INTRODUCTION

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AT BIRTH AND ALONG THE FIRST days and weeks of extrauterine life, several important functional and structural changes occur in the pulmonary circulation to replace the placenta for gas exchange (12). During the first minutes of extrauterine life, as the lung becomes responsible for blood oxygenation, there is an 8- to 10-fold increase in pulmonary blood flow, and pulmonary arterial pressure falls from suprasystemic levels in the fetus to about half of systemic values (10). This acute decline in pulmonary vascular resistance is triggered by lung ventilation, oxygenation, and increased shear stress (12). The endothelium-derived vasodilators nitric oxide (NO) and cyclooxygenase (Cox)-derived metabolites, mainly prostacyclin (PGI₂), are critically involved in these changes (30). In fact, inhibition of Cox or NO synthases attenuates the birth-related decline in pulmonary vascular resistances in lambs (1, 9, 29).

A second postnatal maturational phase of pulmonary vascular resistance reduction takes place over the first 2-3 wk of extrauterine life in pigs and humans to reach the pulmonary pressure values characteristic of the adult life (14). During this period, the pulmonary circulation is highly vulnerable to develop pulmonary hypertension in response to exogenous insults. Several groups have consistently reported an increase in endothelium-dependent vasodilation to ACh or exogenous NO during the first days of extrauterine life in rabbit (21), lamb (2, 15, 32), and piglet (4, 18, 32, 37) pulmonary arteries, which seems to be a primary factor for the postnatal adaptative maturation. This maturational process is specific to the pulmonary circulation (20, 25, 33, 34), but the mechanisms involved in the age-dependent changes are unclear. Although the responses to exogenous NO gas increase with age (34, 37), the vasodilator responses to the NO donor sodium nitroprusside (18) or the cGMP analog 8-bromo-cGMP (34) remain unchanged. These results indicate that changes in the activity of soluble guanylate cyclase and cGMP-dependent phosphodiesterase are not essential to explain the maturation of NO-dependent relaxation. In fact, the activity and expression of soluble guanylate cyclase decrease postnatally in rat lung (3). Because NO and nitroprusside exhibit a different susceptibility to be inactivated by superoxide (19), an attractive hypothesis to explain the age-dependent difference in NO-induced relaxation involves an elevated metabolism of NO resulting from excess tissue levels of superoxide. Therefore, the aim of the present study was to further analyze the maturational changes in the response to exogenous NO and to determine the possible role of superoxide-generating and -metabolizing enzymatic pathways.

	METHODS

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All of the procedures conform with the Guide for the Care and Use of Laboratory Animals (NIH publication no. 85-23, revised 1996).

Tissue preparation. Male piglets of 3-18 h (n = 25) or 15-20 days of age (n = 29) were used in this study. The lungs were rapidly immersed in cold (4°C) Krebs solution (composition in mM: 118 NaCl, 4.75 KCl, 25 NaHCO₃, 1.2 MgSO₄, 2.0 CaCl₂, 1.2 KH₂PO₄, and 11 glucose). The pulmonary arteries (third branch with an internal diameter of ~0.5-1.5 mm) were carefully dissected free of surrounding tissue and cut into rings of 2-3 mm length (26-28). Except where otherwise stated, the endothelium was removed by gently rubbing the intimal surface of the rings with a metal rod. The endothelium removal procedure was verified by the inability of ACh (10⁶ M) to relax arteries precontracted with norepinephrine (10⁶ M). Rings were mounted between two hooks under 0.5 g of tension in a 5-ml organ bath filled with Krebs solution at 37°C gassed with a 95% O₂-5% CO₂ gas mixture as previously described (26-28). The contraction was measured by an isometric force transducer (model PRE 206-4 from Cibertec, Madrid, Spain; or Grass model FT03) using data acquisition software and hardware (REGXPC computer program from Cibertec or Powerlab hardware and Chart version 3.4 software from AD Instruments, Castle Hill, Australia).

