyses of SOD activity

For analyses of SOD activity, frozen lung tissues were homogenised in ice-cold 0.1 M Tris/HCl, pH 7.4 containing 0.5% Triton X-100, 5 mM β-mercaptoethanol and 0.1 mg·mL⁻¹ phenylmethylsulfonyl fluoride. Tissue homogenates were centrifuged at 14 000×g for 10 min at 4°C and the supernatants were harvested. Total protein levels were measured using a bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL, USA). The SOD activity was determined using a SOD activity assay kit (BioVision, Mountain View, CA, USA) according to the manufacturer's instructions. Briefly, this kit utilises the cell proliferation reagent WST-1 that produces a water-soluble formazan dye upon reduction with superoxide anion. The rate of the reduction with superoxide anion is linearly related to the xanthine oxidase activity and is inhibited by SOD. The inhibition activity of SOD can be determined by a colorimetric method. Relative SOD activity (inhibition rate (%)) was normalised by protein amount of each sample.

Statistical analysis

Data are presented as mean±sem. Statistical comparisons were performed with ANOVA with Tukey's post hoc test using the Prism 5 software (GraphPad, San Diego, CA, USA). Differences were considered significant when p<0.05.