Nitric oxide (NO) is thought to play an important role in the regulation of neonatal pulmonary vasculature. It has been suggested that neonates with pulmonary hypertension have a defective NO pathway. Therefore, we measured in 1-day-old piglets exposed to hypoxia (fraction of inspired O2 = 0.10) for 3 or 14 days to induce pulmonary hypertension 1) the activity of NO synthase (NOS) via conversion of l-arginine to l-citrulline and the concentration of the NO precursorl-arginine in isolated pulmonary vessels,2) the vasodilator response to the NO donor 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1) and the cGMP analog 8-bromo-cGMP in isolated perfused lungs, and 3) the production of cGMP in response to SIN-1 in isolated perfused lungs. After 3 days of exposure to hypoxia, endothelial NOS (eNOS) activity was unaffected, whereas, after 14 days of hypoxia, eNOS activity was decreased in the cytosolic fraction of pulmonary artery (P < 0.05) but not of pulmonary vein homogenates. Inducible NOS activity was decreased in the cytosolic fraction of pulmonary artery homogenates after both 3 (P < 0.05) and 14 (P < 0.05) days of hypoxia but was unchanged in pulmonary veins. Pulmonary artery levels ofl-arginine were unaffected by hypoxic exposure. After 3 days of exposure to hypoxia, the reduction in the dilator response to SIN-1 (P < 0.05) coincided with a decrease in cGMP production (P < 0.005), suggesting that soluble guanylate cyclase activity may be altered. When the exposure was prolonged to 14 days, dilation to SIN-1 remained decreased (P < 0.05) and, although cGMP production normalized, the dilator response to 8-bromo-cGMP decreased (P < 0.05), suggesting that, after prolonged exposure to hypoxia, cGMP-dependent mechanisms may also be impaired. In conclusion, neonatal hypoxia-induced pulmonary hypertension is associated with multiple disruptions in the NO pathway.
- endothelial nitric oxide synthase
- inducible nitric oxide synthase
- guanosine 3′,5′-cyclic monophosphate
- isolated perfused lungs
- pulmonary arteries
- pulmonary veins
normal fetal pulmonary circulation is characterized by active vasoconstriction, elevated pulmonary vascular resistance, and low pulmonary blood flow. Successful transition to extrauterine life is associated with a dramatic decrease in pulmonary vascular resistance and pulmonary arterial pressure, with a concurrent increase in pulmonary blood flow (28). There is increasing evidence that nitric oxide (NO) is important in this transition. For example, in the lamb, basal production of NO increases during late gestation and into the neonatal period (29), whereas inhibition of NO production attenuates the normal rise in pulmonary blood flow seen at birth (1, 20). In addition, inhibition of NO production in neonatal piglets is associated with a marked increase in pulmonary vascular resistance (24).
NO is synthesized in vascular endothelial cells froml-arginine by the endothelial isoform of NO synthase (eNOS; see Ref. 22). NO then diffuses to vascular smooth muscle cells where interaction with the heme group of soluble guanylate cyclase results in the formation of cGMP. Subsequent phosphorylation of cGMP-dependent protein kinases results in calcium sequestration and vascular smooth muscle relaxation (16).
Persistent pulmonary hypertension of the newborn (PPHN) represents a failure of the normal birth-related pulmonary vascular changes to occur. There is increasing evidence that the pathogenesis of PPHN involves impairment(s) of the NO pathway, and several laboratories have used different models of PPHN to investigate some of the steps in this pathway. On one hand, eNOS activity is decreased in pulmonary hypertension caused by prenatal ligation of the ductus arteriosus in lambs (31), congenital diaphragmatic hernia in rats (21), and chronic hypoxia in piglets (10). On the other hand, the vasodilator response to NO is reduced in both piglets (36) and calves (7) with hypoxia-induced pulmonary hypertension. However, the little information that is known about the role of guanylate cyclase and more distal cGMP-dependent mechanisms is controversial. For example, NO-mediated cGMP production is unaffected by chronic hypoxia in piglet pulmonary arteries (36) but is decreased in pulmonary arteries of lambs subjected to prenatal ligation of the ductus arteriosus (33). Therefore, systematic examination of the different steps of NO production and action, using the same animal model, is required for better understanding of the pathogenesis of PPHN.