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Western blot analysis. Pulmonary arteries were frozen in liquid nitrogen and stored at 70°C. Frozen arteries were homogenized in a glass potter in a buffer of the following composition: 1 mM sodium vanadate, 1% SDS, and 10 mM Tris. The homogenate was centrifuged at 10,000 revolutions/min for 1 min. The protein content in the supernatant was determined using the Bradford assay (reagents from Bio-Rad). Western blotting was performed with 20 µg protein from the supernatant/lane. SDS-PAGE (12% acrylamide) was performed using the method of Laemmli (16) in a minigel system (Bio-Rad). The proteins were transferred to polyvinylidene difluoride membranes overnight and incubated with rabbit anti-Cu,Zn-SOD polyclonal (1:1,400; StressGen Biotechnologies), goat anti-Cox-1 monoclonal (1:1,000 dilution; SantaCruz Biotechnology), rabbit anti-Cox-2 polyclonal (1:1,000; Cayman Chemical), or mouse anti--smooth muscle-actin monoclonal (1:400; Sigma) antibodies and then with the respective secondary horseradish peroxidase-conjugated antibodies. The bands were visualized by enhanced chemiluminiscence (Amersham) and quantified using image analysis software (NIH Image). The bands of actin in pulmonary arteries were similar in the two experimental groups (15- to 20-day-old animals were 103 ± 10% of those of newborns, P > 0.05) and were used as a reference for the expression of other proteins. Thus the results of SOD, Cox-1, and Cox-2 protein expression were normalized with respect to the bands of actin and expressed as a percentage of the data of 3- to 18-h-old animals.

Drugs. U-46619 was from Alexis Biochemicals (Läufelfingen, Switzerland), ODQ was from Tocris Cookson (Bristol, UK), SKF-525A was from RBI, AA-861 was from Takeda, meclofenamate was from Warner Lambert, and all other drugs were from Sigma Chemical (Alcobendas, Spain). Drugs were dissolved initially in distilled deionized water (except for thapsigargin, AA-861, and ODQ, which were dissolved in dimethyl sulfoxide, indomethacin and meclofenamate in ethanol, and HX in 0.1% NaOH) to prepare a 10² or 10³ M stock solution, and further dilutions were made in Krebs solution.

Statistical analysis. Results are expressed as means ± SE, and n reflects the number of animals from which the arterial rings were obtained. Individual cumulative concentration-response curves were fitted to a logistic equation. The drug concentration exhibiting 30% relaxation was calculated from the fitted concentration-response curves for each ring and expressed as negative log molar (-log [IC₃₀]). The magnitude of the effect of agents modulating the response to NO agonists was quantified by the log dose ratios, which represents the distance between the two curves, i.e., log (dose ratio) = log [IC₃₀ (control)/IC₃₀ (drug)]. Statistically significant differences between groups were calculated by an ANOVA followed by a Newman Keuls test. P < 0.05 was considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Developmental changes in NO- and NO donor-induced vasodilatation. The vasodilator response to exogenously added NO increased significantly with postnatal development, and this effect was similarly observed in arteries preconstricted with KCl, U-46619, and endothelin-1 (Fig. 1). However, in contrast to exogenous NO gas, the NO donor nitroprusside produced a relaxant response in arteries stimulated with KCl, U-46619, or endothelin-1 that was similar in the two age groups (Fig. 2).

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Effects of nitric oxide (NO) in endothelium-denuded pulmonary arteries stimulated with 40 mM KCl (A), 107 M U-46619 (B), or 3 × 109 M endothelin-1 (C) in animals of 3-18 h and 15-20 days (d) of extrauterine life. Results are expressed as means ± SE (n = 8-14). *P < 0.05 vs. 3-18 h. Brackets denote concentration.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Effects of sodium nitroprusside (SNP) in endothelium-denuded pulmonary arteries stimulated with 40 mM KCl (A), 107 M U-46619 (B), or 3 × 109 M endothelin-1 (C) in animals of 3-18 h and 15-20 days of extrauterine life. Results are expressed as means ± SE (n = 8-14).

