Main

RDS, the leading cause of mortality in premature infants, is characterized by insufficient surfactant and excess fluid in the lung (1, 2) and persistent high pulmonary vascular resistance. Glucocorticoid administration to the mother before premature delivery decreases the incidence of RDS (3). Some investigators have reported that antenatal glucocorticoid administration accelerates cell differentiation and surfactant production in the fetal lungs (4, 5). However, the mechanisms by which antenatal glucocorticoid therapy prevents RDS have not been explained sufficiently. Generally, during fetal life, the pulmonary vasculature is a high pressure bed with minimal blood flow through the lung. At birth, pulmonary vascular resistance decreases and blood flow increases as the lungs take over the function of gaseous exchange from the placenta (6). After birth, the pulmonary vascular resistance remains higher in neonates with RDS than in healthy neonates (7). Dubei et al. (8) suggested that fetal lung blood perfusion is increased after maternal corticosteroid administration.

NO is known to be an important factor in a variety of physiologic and pathologic processes. Besides acting as a vasodilatory substance, NO participates in cardiac and respiratory functions (9, 10). Dellinger et al. (11) reported that inhaled NO is associated with a significant improvement in oxygenation over that provided by the placebo. NO is synthesized by NOS from l-arginine in a variety of cell types. Biochemical and molecular studies have identified three isoforms of NOS: neuronal (type I), inducible (type II), and constitutive (type III). Constitutive and neuronal NOS need Ca2+ for their activation. Particularly, large quantities of constitutive NOS are known to be present in the lung (12). Kinsella et al. (13) reported that NO inhalation caused a consistent improvement in pulmonary hemodynamics and gas exchange in experimental hyaline membrane disease of premature lambs. Therefore, we expected that antenatal glucocorticoid therapy might improve pulmonary function in premature infants via the NO-NOS system in the lung.

In the present study, we measured the NOS activity, NOX concentration, and cGMP concentration in the lungs of antenatal glucocorticoid-treated premature and full-term neonates to assess the effect of antenatal glucocorticoid therapy on the NOS-NO system in the respiratory system.

METHODS

Animals and drug treatment.

Studies were performed according to the “Guiding Principles for the Care and Use of Laboratory Animals” of the Japanese Society of Pharmacology. Pregnant Wistar rats were housed in a room with controlled temperature (23° ± 1°C), humidity (55 ± 5%), and lighting (0600–1800 h), and were given free access to food and water. DEX (1 mg/kg/d, s.c.; Wako Junyaku Co., Tokyo, Japan) was administered to the pregnant rats on gestational days 17 and 18 (group A), 19 and 20 (group B), or 21 and 22 (group C). Floros et al. (14) demonstrated that the effect of antenatal DEX treatment on surfactant protein A was maximal at a dose of 2 mg/kg. In the present study, the dose of DEX was chosen on the basis of this study. Control groups were administered equivalent volumes of corn oil on the same three 2-d periods as DEX-treated groups. In groups A and B, fetuses were delivered by cesarean section with pentobarbital anesthesia 1 d after the last drug treatment. In group C, the rats were delivered vaginally on gestational day 22. In groups A and B, neonates were killed immediately after cesarean section, and in group C, neonates were killed 1 d after birth, which was 1 d after the last drug treatment to the mother. Their lungs were removed as soon as possible. The lung tissue was frozen with liquid nitrogen and stored at −80°C. Analysis was performed on lung tissues from six rats of each group.

NOS activity analysis.

NOS activity was measured by the methods of Salter et al. (15) with a slight modification of the concentration of EDTA in buffer (5 mM). We also modified the concentration of l-[2,3-3H]arginine (0.125 nmol/L). In brief, tissues were homogenized in a homogenate buffer (50 mM Tris HCl, pH 7.4) containing 0.1 mM EDTA, 0.1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM DTT, 10 mg/mL leupeptin, 1 mM pepstatin A, pH 7.4. The homogenate was centrifuged at 15,000 ×g for 20 min at 4°C. The supernatant was used for the NOS activity assay. An aliquot (20 mL) of supernatant was added to 200 mL of prewarmed incubation buffer composed of the following: 50 mM Tris, 1.2 mM CaCl2, 1 mM NADPH, 50 mM l-valine, 1 mg/200 mL calmodulin, 10 mM tetrahydrobiopterin, and 0.125 nmol l-[2,3-3H]arginine, pH 7.4. Three aliquots were incubated for 10 min at 37°C: control; 5 mM l-NAME plus 5 mM EDTA and 1 mM EGTA; and 5 mM EDTA plus 1 mM EGTA. The reaction was stopped by adding the sample to Dowex 50W (sodium form) in 20 mM HEPES (pH 5.5) and 2 mM EDTA. Citrulline was eluted from the resin, and the citrulline 3H activity was investigated by comparing the citrulline 3H activity in the control with that in the 5 mM l-NAME plus 5 mM EDTA and 1 mM EGTA-treated sample. The Ca2+-independent NOS activity was investigated by comparing the control with the 5 mM EDTA and 1 mM EGTA-treated sample.

