Figures
- FIGURE 1
Expression of endothelial nitric oxide synthase (eNOS) assessed by Western blot in whole-lung homogenates. a, b) Densitometric values for relative eNOS protein (normalised to β-actin) at a) post-natal day (P) 7 and b) P14. CC: rat pups kept in room air (control group); iNO5: rat pups kept in a Plexiglas chamber continuously filled with NO at 5 ppm (iNO-5 ppm group); iNO20: rat pups kept in a Plexiglas chamber continuously filled with NO at 20 ppm (iNO-20 ppm group). c, d) Representative Western blots for whole-lung protein extracts at c) P7 and d) P14, separated on 7.5% SDS-PAGE, electrophoretically transferred to polyvinylidene difluoride membranes and analysed with a specific antiserum raised again eNOS (upper bands) or β-actin (lower bands). At P7, expression of eNOS was significantly decreased in the iNO-20 ppm group as compared with that in the control group (p<0.05). At P14, there was no significant difference among the three groups of rats (p>0.05). **: p<0.01.
- FIGURE 2
Representative images of endothelial nitric oxide synthase (eNOS) immunohistochemically stained in paraffin-embedded lung sections at post-natal day 7: a–d) bronchial structures and e–h) arterial structures. a, e) Negative control (primary antibody was replaced by mouse IgG serum at the same dilution). b, f) Rat pups kept in room air (control group). c, g) Rat pups kept in a Plexiglas chamber continuously filled with NO at 5 ppm (iNO-5 ppm group). d, h) Rat pups kept in a Plexiglas chamber continuously filled with NO at 20 ppm (iNO-20 ppm group). Scale bar: 50 μm.
- FIGURE 3
Expression of endothelial nitric oxide synthase (eNOS) evaluated by immunostaining in arterial and bronchial sections at a) post-natal day (P) 7 and b) P14. For semiquantitative analyses of eNOS expression, an arbitrary immunohistochemistry (IHC) intensity scoring system was predetermined and evaluated by two observers: no staining (0), focal staining (1), diffuse weak staining (2), diffuse moderate staining (3) and diffuse strong staining (4). CC: rat pups kept in room air (control group); iNO5: rat pups kept in a Plexiglas chamber continuously filled with NO at 5 ppm (iNO-5 ppm group); iNO20: rat pups kept in a Plexiglas chamber continuously filled with NO at 20 ppm (iNO-20 ppm group). a) At P7, eNOS expression was significantly lower in the iNO-20 ppm group than in the control group in arterial (p<0.01) and bronchial (p<0.005) sections. b) At P14, there was no significant difference of eNOS expression among the three groups of rats (p>0.05). **: p<0.01; ***: p<0.005 versus the control group (CC-P7).
- FIGURE 4
Quantification of endothelial nitric oxide synthase (eNOS) mRNA by quantitative reverse transcriptase (RT-PCR) in whole-lung homogenates at a) post-natal day (P) 7 and b) P14. Relative gene expression of eNOS in rat lungs was assessed by quantitative RT-PCR. Results were normalised to the amounts of hypoxanthine-guanine phosphoribosyltransferase (HPRT) mRNA. CC: rat pups kept in room air (control group); iNO5: rat pups kept in a Plexiglas chamber continuously filled with NO at 5 ppm (iNO-5 ppm group); iNO20: rat pups kept in a Plexiglas chamber continuously filled with NO at 20 ppm (iNO-20 ppm group). a) At P7, the amount of lung eNOS mRNA in the iNO-20 ppm group was significantly decreased as compared with that in the control group (p<0.005). b) At P14, the amounts of lung eNOS mRNA were comparable among the three groups of rat (p>0.05). ***: p<0.005 versus the control group (CC-P7).
- FIGURE 5
Measurement of nitric oxide synthase (NOS) and superoxide dismutase (SOD) activities in rat lung homogenates at post-natal day 7 (P7). CC: rat pups kept in room air (control group); iNO5: rat pups kept in a Plexiglas chamber continuously filled with NO at 5 ppm (iNO-5 ppm group); iNO20: rat pups kept in a Plexiglas chamber continuously filled with NO at 20 ppm (iNO-20 ppm group). Total NOS activity was indirectly assessed by determining the concentration of nitrate in rat lung homogenates (µM nitrate·(µg lung tissue protein)–1) and data are presented as mean±sem. The activity of SOD was measured by a colorimetric method and data are presented as the percentage inhibition of formation of formazan as described in the Methods section. The activity of NOS was significantly decreased in the lungs of the iNO-20 ppm group versus the control group (p<0.01), whilst the activity of SOD was significantly increased in the iNO-5 ppm and iNO-20 ppm groups (p<0.01 for both cases) as compared with that of the control group. **: p<0.01 versus the control group for NOS activity; ##: p<0.01 versus the control group for SOD activity.