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Effects of the soluble guanylate cyclase inhibitor ODQ (106 M) and the sacoplasmic Ca2+-ATPase inhibitor thapsigargin (2 × 106 M) on NO-induced relaxation in endothelium-denuded pulmonary arteries (E) and in endothelium-intact arteries (+E) stimulated with 107 M U-46619 in animals of 3-18 h (A) and 15-20 days (B) of extrauterine life. C: log [IC30] values calculated from data in A and B. Results are expressed as means ± SE (n = 6-13). *P < 0.05 vs. control (E) and P < 0.05 vs. 3-18 h.

Role of SOD and increased and decreased superoxide anion. The inhibition of endogenous SOD by DETCA (10³ M) produced a strong inhibitory effect on NO-induced relaxation, whereas addition of exogenous SOD increased it (Fig. 4). A trend for an increased effect of exogenous SOD in 15- to 20-day old animals (log dose ratio = 0.91 ± 0.15) compared with that in 3- to 18-h animals (0.49 ± 0.15) was observed, but these differences did not reach statistical significance. The effects of DETCA were similar at the two ages (log dose ratios = 0.65 ± 0.07 and 0.55 ± 0.2 in arteries from 3- to 18-h- and 15- to 20-day-old animals, respectively, P > 0.05). Therefore, in the presence of DETCA or SOD, the age-dependent increase in NO-induced relaxation was maintained (Fig. 4C). Furthermore, a weak but significant reduction in SOD protein expression was observed at 15-20 days compared with 3-18 h (Fig. 4, D and E). The effects of MnCl₂, which is a membrane-permeable SOD-like compound, on NO-induced relaxation were very similar to those observed for SOD (Fig. 5). A nonsignificant trend for an increased effect of MnCl₂ in 15- to 20-day-old animals (log dose ratio = 0.76 ± 0.13) compared with 3- to 18-h animals (0.40 ± 0.22, P > 0.05) was also observed. Figure 5 also shows that the exogenous superoxide anion-generating system XO plus HX also produced a similar inhibitory effect on NO-induced relaxation at the two developmental stages.

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Effects of superoxide dismutase (SOD, 100 U/ml) and the SOD inhibitor diethyldithiocarbamate (DETCA, 103 M) on NO-induced relaxation in endothelium-denuded pulmonary arteries stimulated with 107 M U-46619 in animals of 3-18 h (A) and 15-20 days (B) of extrauterine life. C: log [IC30] values calculated from data in A and B. Results are expressed as means ± SE (n = 7-8). *P < 0.05 vs. control and P < 0.05 vs. 3-18 h. D: representative Western blot of Cu,Zn-SOD and -actin protein expression. E: averaged SOD protein expression (mean ± SE) at the two developmental stages (n = 6 and 4, respectively) normalized with respect to the expression of -actin and expressed as a percentage of 3-18 h.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Effects of the SOD mimetic (MnCl2) and xanthine oxidase (XO, 5 mU/ml) plus hypoxanthine (HX, 104 M) on NO-induced relaxation in endothelium-denuded pulmonary arteries stimulated with 107 M U-46619 in animals of 3-18 h (A) and 15-20 days (B) of extrauterine life. C: log [IC30] values calculated from data in A and B. Results are expressed as means ± SE (n = 6). *P < 0.05 vs. control and P < 0.05 vs. 3-18 h.

Role of endogenous superoxide-generating systems. The source of superoxide that could be responsible for the destruction of NO was analyzed using pharmacological tools to inhibit different potential enzymatic superoxide-generating systems (7). We used DPI [inhibitor of membrane NAD(P)H oxidase], L-NAME (inhibitor of NO synthase), oxypurinol (inhibitor of XO), and rotenone (inhibitor of complex I of the mitochondrial electron transport chain). DPI significantly potentiated the relaxant effect of NO at the two ages, whereas the other inhibitors had no effect on these responses (Fig. 6). However, DPI-induced potentiation was significantly higher in the 15- to 20-day-old animals (log dose ratio = 0.58 ± 0.07) compared with the 3- to 18-h animals (0.24 ± 0.03, P < 0.05).