NOX analysis.

NOX concentration was measured by Griess methods (Nitrate/Nitrite Colorimetric Assay Kit; Cayman Chemical Co., Ann Arbor, MI, U.S.A.). The tissue was homogenized in 0.25 M sucrose (5 vol) with a Polytron homogenizer (Kinematica, Lucerne, Switzerland). The homogenate was centrifuged at 4°C for 20 min at 15,000 ×g. Eighty milliliters of supernatant was incubated with nitrate reductase for 3 h. After incubation, Griess reagent was added to the sample and detected by OD measurement at 540 nm (U-2000A, Hitachi Co., Tokyo, Japan).

cGMP analysis.

The tissue was homogenized in 0.25 M sucrose (5 vol) with a Polytron homogenizer. The homogenate was centrifuged at 4°C for 20 min at 15,000 ×g. For cGMP measurement, 100 mL of supernatant was subjected to RIA (cGMP assay system, Amersham International, Buckinghamshire, UK): the cross-reactivity with cAMP was <0.1%. The radioactivity was measured by a gamma scintillation counter (ARC-300, Aloka Co., Tokyo, Japan).

Statistical analysis.

Data are presented as mean ± SD. The statistical difference between means was analyzed by two-way ANOVA. A probability <0.05 was considered statistically significant.

RESULTS

Comparison of effects of DEX on Ca2+-dependentNOS activity in lungs of neonatal and fetal rats.

Figure 1 shows the effect of gestational DEX on Ca2+-dependent NOS activity. Also, maturational changes in Ca2+-dependent NOS activity in the lungs of control neonatal and fetal rats are depicted in Figure 1. Ca2+-dependent NOS activity gradually increased with increasing gestational and postnatal age of control groups.

Figure 1
figure 1

Ca2+-dependent NOS activity in the lungs of neonates born at 19 d of gestation (19 d), 21 d of gestation (21 d), and full-term (1 D). Values are mean ± SD. Cont indicates control groups; DEX indicates dexamethasone-treated groups. DEX (1 mg/kg, s.c.) was administered to dams for 2 d [days 17 and 18 (19 d), days 19 and 20 (21 d), and days 21 and 22 (1 D); full-term = 22 d]. Number of rats is shown in parentheses. Detailed methods are presented in Methods.

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Comparison of effects of DEX on NOX concentration in lungs ofneonatal and fetal rats.

Figure 2 shows the effect of DEX on NOX concentration. Also, maturational changes in NOX concentration in the lungs of control neonatal and fetal rats are depicted in Figure 2. NOX concentration in the lungs was gradually increased according to gestational and postnatal age of control groups. DEX significantly increased NOX concentration of these rats.

Figure 2
figure 2

NOX concentration in the lungs of neonates born at 19 d of gestation (19 d), 21 d of gestation (21 d), and full-term (1 D). Values are mean ± SD. Cont indicates control groups; DEX indicates dexamethasone-treated groups. DEX (1 mg/kg, s.c.) was administered to dams for 2 d [days 17 and 18 (19 d), days 19 and 20 (21 d), and days 21 and 22 (1 D); full-term = 22 d]. Number of rats is shown in parentheses. Detailed methods are presented in Methods.

Full size image

Comparison of effect of DEX on cGMP concentration in lungs ofneonatal and fetal rats.

Figure 3 shows the effect of DEX on cGMP concentration. Also, maturational changes in cGMP concentration in the lungs of control neonatal and fetal rats are depicted in Figure 3. cGMP concentration in the lungs increased gradually with increasing gestational and postnatal age of control groups. DEX significantly increased cGMP concentration of these rats.

Figure 3
figure 3

cGMP concentration in the lungs of neonates born at 19 d of gestation (19 d), 21 d of gestation (21 d), and full-term (1 D). Values are mean ± SD. Cont indicates control groups; DEX indicates dexamethasone-treated groups. DEX (1 mg/kg, s.c.) was administered to dams for 2 d [days 17 and 18 (19 d), days 19 and 20 (21 d), and days 21 and 22 (1 D); full-term = 22 d]. Number of rats is shown in parentheses. Detailed methods are presented in Methods.

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DISCUSSION

NO stimulates guanylyl cyclase to produce cGMP. Our present data suggest that DEX increased NOX and cGMP concentrations through increases in NOS.

In the present study, developmental increases were observed in Ca2+-dependent NOS activity in the lungs of both late-gestation and full-term neonates. North et al. (16) reported that type III NOS gene expression in the fetal lung increases during late gestation to reach maximal amounts near term, which agrees with the changes in NOS activity we observed. They also demonstrated that type I NOS gene expression in the fetal lung decreases during late gestation (16). The developmental changes in Ca2+-dependent NOS activity that we measured may reflect type III NOS activity and may relate to type III NOS gene expression.