We hypothesized that, in piglets with hypoxia-induced pulmonary hypertension, there is a reduction in the production of and responsiveness to NO, and this is accompanied by a reduction in the release of and responsiveness to cGMP. Therefore, newborn piglets were exposed to short (3-day)- or long (14-day)-term hypoxia to induce pulmonary hypertension. Our objectives were to1) determine if nitric oxide synthase (NOS) synthetic function is altered in chronic hypoxia by measuring both NOS activity and l-arginine levels in plasma and pulmonary vessels, 2) determine if chronic hypoxia alters the pulmonary vascular response to NO in isolated perfused lungs, and 3) determine if chronic hypoxia alters the production of and/or pulmonary vascular response to cGMP in isolated perfused lungs.
MATERIALS AND METHODS
Experiments were performed on 1-day-old Yorkshire-Landrace piglets (1.62 ± 0.30 kg) in accordance with the McGill University guidelines for the use of experimental animals. Piglets were maintained in a 440-liter Plexiglas chamber with a fraction of inspired O2( ) of 0.10 ± 0.005 (hypoxia) or in room air (control) for a period of either 3 or 14 days. Hypoxia was achieved by a continuous mixture of separate sources of air and N2 (liquid N2 reservoir; Megs Specialty Gases, St. Laurent, PQ, Canada). and the fraction of inspired CO2( ) were measured at least three times per day with an electrochemical cell O2 analyzer (model S3-A/1) and sensor (model N-22M; Ametek, Pittsburgh, PA) and an infrared CO2 analyzer (model CD-3A) and sensor (model P-61B; Ametek). was also continuously monitored with an Oxychek oxymeter (Critikon, Tampa, FL). was kept below 0.005 via adjustment of total gas flow. Piglets were maintained in a thermoneutral environment, and temperature was adjusted (26–36°C) according to age using an Air Shields (Air Shields, Hatboro, PA) heater with servo-control. Humidity level was maintained at less than 70% using a condensing coil as a dehumidifier. A 12:12-h dark-light cycle was established, and animals were fed ad libitum with balanced artificial milk (Wet Nurser; Jefo Import Export, St. Hyacinthe, PQ, Canada). Daily care of the animals was done without interruption of hypoxia. Control piglets were raised under identical conditions, except that was maintained at 0.21. All piglets received an intramuscular injection of iron on arrival. Animals displaying any evidence of cardiorespiratory disorder were excluded. Animals from the four study groups (3-day normoxia, 3-day hypoxia, 14-day normoxia, and 14-day hypoxia) were used for either lung perfusion,l-arginine measurement, or pulmonary vessel preparation for measurement of NOS activity in addition to physiological measurements.
The Krebs solution for the isolated lung experiments had the following composition (in mM): 119 NaCl, 4.0 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.5 CaCl2, 5.5 dextrose, and 25 NaHCO3. The solution was supplemented with 50 g/l BSA.
The following compounds were used: BSA, l-arginine,l-valine, l-citrulline, glucosaminic acid, 8-bromo-cGMP, phenylmethylsulfonyl fluoride (PMSF), dithiothreitol (DTT), aprotinin, calmodulin, tetrahydrobiopterin (BH4), FAD, FMN, NADPH, isobutyl methylxanthine (IBMX) (Sigma Chemical, St. Louis, MO), EDTA (Fisher Scientific, Nepean, ON, Canada), 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1 hydrochloride; Calbiochem, San Diego, CA), and the stable endoperoxide analog 9,11-dideoxy-11α,9α-methanoepoxy-PGF2α(U-46619; Biomol Research Laboratories, Plymouth Meeting, PA).l-[2,3,4,5-3H]arginine was purchased from Amersham Life Sciences (Amersham, UK).
