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J Appl Physiol 104: 1786-1792, 2008. First published April 3, 2008; doi:10.1152/japplphysiol.01314.2007
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Effect of long-term high-altitude hypoxia on fetal pulmonary vascular contractility

Qin Xue, Charles A. Ducsay, Lawrence D. Longo, and Lubo Zhang

Department of Physiology and Pharmacology, Center for Perinatal Biology, Loma Linda University School of Medicine, Loma Linda, California

Submitted 12 December 2007 ; accepted in final form 31 March 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hypoxia in the fetus and/or newborn is associated with an increased risk of pulmonary hypertension. The present study tested the hypothesis that long-term high-altitude hypoxemia differentially regulates contractility of fetal pulmonary arteries (PA) and veins (PV) mediated by differences in endothelial NO synthase (eNOS). PA and PV were isolated from near-term fetuses of pregnant ewes maintained at sea level (300 m) or high altitude of 3,801 m for 110 days (arterial PO2 of 60 Torr). Hypoxia had no effect on the medial wall thickness of pulmonary vessels and did not alter KCl-induced contractions. In PA, hypoxia significantly increased norepinephrine (NE)-induced contractions, which were not affected by eNOS inhibitor NG-nitro-L-arginine (L-NNA). In PV, hypoxia had no effect on NE-induced contractions in the absence of L-NNA. L-NNA significantly increased NE-induced contractions in both control and hypoxic PV. In the presence of L-NNA, NE-induced contractions of PV were significantly decreased in hypoxic lambs compared with normoxic animals. Acetylcholine caused relaxations of PV but not PA, and hypoxia significantly decreased both pD2 and the maximal response of acetylcholine-induced relaxation in PV. Additionally, hypoxia significantly decreased the maximal response of sodium nitroprusside-induced relaxations of both PA and PV. eNOS was detected in the endothelium of both PA and PV, and eNOS protein levels were significantly higher in PV than in PA in normoxic lambs. Hypoxia had no significant effect on eNOS levels in either PA or PV. The results demonstrate heterogeneity of fetal pulmonary arteries and veins in response to long-term high-altitude hypoxia and suggest a likely common mechanism downstream of NO in fetal pulmonary vessel response to chronic hypoxia in utero.

endothelial nitric oxide synthase; pulmonary arteries

OF ALL THE STRESSES TO WHICH the fetus is subjected, perhaps the most important and clinically relevant is that of hypoxia. The fetus may experience prolonged hypoxic stress under many different conditions, including pregnancy at high altitude. Nearly 140 million people residing at over 2,500 m above the sea level are permanently exposed to chronic hypoxic conditions (19). Pulmonary vasoconstriction and high pulmonary vascular resistance are hallmarks of the fetal circulation. Following birth, pulmonary vascular resistance falls and pulmonary blood flow increases immediately as the lung expands with air and functions in oxygen exchange. Oxygen plays a vital role in the transition of high pulmonary resistance in the fetus to low pulmonary resistance in the newborn (4–6, 8, 23). Chronic hypoxia in utero is one of the major factors associated with persistent pulmonary hypertension in the newborn (1, 22). It has been shown that newborns at high altitude have elevated pulmonary vascular resistance (15, 21, 22).

Among other mechanisms, oxygen-induced changes in endothelial NO production play a key role and contribute significantly to pulmonary vasodilation after birth (4, 5, 8, 27). Although there are many studies of NO and pulmonary vascular reactivity in the newborn and adult, to our knowledge, studies of the effect of chronic hypoxia in utero on contractility and endothelium-dependent relaxation of fetal pulmonary vessels are limited. This deficit is partly due to a limit of animal models of chronic in utero hypoxia, in which small fetal pulmonary vessels can be isolated and studied in an organ bath. In the well-defined animal model of pregnant sheep maintained at a high altitude of 3,801 m for 110 days during gestation [maternal arterial PO2 (PaO2) of 60 Torr and fetal PaO2 of 19 Torr], a recent study demonstrated that chronic hypoxia in utero attenuated PKG-mediated relaxation in pulmonary arteries in near-term fetal lambs, which was due in part to inhibited cGMP-dependent protein kinase activity and enhanced Rho kinase activity (10). Nonetheless, it is unknown to what extent chronic hypoxia affects the upstream mechanisms at endothelial NO synthase (eNOS) levels and endothelium-dependent relaxation in pulmonary vessels in the fetus.