The physiologic role of the NOS-NO-cGMP system in the lung during the developmental stages is unknown. Moore et al. (17) suggested that endothelium-derived relaxing factor decreases pulmonary arterial resistance and increases pulmonary blood flow. Lipsitz et al. (18) observed that l-NAME, which is a known NOS inhibitor, increases pulmonary arterial pressure. Abman et al. (19) reported that infusion of nitro-l-arginine, a different NOS inhibitor, to pregnant ewes decreases fetal pulmonary blood flow and increases pulmonary artery pressure. In addition, Gommers et al. (20) suggested that NO in the lung increases arterial oxygenation. Kinsella et al. (13) found that inhaled NO reduces pulmonary vascular resistance, and increases left pulmonary artery blood flow and arterial Pco2 in severe experimental hyaline membrane disease of premature lambs. Roberts et al. (21) reported that NO inhalation increased oxygen saturation and oxygen tension without systemic hypotension in infants with persistent pulmonary hypertension. The improvement in oxygenation with inhaled NO is thought to occur through a reduction in pulmonary vascular resistance and a decreased right-to-left shunting (22). Indeed, Skimming et al. (23) reported that inhaled NO reduced right-to-left intracardiac shunting. Further, Roze et al. (24) suggested that inhaled NO improves ventilation-perfusion matching. These events might be related to the improvement in oxygen partial pressure in arterial blood by NO inhalation. Recently, Kabbani and Cassin (25) demonstrated that cGMP decreases lung liquid production and increases pulmonary blood flow. These findings, combined with our data, suggest that the NOS-NO-cGMP system in the lung can participate in an acute, dramatic reduction in pulmonary vascular resistance associated with the transition from fetal to postnatal life. Further study is needed to assess this hypothesis.

In the present study, we demonstrated that NOS activity in the lung of preterm rats in the control group was lower compared with that of full-term neonates. Our finding suggests that the NOS-NO system in the lungs of a preterm neonate does not reach the amount of a full-term neonate. Dellinger et al. (11) reported that NO facilitates pulmonary functions. Furthermore, NO plays an important role in the alteration of the pulmonary circulation during the transition at birth (16, 26). Therefore, the presence of an immature NOS system in the lungs might be an important factor in the mechanism of RDS in the human infant.

We demonstrated that NOS activity in the lungs of neonates exposed to DEX was significantly higher than that of nonexposed controls. Okoye et al. (27) reported that antenatal DEX treatment in rats of various ages with experimental congenital diaphragmatic hernias increased endothelial (type III) NOS protein immunoreactivity in small pulmonary arteries. Therefore, our observed Ca2+-dependent NOS activity may have been derived from endothelial cells in the lung.

Yu et al. (28) suggested that the vasodilatory effect of surfactant instillation in newborn piglets with surfactant deficiency is associated with activation of NOS. Indeed, numerous reports demonstrated that DEX increased surfactant in lungs of premature subjects. Therefore, our observations of DEX-induced increments of NOS-NO-cGMP may be related to surfactant increases by DEX treatment. Further study will be needed to evaluate this hypothesis.

NOS generates NO as a substrate of l-arginine, and NO stimulates cGMP production via activation of guanylyl cyclase. NOX is a metabolite of NO. Therefore, our present data suggested that increased NO, which is mediated by activation of NOS activity with DEX treatment, stimulates cGMP production. Antenatal glucocorticoid therapy is known to improve pulmonary function in immature neonates (3, 29). The increased NOS in the fetal lung after antenatal DEX treatment may lead to an early functional maturation through the NOS-NO-cGMP system. This increased NOS-NO-cGMP system may be responsible, in part, for the resistance to RDS produced by antenatal glucocorticoid therapy. Gao et al. (30) and Zhou et al. (31) reported that antenatal betamethasone therapy increased NO-mediated relaxation of coronary arteries and pulmonary veins, probably by increasing soluble guanylate cyclase activity. These findings suggest that the mechanisms of improved pulmonary function by antenatal glucocorticoid treatment involve not only increases of NO but also enhancement of NO-mediated vascular relaxation.

Many beneficial extrapulmonary effects of antenatal glucocorticoid therapy have been documented, for example, the improvement of cardiac, endocrine, and renal functions (32). At birth, the production of toxic oxygen free radicals is associated with the sudden increase in oxygen tension (33). The oxygen free radicals cause cellular damage in the cardiac muscle, and NO exerts a cardioprotective effect against several free radicals in the cardiomyocyte (34). If the NOS-NO system in the heart of a fetus is also relatively undeveloped, as is that in the lungs of neonates in our present study, it is possible that increased oxygen free radicals after birth exert toxic cardiac effects in the premature neonate. The beneficial effects of antenatal glucocorticoid therapy on cardiac function in neonates with RDS also may be related to the NOS-NO system. Further study is needed to assess the possibility that NO participates in these effects.

In conclusion, we showed that antenatal DEX treatment increases development of the NOS-NO-cGMP system in the lung of the neonatal rat and that antenatal DEX treatment increased the amounts of NOS activity in the lung of the preterm and full-term rat neonate. These findings suggest that the increase in the NOS-NO-cGMP system by antenatal DEX treatment may be related to the improvement in pulmonary function that is obtained by antenatal glucocorticoid therapy.