The animals were weighed at the beginning and at the end of exposure. Weight gain was then determined. Blood was sampled from the left ventricle for measurement of hemoglobin concentration with a hemoximeter (OSM2b; Radiometer, Copenhagen, Denmark). In animals that were not used for lung perfusion, the heart was removed and dissected into right ventricle (RV) and left ventricle plus septum (LV+S). The ratio of RV-to-LV+S weight was then calculated as an index of right ventricular hypertrophy.
Isolated Perfused Lung
Animals were anesthetized with pentobarbital sodium (65 mg/kg ip) and tracheostomized. Lungs were ventilated with 25% O2-5% CO2 in N2 with a tidal volume of 20 ml/kg. The isolated perfused lung was set up as previously described (13). Briefly, a cannula was placed in the pulmonary artery, and the lungs were perfused with Krebs solution supplemented with 5% BSA at a constant flow of 30 ml ⋅ min−1 ⋅ kg body wt−1 using a peristaltic pump (Masterflex model 7553–30; Cole-Parmer, Chicago, IL). Perfusate drained by gravity through a cannula placed in the left ventricle. Venous pressure was then equal to atmospheric pressure. Throughout the experiments, perfusion pressure, which reflects pulmonary artery inflow pressure, was monitored with a transducer (model P2310; Gould Instruments, Cleveland, OH) and a chart recorder (model 7E; Grass Instruments, Quincy, MA). Baseline perfusion pressure has been shown previously to be stable for at least 2.5 h. Integrity of the preparation was assessed by the stability of the baseline perfusion pressure and/or by the presence of edema as revealed by macroscopic examination. No preparations were discarded due to lack or loss of integrity.
Lung preparations were allowed to stabilize for 30 min after being mounted and perfused inside the chamber. To achieve comparable baseline perfusion pressures among all four groups, vascular tone was raised as needed by adding the endoperoxide analog U-46619 to the perfusate after the stabilization period. The NO donor SIN-1 or the cGMP analog 8-bromo-cGMP was then added to the perfusion bath and was continuously recirculated through the preparation. An interval of at least 20 min was allowed between each concentration of SIN-1 and 8-bromo-cGMP tested. At least five different preparations were used for each of the study groups.
Measurement of l-Arginine Levels
Plasma. After thoracotomy and before lung excision, blood was sampled from the left ventricle and was placed in chilled tubes with heparin. Blood was immediately centrifuged at 3,000 g at 4°C for 20 min, and the plasma was stored at −70°C until analyzed.
Plasma samples were prepared according to the method of Slocum and Cummings (32) with the following modifications. After being thawed, 200 μl of plasma were deproteinized with 20 μl of 35% (wt/vol) sulfosalicylic acid, and then glucosaminic acid in LiS buffer was added as an internal standard. Samples were vortexed for 5 s, left standing for 5 min at room temperature, and centrifuged at 11,200 g for 5 min. The supernatant was then analyzed with a Beckman 6300 amino acid analyzer (Beckman Instruments, Palo Alto, CA). The detection limits of this analyzer are 1 μM to 1 mM.
Tissue. As described previously (11), lungs were excised and immersed in cold (4°C) HEPES-buffered salt solution that had the following composition (in mM): 20 HEPES, 135 NaCl, 2.68 KCl, 1.8 CaCl2, and 2.05 MgCl2. Arteries were dissected from the hilum down to a 100-μm diameter, snap-frozen in liquid N2, and stored at −70°C. Samples of pulmonary artery (400–600 mg) were dissected and homogenized two times in 5.5 ml of LiS buffer with a Polytron PT-3000 homogenizer (Brinkman Instruments Canada, Rexdale, ON, Canada) at a speed of 13,500 rpm for 1 min. Homogenate preparation and amino acid content determination were performed as above. Protein concentration of the homogenate was determined by the Bradford (4) method using BSA as a standard. Values are means of triplicate determinations and are expressed as micrograms per milligram of protein.