The present study was designed to test the hypothesis that chronic hypoxia during gestation differentially regulates pulmonary vascular contractility and relaxation in near-term fetal lambs. We determined the effect of chronic hypoxia on KCl- and norepinephrine-induced contractions, endothelium-dependent and -independent relaxations, and eNOS protein levels in pulmonary vessels of fetal lambs. Because both pulmonary arteries and veins contribute significantly to pulmonary vascular resistance in the fetus and newborn (11), we studied the effect of hypoxia on both pulmonary arteries and veins in fetal lambs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental animals.   Pregnant sheep of the same age and breed were obtained from the Nebeker Ranch in Lancaster, CA. Normoxic controls were maintained at the Nebeker Ranch (altitude: ~300 m; PaO2: 102 ± 2 Torr). For hypoxic exposure, pregnant sheep were transported to the Barcroft Laboratory, White Mountain Research Station, Bishop, CA (3,801-m altitude; PaO2: 60 ± 2 Torr) at 30 days of gestation and maintained at high altitude for ~110 days, as previously described (33, 34). Near-term pregnant sheep (~140 days of gestation; term being 147 days) were transported to the Animal Care Facility at Loma Linda University. Ewes were anesthetized with thiamylal (10 mg/kg) administered via an external jugular vein, and anesthesia was maintained on 1.5–2.0% halothane in oxygen throughout surgery. Pulmonary arteries and veins were obtained from near-term fetuses of both normoxic control and chronically hypoxic pregnant sheep. All procedures and protocols used in the present study were approved by the Institutional Animal Care and Use Committee of Loma Linda University and followed the guidelines by the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Tissue preparation and contraction studies.   Preparation of pulmonary vessels was conducted in ice-cold Krebs solution (pH 7.4) of the following composition (in mM): 115.21 NaCl, 4.7 KCl, 1.80 CaCl2, 1.16 MgSO4, 1.18 KH2PO4, 22.14 NaHCO3, 0.03 EDTA, and 7.88 dextrose. The Krebs solution was oxygenated with a mixture of 95% O2 and 5% CO2. Fourth-generation pulmonary arteries and veins were dissected and cut into rings of 4 mm in length (diameter of 1.5–2.0 mm for artery and 0.8–1.3 mm for vein). Isometric tensions of vessel rings were measured in Krebs solution in tissue baths at 37°C, as described previously (33, 34). After 60 min of equilibration in the tissue bath, each ring was stretched to the optimal resting tension, as determined by the tension developed in response to 120 mM KCl added at each stretch level. Norepinephrine-induced concentration-dependent contraction curves were determined by cumulative addition of the agonist in approximate one-half log increments in the absence or presence of eNOS inhibitor NG-nitro-L-arginine (L-NNA; 100 µM, pretreatment for 20 min). For relaxation studies, tissues were precontracted with submaximal concentration (3 µM) of norepinephrine, followed by acetylcholine and sodium nitroprusside added in a cumulative manner, respectively. The relaxation responses to acetylcholine and sodium nitroprusside were expressed as the percentage of norepinephrine precontractions.

Immunoblotting.   Protein levels of eNOS were determined by Western blot analysis, as described previously (33). Briefly, pooled segments of fourth-generation pulmonary arteries and veins, respectively, were homogenized in a lysis buffer containing 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, pH 7.4. Homogenates were then centrifuged at 4°C for 10 min at 12,000 g, and the supernatants were collected. Protein was quantified in the supernatant using a protein assay kit from Bio-Rad. Samples with equal protein were loaded on 7.5% polyacrylamide gel with 0.1% sodium dodecyl sulfate and separated by electrophoresis at 100 V for 90 min. Proteins then were transferred onto nitrocellulose membranes. Nonspecific binding sites on the membranes were blocked in a Tris-buffered saline solution containing 5% dry milk for 1 h at room temperature. The membranes were incubated with mouse eNOS monoclonal antibody (1:500) overnight at 4°C. The membranes then were incubated with secondary horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (1:2,000). Proteins were visualized with enhanced chemiluminescence reagents, and the blots were exposed to Hyperfilm. For comparison of eNOS between arteries and veins, the bands were measured as absolute density values. For comparison of eNOS between normoxia and hypoxia for a given vessel type, eNOS bands were normalized to those of actin used as a loading control. Results were quantified by the Kodak electrophoresis documentation and analysis system and Kodak ID image analysis software.