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Effects of inhibitors of potential enzymatic sources of superoxide anion: nitro-L-arginine methyl ester (L-NAME, 104 M, NO synthase inhibitor), oxypurinol (105 M, inhibitor of xanthine oxidase), rotenone (105 M, inhibitor of complex I of the mitochondrial electron transport chain), and diphenyleneiodonium (DPI, 105 M, inhibitor of the membrane NADPH oxidase) on NO-induced relaxation in endothelium-denuded pulmonary arteries stimulated with 107 M U-46619 in animals of 3-18 h (A) and 15-20 days (B) of extrauterine life. C: log [IC30] values calculated from data in A and B. Results are expressed as means ± SE (n = 5-6). *P < 0.05 vs. control and P < 0.05 vs. 3-18 h.

View larger version (36K): [in this window] [in a new window]   Fig. 7.   Effects of arachidonic acid (105 M) and inhibitors of arachidonic acid metabolism [indomethacin (105 M, cyclooxygenase inhibitor), AA-861 (105 M, inhibitor of 5-lipooxygenase), and SKF-525A (105 M, inhibitor of cytochrome P-450)] on NO-induced relaxation in endothelium-denuded pulmonary arteries stimulated with 107 M U-46619 in animals of 3-18 h (A) and 15-20 days (B) of extrauterine life. C: log [IC30] values calculated from data in A and B. D: lack of effect of indomethacin on nitroprusside-induced relaxation in arteries from both age groups stimulated with U-46619. Results are expressed as means ± SE (n = 5-7). *P < 0.05 vs. control and P < 0.05 vs. 3-18 h.

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View larger version (19K): [in this window] [in a new window]   Fig. 8.   Effects of indomethacin (105 M) on NO (A)- and nitroprusside (B)-induced relaxation in endothelium-denuded pulmonary arteries stimulated with 3 × 109 M endothelin-1 from animals of 3-18 h and 15-20 days of extrauterine life. Results are expressed as means ± SE (n = 5-6). *P < 0.05 vs. control.

Isoforms of Cox involved. To further determine the role of Cox isoforms (Cox-1 and Cox-2) involved in the reduced effect of NO, we studied the effects of meclofenamate, another nonselective Cox inhibitor chemically unrelated to indomethacin, aspirin, which at 5 × 10⁵ M is a fairly selective Cox-1 inhibitor, and NS-398, a selective Cox-2 inhibitor. None of these drugs produced any effect on NO-induced effects in the older animals (Fig. 9B). Meclofenamate and aspirin, however, significantly potentiated NO responses in neonates (log dose ratio 0.34 ± 0.06 and 0.35 ± 0.14, respectively, Fig. 9A) to a similar extent to that observed with indomethacin so that both drugs abolished the maturational differences (Fig. 9C). In contrast, NS-398 had no significant effect.

View larger version (32K): [in this window] [in a new window]   Fig. 9.   Effects of the cyclooxygenase (Cox) inhibitors meclofenamate (105 M, nonselective), aspirin (5 × 105 M, fairly selective Cox-1 inhibitor), and NS-398 (105 M, selective Cox-2 inhibitor) on NO-induced relaxation in endothelium-denuded pulmonary arteries stimulated with 107 M U-46619 in animals of 3-18 h (A) and 15-20 days (B) of extrauterine life. C: log [IC30] values calculated from data in A and B. Results are expressed as means ± SE (n = 5-8). *P < 0.05 vs. control and P < 0.05 vs. 3-18 h. D and E: representative Western blots of Cox-1, Cox-2, and -actin. F: Cox-1 and Cox-2 protein expression (mean ± SE) at the two developmental stages (n = 6 and 4, respectively) normalized with respect to the expression of -actin and expressed as a percentage of 3-18 h.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In the present paper, we found that, in isolated pulmonary arteries, the relaxant responses to exogenous NO increased during the first days of extrauterine life, whereas the responses to the NO donor sodium nitroprusside remained unchanged. All of these results are consistent with data previously published in piglet, rabbit, and lamb pulmonary arteries (2, 4, 15, 18, 21, 32, 34, 37). The most interesting finding of this study is that the inhibitors of Cox-1 specifically potentiated the responses to NO in the newborn animals so that, in their presence, the age-dependent changes were no longer observed. Other attempts to modulate the response to exogenous NO produced similar effects in newborns than in older animals. In addition, the maturation of the response to NO was not related to changes in the patterns of expression of SOD, Cox-1, and Cox-2 proteins.