To ensure that the final concentration of l-arginine was well within the detection limits of the analyzer,l-arginine (final concentration 3 μM) was added to the homogenate before measurement of amino acid content. This value was then subtracted from the results before reporting.
Measurement of NOS Activity
Pulmonary arteries and veins were dissected as described above. Arteries or veins were homogenized in ice-cold HEPES buffer (20 mM, pH 7.4) containing 0.32 M sucrose, 1 μM aprotinin, 0.1 mM DTT, 0.5 mM EDTA, and 0.5 mM PMSF with a Polytron PT-3000 homogenizer (Brinkman Instruments Canada) at a speed of 13,500 rpm, with six bursts of 20 s separated by 10-s cooling intervals. The homogenate was centrifuged at 16,000 g for 30 min at 4°C. The supernatant was filtered through a 100-μm mesh filter (Nytex, Zurich, Switzerland) before being centrifuged at 100,000g for 60 min to constitute the cytosolic fraction. For each of the pulmonary artery cytosolic preparations, tissue from one and occasionally two animals was used. For each of the vein cytosolic preparations, tissues from two to four animals were combined. Pulmonary artery membrane preparations required pooling of pellets from two to five animals. Membrane and cytosolic fractions were stored at −70°C to be used for later measurement of eNOS activity. Protein content of each fraction was determined by the Bradford (4) method using BSA as a standard.
NOS activity was determined by the method of Bredt and Snyder (5) with the following modifications. Membrane or cytosolic fractions (50 μg protein) were incubated for 20 min at 37°C in a 400-μl final volume of HEPES buffer (20 mM, pH 7.4) containing 200 nMl-[2,3,4,5-3H]arginine, 800 nM cold l-arginine, 0.5 mM CaCl2, 100 nM calmodulin, 5 μM BH4, 5 μM FAD, 5 μM FMN, 0.5 mM NADPH, 1 mM MgCl2, 50 mM l-valine, and 1 mM l-citrulline. The reaction was terminated by the addition of 2 ml of ice-cold HEPES buffer (20 mM, pH 5.5) containing 2 mM EGTA and 2 mM EDTA. The incubation mixture was applied to 2-ml Dowex AG50W-X8 (Na+ form) chromatography columns, and the resulting [3H]citrulline was eluted with water. Radiolabeled citrulline was quantified by liquid scintillation counting (1216 RackBeta II; LKB Wallac, Turku, Finland). Values are means of triplicate determinations, representing the calcium-dependent activity, and are expressed per milligram of protein per minute. Measurement of inducible (calcium-independent) NOS (iNOS) activity was performed by adding EGTA to a calcium-free medium. Control values were obtained in the absence of enzyme extract. eNOS activity was then calculated as the difference between calcium-dependent and calcium-independent activities.
Measurement of cGMP Production
cGMP production, basal and in response to SIN-1 (10−4 M), was measured in the perfusate of isolated perfused lungs using a125I-labeled cGMP RIA kit (Amersham Life Science). Briefly, 5-ml aliquots of perfusate were collected before and 5 min after the addition of SIN-1 and were frozen at −70°C in tubes containing 100 μl of 0.025 M IBMX. Samples were extracted with ethanol and centrifuged at 2,000g for 15 min. Supernatants were lyophilized, reconstituted in acetate buffer (0.05 M, pH 5.8) with 0.01% sodium azide, and acetylated by addition of acetic anhydride and triethylamine. cGMP concentrations were determined as per the manufacturer's recommendations. Values are means of duplicate measurements and are expressed as nanomolar. Nonspecific binding was measured as above without addition of the cGMP-specific antibody.
Data are presented as means ± SD (Tables 1-6) or means ± SE (Figs. 1-3). Statistical analysis of physiological data, eNOS activity, l-arginine levels, and cGMP levels was performed using ANOVA, with a P value of <0.05 being considered significant.