Histological analysis and immunohistochemistry.   Pulmonary vessel rings were fixed in 10% neutral buffered formalin and embedded in paraffin. Immunohistochemical detection of eNOS and Von Willebrand Factor (vWF) was performed using Pharmingen Anti-Ig HRP Detection Kit, as described previously (35). Briefly, tissue slices (4 µm thick) of vessel rings were incubated with primary antibodies against eNOS (1:100) or vWF (1:200) for 60 min at room temperature. After rinsing the slices three times in phosphate-buffered saline for 15 min, the slices were incubated with biotinylated goat anti-mouse IgG or anti-rabbit Igs (1:50) for 60 min at room temperature. The samples were then exposed to streptravidin-HRP and reacted with diaminobenzidine substrate solution according to the manufacture's recommendations and counterstained with hematoxylin. The negative control of eNOS staining was performed in the absence of eNOS antibody. For histological analysis of medial wall thickness, tissue slides were stained with hematoxylin and eosin. The slices were viewed with an Olympus BH-2 microscope, and images were captured with an attached SPOT digital camera imaging system.

Materials.   Norepinephrine, L-NNA, vWF antibody, and sodium nitroprusside were obtained from Sigma (St. Louis, MO). Mouse anti-eNOS monoclonal antibody was from Transduction Laboratory (Lexington, KY). Anti-Ig HRP Detection Kits were from BD Biosciences (San Diego, CA). Electrophoresis and Western blotting reagents were from Bio-Rad (Hercules, CA).

Data analysis.   Concentration-response curves were analyzed by computer-assisted nonlinear regression to fit the data using GraphPad Prism (GraphPad Software, San Diego, CA) to obtain the values of pD2 (–log EC50) and the maximum response. Results were expressed as means ± SE, and the differences were evaluated for statistical significance (P < 0.05) by Student's t-test or two-way ANOVA followed by Bonferroni posttests.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of chronic hypoxia on medial wall thickness of pulmonary vessels.   Figure 1 shows the effect of hypoxia on medial wall thickness of fourth-generation pulmonary arteries and veins in near-term fetal lambs. The medial wall was significantly thicker in pulmonary arteries than that in pulmonary veins. Chronic hypoxia showed no significant effect on medial wall thickness in either pulmonary arteries or veins in fetal lambs (Fig. 1).


Figure 1
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  		Fig. 1. Effect of chronic hypoxia on medial wall thickness of fetal pulmonary arteries and veins. Bar graphs show medial wall thickness of pulmonary arteries and veins obtained from near-term fetal lambs of normoxic control and hypoxic ewes. Data were analyzed by two-way ANOVA with vessel type as one factor and hypoxia as the other. *Significant difference (P < 0.05) from artery (n = 4).

 
Effect of chronic hypoxia on KCl- and norepinephrine-induced contractions.   The effect of hypoxia on KCl-induced contractions is illustrated in Fig. 2. In both normoxic and hypoxic lambs, KCl-induced contractions were significantly greater in pulmonary veins than in arteries. Chronic hypoxia had no significant effect on KCl-induced contractions in either pulmonary arteries or veins (Fig. 2).


Figure 2
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  		Fig. 2. Effect of chronic hypoxia on KCl-induced contractions of fetal pulmonary arteries and veins. Bar graphs show KCl-induced contractions of pulmonary arteries and veins obtained from near-term fetal lambs of normoxic control and hypoxic ewes. Data were analyzed by two-way ANOVA with vessel type as one factor and hypoxia as the other. *Significant difference (P < 0.05) from vein (n = 5).

 
Figure 3 shows the effect of hypoxia on norepinephrine-induced concentration-dependent contractions of fetal pulmonary vessels in the absence or presence of the eNOS inhibitor L-NNA. In pulmonary arteries, L-NNA had no significant effect on norepinephrine-induced contractions in either control or hypoxic vessels (Fig. 3, top). Chronic hypoxia significantly increased the maximal response of norepinephrine-induced contractions in pulmonary arteries regardless of L-NNA treatment (Fig. 3, bottom, and Table 1). In contrast to arteries, L-NNA significantly increased norepinephrine-induced maximal contractions of pulmonary veins from both control and hypoxic fetal lambs, but the effect of L-NNA was significantly reduced in hypoxic fetal lambs (Fig. 3, bottom, and Table 1).


Figure 3
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  		Fig. 3. Effect of chronic hypoxia on norepinephrine-induced contractions of fetal pulmonary arteries (top) and veins (bottom). Norepinephrine (NE)-induced contractions were determined in the absence or presence of NG-nitro-L-arginine (L-NNA; 100 µM, 20 min) in pulmonary arteries and veins obtained obtained from near-term fetal lambs of normoxic control and hypoxic ewes. Data are expressed as percent of KCl (120 mM)-induced contractions and are means ± SE of tissues from 5 to 6 animals. The pD2 values and the maximal responses are presented in Table 1.