NO- and nitroprusside-induced vasodilatation in piglet pulmonary arteries has been related to a cGMP-dependent activation of the sarcoplasmic reticulum Ca²⁺-ATPase (8). The vasodilator effects of NO in the two age groups were similarly inhibited by ODQ and thapsigargin (inhibitors of guanylate cyclase and the sarcoplasmic reticulum Ca²⁺-ATPase, respectively). Therefore, these results indicate that no differences in the NO/cGMP pathway beyond the activation of guanylate cyclase are responsible for the maturation of the NO response. Early maturational changes in phosphodiesterase 5 (PDE5) expression in mouse and sheep lung tissue have been proposed to account for the postnatal decrease in pulmonary vascular resistance (13). However, unchanged age-dependent responses to nitroprusside as described herein and by other authors (18) are not consistent with NO-dependent increases being the result of changes in PDE5 activity.

In contrast to NO, the responses to nitroprusside are unaffected by basal or exogenously stimulated superoxide production (19). Therefore, we hypothesized that age-dependent differences in NO- but not in nitroprusside-induced relaxation could be related to changes in the reactive oxidant species. Likewise, Morecroft and McLean (21) reported that the ACh-induced vasodilatation was preferentially increased by SOD in neonatal rabbits compared with older animals and suggested that the age-dependent differences could be the result of reduced SOD activity at birth. However, in piglets, the potentiation of the effects of exogenous SOD was, if any, higher in older animals (present results and Ref. 34). In the present study, the membrane-permeable SOD mimetic MnCl₂ also potentiated the vasodilator effects of NO, but these effects were similar to those of SOD. It is likely that the concentration of MnCl₂ used was not maximally effective, but the use of higher concentrations was limited by its solubility in Krebs solution. Conversely, Cu,Zn-SOD inhibition by DETCA produced similar inhibitory effects in both groups. Moreover, the inhibitory effects of the superoxide-generating system XO plus HX were also similar in the two groups. In addition, the expression of Cu,Zn-SOD protein was lower in older animals. Thus there is a maturation in the expression of Cu,Zn-SOD protein in piglet pulmonary arteries. However, these age-dependent changes in SOD expression and the effects of SOD mimetics and inhibitors cannot explain those of the responses to NO. Nevertheless, in the present study, we did not address a possible role of other isoforms of SOD (e.g., the mitochondrial Mn-SOD isoenzyme) or nonenzymatic scavengers of superoxide. Therefore, these results clearly indicate that a reduced Cu,Zn-SOD activity does not account for the reduced NO activity at birth but do not fully exclude an increased tissue superoxide in newborn animals.