The dilator responses to SIN-1 and to 8-bromo-cGMP were evaluated by measuring the lowest perfusion pressure reached for each concentration tested. The significance of the effect of each drug on perfusion pressure at different concentrations was assessed statistically using ANOVA for repeated measures, with a Pvalue of <0.05 being considered significant.
In the conditions described above, the animals raised in normoxia had an excellent weight gain (Table 1). However, animals raised in hypoxia experienced a lower weight gain compared with those raised in normoxia, with significance being reached after 14 days of exposure (P < 0.001). As expected in normoxia, the RV-to-LV+S ratio decreased fromday 3 to day 14 (P < 0.001). The RV-to-LV+S weight ratio was significantly increased after 3 days of exposure to hypoxia compared with normoxia (P < 0.001). After 14 days of hypoxia, this ratio was increased even further compared with the 3-day hypoxia group (P < 0.05) and was significantly greater than in the 14-day normoxia group (P < 0.001). In normoxic exposure, there was a decrease in hemoglobin concentration with age (P < 0.01). In hypoxia, however, the hemoglobin concentration did not decrease and was significantly elevated compared with normoxic controls at both 3 (P < 0.05) and 14 (P < 0.001) days.
In preliminary experiments, we found that NOS activity could be abolished in a dose-dependent manner by addition of the NOS inhibitorN-nitro-l-arginine (l-NNA), confirming the specificity of the reaction (data not shown). Nonspecific conversion of l-arginine tol-citrulline was 1.8%. The recovery of radiolabeled citrulline was 78%.
The activity of eNOS in the cytosolic fraction of pulmonary artery homogenates is summarized in Table 2. In normoxia, there was an age-related increase in eNOS activity (P < 0.05). Exposure to hypoxia for 3 days did not alter eNOS activity. However, after 14 days of exposure, eNOS activity was significantly decreased in hypoxia compared with normoxia (P < 0.05).
We then measured eNOS activity in the membrane fraction of pulmonary artery homogenates (Table 3). eNOS activity was roughly 10 times greater in the membrane compared with the cytosolic fraction. Exposure to hypoxia had no significant effect on eNOS activity in this fraction.
In contrast to the arteries, eNOS activity in the cytosolic fraction of pulmonary vein homogenates decreased significantly with age in normoxia (P < 0.01) and almost reached significance in hypoxia (P = 0.07, Table 4). However, exposure to hypoxia did not alter eNOS activity in pulmonary veins at either age.
Calcium-dependent (iNOS) activity was also measured in pulmonary artery and vein homogenates. iNOS activity in the cytosolic fraction of pulmonary artery homogenates is summarized in Table 2. In contrast to eNOS activity, iNOS activity did not increase with age in normoxia. In hypoxia, iNOS activity was significantly decreased compared with normoxia at both ages (P < 0.05).
iNOS activity in the membrane fraction of pulmonary artery homogenates is shown in Table 3. As with eNOS, iNOS activity was roughly 10 times greater in the membrane compared with the cytosolic fraction but was not altered by exposure to hypoxia. In the cytosolic fraction of pulmonary vein homogenates, iNOS activity increased with age, but significance was only reached in hypoxia (P < 0.05, Table 4).
To determine if the decrease in eNOS activity in hypoxia was due to substrate limitation, we measured the level of l-arginine in both plasma and pulmonary arteries. l-Arginine levels in both plasma and pulmonary arteries were unaffected by exposure to hypoxia (Table 5).
Pulmonary Vascular Response to SIN-1
Baseline perfusion pressure was increased in animals raised in hypoxia compared with those in normoxia at both ages (P < 0.001) and more so in the 14-day than in the 3-day hypoxia group (P < 0.001; Fig.1).
SIN-1 caused a dose-dependent decrease in perfusion pressure in all four study groups (Fig. 2). In normoxia, there was an age-related decrease in dilator response (P < 0.05). In hypoxia, the dilator response was significantly reduced compared with normoxic controls at both ages (P < 0.05).