 

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  		Table 1. Effect of chronic hypoxia on norepinephrine-induced contractions of pulmonary arteries and veins from fetal lambs in the absence or presence of L-NNA

 
Effect of chronic hypoxia on endothelium-dependent and -independent relaxations.   The endothelium-dependent relaxations induced by acetylcholine were determined in pulmonary vessels precontracted with 3 µM norepinephrine. Acetylcholine had no effect in pulmonary arteries but produced concentration-dependent relaxations of pulmonary veins in both control and hypoxic fetal lambs (Fig. 4). Hypoxia significantly decreased pD2 (6.10 ± 0.08 vs. 6.43 ± 0.10; P < 0.05) and the maximal relaxation (41.6 ± 2.1 vs. 49.7 ± 2.4%; P < 0.05) induced by acetylcholine in pulmonary veins (Fig. 4). Unlike acetylcholine, sodium nitroprusside produced concentration-dependent relaxations in both pulmonary arteries and veins. Hypoxia significantly decreased sodium nitroprusside-induced relaxations in both pulmonary arteries and veins (Fig. 5 and Table 2).


Figure 4
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  		Fig. 4. Effect of chronic hypoxia on acetylcholine-induced relaxations in fetal pulmonary arteries (top) and veins (bottom). Acetylcholine (ACh)-induced relaxations were determined in pulmonary arteries and veins (precontracted with 3 µM norepinephrine) obtained from near-term fetal lambs of normoxic control and hypoxic ewes. Data are means ± SE of tissues from 5 to 6 animals. The pD2 value and the maximal response are presented in the text.

 

Figure 5
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  		Fig. 5. Effect of chronic hypoxia on sodium nitroprusside (SNP)-induced relaxations in fetal pulmonary arteries (top) and veins (bottom). SNP-induced relaxations were determined in pulmonary arteries and veins (precontracted with 3 µM norepinephrine) obtained from near-term fetal lambs of normoxic control and hypoxic ewes. Data are means ± SE of tissues from 5 to 6 animals. The pD2 value and the maximal response are presented in Table 2.

 

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  		Table 2. Effect of chronic hypoxia on sodium nitroprusside-induced relaxations in pulmonary arteries and veins from fetal lambs

 
Effect of chronic hypoxia on eNOS expression.   eNOS protein levels and distribution in fetal pulmonary vessels were determined with immunohistochemistry and immunoblotting. As shown in Fig. 6, eNOS immunoreactivity was primarily detected in the endothelium of the pulmonary arteries and veins. Additionally, the endothelium was labeled with vWF (Fig. 6). Both immunoblotting and immunohistochemistry analyses showed significantly higher levels of eNOS expression in pulmonary veins than in arteries (Fig. 6). In either pulmonary arteries or veins, eNOS protein levels were not significantly different between the control and hypoxic fetal lambs (Fig. 7).


Figure 6
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  		Fig. 6. Localization and density of eNOS in fetal pulmonary arteries and veins. eNOS localization and density were determined in fetal pulmonary arteries and veins by immunohistochemical staining (top) and immunoblotting (bottom), respectively. eNOS expression was detected in the endothelium of fetal pulmonary arteries and veins. Additionally, the endothelium was labeled with vWF. Western blot illustrates eNOS bands detected by the monoclonal antibody at the expected size of ~140 kDa. Data are means ± SE of tissues from 4 animals in each group. *Significant difference (P < 0.05) from artery.

 

Figure 7
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  		Fig. 7. Effect of chronic hypoxia on eNOS protein levels in fetal pulmonary arteries and veins. eNOS protein levels were determined by Western blot in pulmonary arteries and veins obtained from near-term fetal lambs of normoxic control and hypoxic ewes. Data are means ± SE of tissues from 4 animals in each group.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major findings of the present study are that chronic hypoxia in utero differentially regulates contractions and relaxations of small pulmonary arteries and veins in near-term fetal lambs by 1) increasing norepinephrine-induced contractions of pulmonary arteries; 2) decreasing norepinephrine-induced contractions of pulmonary veins in a NO-dependent manner; 3) reducing endothelium-dependent relaxations selectively in pulmonary veins; 4) decreasing endothelium-independent relaxations of both pulmonary arteries and veins; and 5) having no significant effect on eNOS protein levels in either pulmonary arteries or veins. Additionally, chronic hypoxia has no significant effect on KCl-induced contractions and medial wall thickness in either pulmonary arteries or veins in fetal lambs.