In the search for a maturational change in the source of superoxide, we investigated the effects of inhibitors of several potential enzymatic sources of superoxide. In pulmonary arteries, XO, NO synthases, complex I of the respiratory electron transport chain, 5-lypoxygenase, and cytochrome P-450 are not important sources of superoxide, as indicated by the lack of inhibitory effect of oxypurinol, L-NAME, rotenone, AA-861, and SKF-525A, respectively. In contrast, DPI potentiated NO-induced relaxation. DPI is a membrane NADPH oxidase inhibitor but also inhibits complex I of the mitochodrial electron transport chain. Because rotenone (which does not affect NADPH oxidase) failed to modify NO-induced relaxation, the effects of DPI should be attributed to its inhibitory effect on NADPH oxidase. This is consistent with previous findings that indicate that NADPH oxidase is a major source of oxidative stress in pulmonary and systemic vessels under physiological and pathological conditions (11, 19, 36). However, the potentiation induced by DPI was greater in the older animals. Therefore, none of the above-mentioned enzymatic systems is involved in the maturation of the responses to NO. In contrast, the nonselective Cox inhibitor indomethacin specifically potentiated the responses to NO in newborns so that in its presence NO-induced relaxation was similar in neonates and in older animals. We then tested the effects of meclofenamate, another nonselective Cox inhibitor structurally unrelated to indomethacin; NS-398, a selective Cox-2 inhibitor; and aspirin, which at the concentration used (5 × 10⁵ M) is a fairly selective Cox-1 inhibitor (35). Both meclofenamate and aspirin enhanced NO relaxation specifically in the newborn, whereas NS-398 was without effect. These results suggested that Cox-1 is responsible for a reduced vasodilator activity of NO during the first hours of extrauterine life. This suggestion was also supported by the further inhibitory effect of arachidonic acid (the substrate of Cox), specifically in neonates. The fact that the AA-861, an inhibitor of 5-lipooxygenase (which also uses arachidonic acid as its main substrate), also inhibited NO-induced relaxation specifically in neonates might be explained also through an increased substrate for Cox-1. The Cox-1 responsible for the reduced response of NO is not located in the endothelium because endothelium removal did not affect the response to NO. Cox-1 produces reactive oxygen species as byproducts of the synthesis of endoperoxide prostaglandins (31), which may account for the reduced response to NO in newborns reported herein. A role for Cox-1-derived superoxide has been recently suggested to play a major role in the regulation of cerebral circulation (22). In addition, indomethacin-sensitive Cox-1-dependent NO consumption during prostaglandin synthesis has been reported to play a physiological role in platelet function (24). However, prostaglandins or thromboxane synthesized by Cox could also inhibit NO-induced relaxation, even when thromboxane is unlikely to be involved, since the preparations were contracted by a thromboxane A₂ analog at a concentration that produced 70-90% of the maximal response.

We then tested whether the differences in the effects of Cox inhibitors were associated with changes in the expression of Cox proteins. In the late-gestation ovine fetus, pulmonary PGI₂ increases acutely with ventilation and enhanced oxygenation (17) resulting from rapid changes in the expression of Cox-1 (22). The lung mRNA and protein expression of the constitutive isoform of Cox (Cox-1) increases six- and twofold, respectively, from fetal to 1-wk-old newborn lambs (6). However, in piglets, we did not find any change in Cox-1 protein expression in the pulmonary arteries. The ovine lung Cox-2 mRNA also increased during the early postnatal period, even when Cox-2 protein was not detected (5). In contrast, Cox-2 was expressed constitutively in piglet cerebral arteries (25) and pulmonary arteries (present results) in both neonates and older animals. A nonsignificant trend for increased expression in the pulmonary arteries from older animals was observed. All these results indicate that the expression of Cox isoforms in the ovine and porcine pulmonary circulations does not change or increases postnatally. Therefore, the Cox-dependent maturation of the responses to NO in newborns reported herein appears to be related to changes in Cox activity, which is not associated with differences in the expression of Cox protein isoforms.

Cox are fundamental enzymes in vascular biology, playing a key role in the pulmonary adaptation to the postnatal circulation (23, 30, 31). Cox-1-dependent inhibition of NO-induced relaxation as described herein is likely to be only part of the role of Cox in the maturation of the pulmonary circulation. In fact, inhibition of Cox in the prenatal period in healthy subjects results in unpaired circulatory adaptation at birth (30) rather than in accelerated maturation. The physiological maturation of endothelial NO is probably more complex that the maturation of the responses to exogenously added NO. However, increased Cox activity may play a role in the development of persistent pulmonary hypertension of the newborn, and it is involved in several experimental models of newborn and adult pulmonary hypertension (30). In addition, Cox-dependent inhibition of NO might participate in the therapeutic failure of inhaled NO.

In conclusion, the present results indicate that, in the pulmonary arteries from newborn piglets, the activity of Cox-1 reduces the vasodilator response to NO, but this mechanism does not operate later in life. Therefore, the present data suggest that the maturational increase in the vasodilator response to NO in the pulmonary arteries during the first days of extrauterine life involves Cox activity.