Pulmonary Vascular Response to 8-Bromo-cGMP
To determine the level at which hypoxia alters the response to SIN-1, we also measured the dilator response to the cGMP analog 8-bromo-cGMP in isolated perfused lungs (Fig. 3). As with SIN-1, 8-bromo-cGMP caused a dose-dependent decrease in perfusion pressure in all four study groups. The dilator response in hypoxia was reduced compared with that in normoxia, with significance being reached only after 14 days of exposure (P < 0.005).
To determine if the activity of soluble guanylate cyclase is altered by exposure to chronic hypoxia, we measured the production of cGMP, both basal and in response to SIN-1 (10−4 M), in isolated perfused lungs (Table 6). Basal production of cGMP was decreased in the 3-day hypoxia group compared with that in normoxic controls (P < 0.005). Similarly, cGMP production in response to SIN-1 was decreased in the 3-day hypoxia group compared with that in 3-day normoxia group (P < 0.005). Of interest, the decrease in both the basal and stimulated release of cGMP seen after 3 days of exposure to hypoxia recovered when hypoxic exposure was prolonged to 14 days (P < 0.005). There was an age-related increase in stimulated cGMP production in both normoxia (P < 0.05) and hypoxia (P < 0.01).
This work suggests that, in neonatal piglet pulmonary vasculature, chronic hypoxia is associated with multiple disruptions in the integrity of the NO pathway. On one hand, the decrease in both eNOS and iNOS activity suggests that hypoxia may decrease NO production, and this does not appear to be due to decreased substrate availability. On the other hand, the vasodilator response to an NO donor is decreased. The fact that the basal and stimulated release of cGMP is decreased after 3 days of hypoxia suggests that guanylate cyclase activity may be altered, whereas the additional finding of decreased vasodilator response to 8-bromo-cGMP suggests that chronic hypoxia is, at minimum, associated with a disruption in cGMP-dependent mechanisms.
As we have previously shown (13), this model is effective in producing significant pulmonary hypertension. The presence of an increased RV-to-LV+S ratio after only 3 days of hypoxic exposure further demonstrates that these changes occur rapidly. In agreement with the work of others (9), we also found that chronic hypoxia is associated with decreased weight gain and increased hemoglobin concentration.
We have shown that chronic hypoxia is associated with a decrease in eNOS activity in neonatal piglet pulmonary vasculature. This finding is consistent with other models of neonatal pulmonary hypertension. Both Shaul et al. (31) and Villamor et al. (37) have shown that eNOS activity is decreased in whole lung homogenates of lambs with pulmonary hypertension caused by prenatal ligation of the ductus arteriosus, whereas Fike et al. (10) recently showed that chronic hypoxia in piglets is associated with decreased basal and ACh-stimulated eNOS activity as measured by plasma concentration. Either a decrease in eNOS gene and/or protein expression or a reduction in substrate availability could explain this decrease in eNOS activity. There is growing evidence that a decrease in eNOS gene expression is involved in the pathogenesis of neonatal pulmonary hypertension. In neonatal rats with pulmonary hypertension secondary to congenital diaphragmatic hernia, both eNOS protein and mRNA levels are decreased (21). In lambs with pulmonary hypertension caused by prenatal ligation of the ductus arteriosus, decreased eNOS enzyme activity is accompanied by diminished eNOS protein and mRNA levels (31, 37), and, in piglets with hypoxia-induced pulmonary hypertension, both eNOS protein and enzyme activity are similarly decreased (10). McQuillan and associates (17) have further shown that this decrease in eNOS mRNA is a function of both altered gene transcription and decreased mRNA stability. It remains unclear whether altered translational processes also contribute to the decrease in eNOS protein expression.