In the present study, we found no significant difference in medial wall thickness of small pulmonary vessels between normoxic and hypoxic fetal lambs. Although it is unlikely that samples of hypoxic and normoxic vessels shrink differently during tissue fixation and thus prevent the observation of treatment differences in medial wall thickness, this possibility cannot be excluded. Nonetheless, the present finding is consistent with previous studies in rats and guinea pigs, in which chronic in utero hypoxemia did not change pulmonary arterial structure (13, 20). Additionally, hypoxia did not cause hyperplasia or hypertrophy of the media of pulmonary arteries in near-term bovine fetuses (3). This is further supported with the finding that KCl-induced contractions were not significantly different in pulmonary vessels obtained from control and hypoxic fetal lambs, suggesting the same vascular smooth muscle mass in control and hypoxic pulmonary vessels. Additionally, the finding suggests that hypoxia had no significant effect on voltage-gated calcium channel density in fetal pulmonary vessels. A recent study showed increased KCl-induced contractions of fourth-generation pulmonary arteries from highland (3,600 m above sea level) newborn (average 10 days of age) lambs compared with lowland (580 m above sea level) newborn lambs (15). Although medial wall thickness was not measured, the authors suggested a greater vascular smooth muscle mass in highland newborn lambs. Taken together, these studies suggest that pulmonary vascular remodeling in hypoxic infants may not occur during fetal life but rather in the transition from prenatal to postnatal life and during the early neonatal period, when changes in pulmonary structure and function are particularly sensitive to hypoxia (21, 22). Whether hypoxic fetuses are more vulnerable to pulmonary vascular remodeling during the early neonatal period is not clear and remains an intriguing area for future investigation.

Despite the thinner media, fetal pulmonary veins showed greater contractions to KCl compared with arteries. This is consistent with previous findings in the pulmonary circulation that, during the perinatal period, veins exhibit greater contractions than arteries in response to a variety of stimuli (2, 11). The present study has demonstrated a complex relationship between the effects of chronic hypoxia on fetal pulmonary arteries vs. veins and the role of NO in utero vs. in vitro. In the absence of L-NNA, hypoxia significantly increased the maximal response of norepinephrine-induced contractions in pulmonary arteries, but not veins, in fetal lambs. This suggests that in utero, when NO is present, the site of resistance under conditions of hypoxia in the intact pulmonary circulation is greater in pulmonary arteries than in veins. It has been demonstrated that chronic hypoxia produces an increase in {alpha}1-adrenoreceptor gene expression and increases pulmonary vascular smooth muscle contractile sensitivity, which is thought to play an important role in the development of pulmonary hypertension (17, 25). Fetal pulmonary vascular beds are under {alpha}-adrenergic control, and fetal pulmonary arteries contract to norepinephrine (16, 24, 28). The finding that norepinephrine-induced contractions in the presence of L-NNA were significantly decreased in pulmonary veins of hypoxic fetal lambs is intriguing and suggests a compensatory adaptation mechanism in vascular contractility of pulmonary veins to chronic in utero hypoxia. Given that both arteries and veins contribute almost equally to pulmonary vascular resistance during perinatal development (11), the opposite changes in vascular contractility of pulmonary arteries and veins, demonstrated in the present study, may result in minimal changes in pulmonary vascular resistance in near-term fetal lambs in response to chronic in utero hypoxia.