Alternatively, decreased substrate availability could account for altered enzyme activity. In fact, l-arginine administration has been found to augment endothelium-dependent relaxation in pulmonary artery rings of ovine fetuses (2) and to restore endothelium-dependent pulmonary vasodilation in chronically hypoxic rats (8). However, in normoxic and hypoxic conditions, our piglets had plasmal-arginine levels in the micromolar range, which is consistent with other reported levels (3, 11, 18, 19). Knowing that the Michaelis constant of eNOS for l-arginine is in the range of 1–10 μM (19), our animals would appear to have a surplus of substrate. It is also possible that decreases in other NOS cofactors could be responsible for alterations in NOS activity.
In this light, it is important to note that we have only shown an effect of hypoxia on eNOS activity in the cytosolic fraction of pulmonary arteries, since previous work suggests that the majority of eNOS activity should lie within the membrane fraction (12). Although we did see a trend toward decreased eNOS activity in this fraction, this difference was not significant. However, it has been proposed that subcellular localization represents a mechanism for the regulation of eNOS activity. In support of this are the findings that eNOS can be both myristylated (26) and phosphorylated (25), processes that both appear to direct subcellular localization of the enzyme. The effect of hypoxia on these processes has not been evaluated, but it is possible that a disturbance in one or both of these represents an additional posttranslational mechanism for hypoxia-induced effects on eNOS activity.
It may also be argued that the vascular remodeling that occurs during chronic hypoxia alters the relative contribution of endothelial cell proteins in our preparation and that, therefore, the decrease in eNOS activity is merely artifactual. However, factor VIII-associated antigen levels as a direct measurement of endothelial cell mass are unaltered in whole lung homogenates of neonatal pulmonary hypertensive rats (21) and lambs (31), thus making this explanation unlikely.
It is very interesting that the effects of hypoxia on eNOS activity appear to be limited to the arterial tree, and, in fact, this is the first study in the newborn to examine the activity of eNOS in each pulmonary vascular compartment. Our findings are consistent with those of Resta et al. (27), who demonstrated by quantitative immunocytochemistry that pulmonary venous expression of eNOS in adult rats is unaffected by chronic hypoxia. These findings also highlight the importance of studying isolated vessels as opposed to whole lung preparations for understanding regulatory mechanisms in which changes may be small and/or compartment specific.
Our findings contrast with adult models of pulmonary hypertension in which eNOS expression and activity appear to increase after exposure to chronic hypoxia (14, 30). This discrepancy is likely a reflection of age-related maturational changes within the pulmonary vasculature. In support of this, piglets develop a rapid increase in the vasodilator response to ACh in the neonatal period, which then decreases into adulthood (15). Furthermore, inhibition of NO production byl-NNA causes a greater increase in pulmonary vascular resistance in 1-day- compared with 7-day-old piglets (24). Normal neonatal pulmonary circulation is characterized by active vasodilation and vascular remodeling as the animal adapts to extrauterine life (28), and pulmonary hypertension in this setting appears to represent a failure of this adaptation. Given the importance of NO in this transition (1, 20), it is quite plausible that pulmonary hypertension in this setting is due, at least in part, to an inadequacy of NO production. In contrast, pulmonary hypertension in adults develops after a period of normal pulmonary vascular pressure and therefore represents vascular constriction and/or remodeling from a previously dilated state. Hence, one could postulate that the increase in eNOS expression and activity in this setting might merely be a secondary phenomenon.
Our findings also indicate that the negative effect of chronic hypoxia on NOS activity is not limited to the endothelial isoform. In fact, this study is the first to investigate the effects of chronic hypoxia on iNOS activity within neonatal pulmonary vasculature. As with eNOS, our findings are in contrast to those in adult rat pulmonary vasculature in which chronic hypoxia is associated with a modest increase in iNOS gene expression (6, 14, 38) and enzyme activity (6). The failure of hypoxia to upregulate iNOS expression in our model suggests that this process may be developmentally regulated.
Neonatal models have consistently shown a disruption in NO-mediated pulmonary vasodilation in chronic hypoxia (7, 9). It is important to determine if this altered vasodilation is merely a function of decreased eNOS activity or if the response to NO itself is also altered. Our finding of a decreased vasodilator response to SIN-1 suggests that chronic hypoxia is, indeed, associated with a fundamental alteration in the vascular smooth muscle response to NO. This is in agreement with Tulloh et al. (36), who demonstrated decreased dilation of isolated porcine pulmonary arterial rings to NO, and Fike and Kaplowitz (9), who found reduced dilation to NO in isolated perfused lungs of chronically hypoxic piglets.
The decreased response to SIN-1 could represent either an alteration in soluble guanylate cyclase activity or a disruption of more distal cGMP-dependent mechanisms. Our finding of decreased basal and stimulated cGMP production after 3 days of hypoxic exposure suggests that soluble guanylate cyclase activity is indeed altered. The finding that cGMP production and soluble guanylate cyclase levels are also decreased in pulmonary arteries of lambs with non-hypoxia-induced pulmonary hypertension (34) further suggests that this decrease may be an inherent component of neonatal pulmonary hypertension. In this light, it is very interesting that increasing hypoxic exposure to 14 days was associated with an apparent recovery in cGMP production. The reason for this is unclear but could represent either an attempt to compensate for ongoing pulmonary hypertension or maturational changes in soluble guanylate cyclase activity. In support of the latter, Tulloh et al. (36) found that, whereas short-term hypoxia was associated with an increase in NO-mediated cGMP production, this response only occurred in older animals. Obviously, the contribution of other systems like phosphodiesterases and particulate guanylate cyclase in the regulation of cGMP levels cannot be ignored. In fact, Perreault et al. (23) showed that atrial natriuretic factor levels are increased significantly after exposure to 14 days of hypoxia.
Concern may be raised about the use of the NO donor SIN-1 in evaluating endothelium-independent vasodilation because, in addition to the liberation of NO, SIN-1 could release peroxynitrite, which may itself stimulate soluble guanylate cyclase (35). However, our results are in agreement with Tulloh et al. (36), who showed that vasodilation to NO itself is decreased in neonatal piglet pulmonary vasculature after chronic hypoxia. Therefore, although the presence of peroxynitrite in these experiments cannot be ruled out, it is unlikely to account for the observed effects.
Our finding that the vasodilator response to 8-bromo-cGMP is decreased suggests that chronic hypoxia is also associated with impairment of distal cGMP-dependent mechanisms. This is consistent with the results of Tulloh et al. (36) who found that dilation to the phosphodiesterase inhibitor zaprinast was decreased in chronically hypoxic piglets. This appears to contrast with the findings of Steinhorn et al. (33), who showed that pulmonary arterial dilation to 8-bromo-cGMP was unaffected in lambs with pulmonary hypertension caused by prenatal ligation of the ductus arteriosus. However, in this study, responses were evaluated only in the immediate newborn period. Because our results showed a trend toward decreased dilation with age, it is possible that this finding is an age-related phenomenon and therefore would not have been assessed in this study. Alternatively, this may represent a species- or model-related difference.
In conclusion, this study suggests that piglets with hypoxia-induced pulmonary hypertension have multiple disruptions in the integrity of the NO pathway. The finding that both eNOS and iNOS activity are decreased adds support to the hypothesis that neonatal, as opposed to adult, pulmonary hypertension is associated with decreased NO production. The fact that the response to NO is altered and that distal cGMP-dependent mechanisms are impaired, may provide some understanding of why some neonates with pulmonary hypertension fail to respond to inhaled NO therapy.
This work was supported by the Medical Research Council (MT-12973) and La Fondation du Québec des Maladies du Coeur.
Address for reprint requests and other correspondence: T. Perreault, Montreal Children's Hospital, Newborn Medicine, 2300 Tupper St., Montreal, Quebec, Canada H3H 1P3 (E-mail:).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
- Copyright © 2000 the American Physiological Society