The finding that L-NNA had no effect on norepinephrine-mediated contractions of fetal pulmonary arteries suggests a lack of basal inhibitory effect of eNOS in the regulation of pulmonary arterial contractility in near-term fetal lambs. This is supported by the findings of the minimal eNOS levels and the lack of acetylcholine-induced relaxations in pulmonary arteries. This is consistent with the previous study in fetal lambs showing a lack of acetylcholine-induced relaxations in pulmonary arteries (12, 27). Additionally, it was demonstrated that calcium ionophore A23187 [GenBank] failed to relax pulmonary arteries in fetal lambs (16). Taken together, these studies have demonstrated that the endothelium is not functional in NO-mediated relaxation in pulmonary arteries in near-term fetal lambs resulting from minimal eNOS protein levels. Nevertheless, sodium nitroprusside produced concentration-dependent relaxations in pulmonary arteries. Because sodium nitroprusside is an NO donor and relaxes vascular smooth muscle via activation of guanylate cyclase and increasing cGMP, the finding suggests that the downstream pathway of cGMP-dependent protein kinase is fully functional in fetal pulmonary arteries, as demonstrated in the present as well as in previous studies (9, 10, 16, 27). The present study demonstrated that chronic hypoxia had no effect on eNOS protein levels and endothelium-dependent relaxation in fetal pulmonary arteries, albeit it decreased the downstream pathway of cGMP-dependent relaxations. The similar finding of decreased cGMP-dependent relaxations in fetal pulmonary arteries was obtained in a recent study (10). Because of the lack of NO-dependent relaxation and the lack of effect of chronic hypoxia on eNOS, the decreased downstream cGMP-dependent relaxations may minimally affect pulmonary arterial tone in the fetus but may be detrimental in the transition of pulmonary arterial contractility and structure from prenatal to postnatal life, in which eNOS/NO becomes a key mechanism in the regulation of pulmonary arterial reactivity.

In contrast to pulmonary arteries, pulmonary veins in near-term fetal lambs have much higher levels of eNOS in the endothelium and relax significantly to acetylcholine. Additionally, inhibition of eNOS by L-NNA significantly increased norepinephrine-induced contractions, suggesting a significant component of basal eNOS activity in the inhibition of pulmonary vein contractility. This is consistent with previous studies (12, 27, 30). It has been demonstrated in intact fetal sheep that acetylcholine produces a decrease in pulmonary vascular resistance and an increase in pulmonary blood flow, which are blocked by eNOS inhibitors L-NNA and L-NMMA (1, 29). Given the lack of eNOS-mediated relaxation in pulmonary arteries, eNOS/NO-mediated regulation of fetal pulmonary vascular resistance and pulmonary blood flow resides primarily in pulmonary veins. In the present study, we have shown that chronic in utero hypoxia results in a significant decrease in NO-mediated relaxations in fetal pulmonary veins. This is supported by the finding that acetylcholine-induced relaxation of pulmonary vein was significantly decreased in hypoxic fetal lambs. Additionally, the effect of eNOS inhibitor L-NNA in increasing norepinephrine-induced contractions was significantly decreased in pulmonary vein of hypoxic compared with normoxic fetal lambs. The findings that hypoxia had no significant effect on eNOS protein levels in pulmonary veins suggests that the inhibition may occur at downstream pathways. Consistent with this notion, the present study demonstrated that sodium nitroprusside-induced relaxations were significantly decreased by chronic hypoxia in both pulmonary veins and arteries. In the same animal model, a recent study demonstrated that 8-Br-cGMP (stimulator of PKG) caused a similar relaxation of pulmonary veins obtained from control and hypoxic fetal lambs (Longo LD, personal communication). Taken together, these findings suggest a likely mechanism of decreased soluble guanylate cyclase (sGC) and reduced cGMP production in fetal pulmonary vessels in response to in utero chronic hypoxia. Indeed, long-term high-altitude hypoxemia significantly decreased sGC abundance and catalytic activity in carotid arteries of fetal lambs (32). Additionally, chronic hypoxia decreased expression of sGC in rat pulmonary artery smooth muscle cells (14).

In conclusion, we have demonstrated heterogeneity in responses of pulmonary arteries and veins in near-term fetal lambs to long-term high-altitude hypoxemia. Although chronic hypoxia in utero has no significant effect on the medial wall thickness of pulmonary vessels in fetal lambs, it significantly increases vasoconstriction of pulmonary arteries and decreases vasorelaxation of pulmonary veins. Furthermore, our studies demonstrate that chronic hypoxia has no significant effect on eNOS protein levels in either pulmonary arteries or veins in near-term fetal lambs and point to a likely common mechanism of decreased sGC in fetal pulmonary arteries and veins in response to chronic hypoxia in utero. This is consistent with the growing literature demonstrating the importance of sGC in addition to eNOS in the regulation of perinatal as well as adult pulmonary circulation (7, 30, 31). Future studies are needed to determine the effect of chronic hypoxia on sGC protein levels and catalytic activity in fetal pulmonary vessels.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by National Institutes of Health Grants HD-31226 and HL-57787, and by the Loma Linda University School of Medicine.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. Zhang, Center for Perinatal Biology, Dept. of Physiology & Pharmacology, Loma Linda Univ. School of Medicine, Loma Linda, CA 92350 (e-mail: lzhang{at}llu.edu)

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. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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