We performed studies to determine whether chronic hypoxia impairs nitric oxide (NO) signaling in resistance level pulmonary arteries (PAs) of newborn piglets. Piglets were maintained in room air (control) or hypoxia (11% O2) for either 3 (shorter exposure) or 10 (longer exposure) days. Responses of PAs to a nonselective NO synthase (NOS) antagonist, Nω-nitro-l-arginine methylester (l-NAME), a NOS-2-selective antagonist, aminoguanidine, and 7-nitroindazole, a NOS-1-selective antagonist, were measured. Levels of NOS isoforms and of two proteins involved in NOS signaling, heat shock protein (HSP) 90 and caveolin-1, were assessed in PA homogenates. PAs from all groups constricted to l-NAME but not to aminoguanidine or 7-nitroindazole. The magnitude of constriction to l-NAME was similar for PAs from control and hypoxic piglets of the shorter exposure period but was diminished for PAs from hypoxic compared with control piglets of the longer exposure period. NOS-3, HSP90, and caveolin-1 levels were similar in hypoxic and control PAs. These findings indicate that NOS-3, but not-NOS 2 or NOS-1, is involved with basal NO production in PAs from both control and hypoxic piglets. After 10 days of hypoxia, NO function is impaired in PAs despite preserved levels of NOS-3, HSP90, and caveolin-1. The development of NOS-3 dysfunction in resistance level PAs may contribute to the progression of chronic hypoxia-induced pulmonary hypertension in newborn piglets.
- nitric oxide synthase isoforms
- cyclic GMP
- heat shock protein 90
pulmonary hypertension develops when newborn piglets are exposed to chronic hypoxia. A number of laboratories, including ours, have provided evidence that alterations in the nitric oxide (NO) pathway occur in lungs of newborns with chronic hypoxia-induced pulmonary hypertension (4, 7, 14, 17, 24, 41). However, to date, all studies evaluating the functional role of NO in chronic hypoxia-induced pulmonary hypertension in newborns have been performed in whole lungs, large conduit-level vessels, or mixtures of large and small vessels. Derangements in NO signaling pathways in small resistance-level pulmonary arteries have not been reported. Moreover, although all three NO synthase (NOS) isoforms, NOS-1 (neuronal NOS), NOS-2 (inducible NOS), and NOS-3 (endothelial NOS) are expressed in mammalian lungs (12, 23, 28, 34, 43, 47, 49), data are scarce regarding the roles of NOS-1 or NOS-2 in newborn models of pulmonary hypertension.
Because of the critical role played by resistance-level pulmonary arteries in the regulation of pulmonary vascular tone, this study was designed to test the hypothesis that alterations in the NO pathway occur in resistance-level pulmonary arteries of newborn piglets during in vivo exposure to chronic hypoxia. We also designed studies to evaluate which of the three NOS isoforms are involved with regulation of basal tone in resistance-level pulmonary arteries from normal piglets and from piglets with pulmonary hypertension. Therefore, we measured responses of small pulmonary arteries to NOS-1-selective antagonist 7-nitroindazole, NOS-2-selective antagonist aminoguanidine, and nonselective NOS antagonist Nω-nitro-l-arginine methylester (l-NAME). We also performed studies to determine potential mechanisms underlying derangements in NOS signaling, including assessment of protein abundance of NOS isoforms and two proteins involved in NOS signal transduction, caveolin-1 and heat shock protein (HSP) 90 (2, 19, 20). Furthermore, because it is likely that the mechanisms underlying the development of pulmonary hypertension change with the length of exposure to hypoxia (14, 17, 26, 47), studies were performed with small pulmonary arteries from piglets exposed to 3 days of hypoxia and their comparable age-control piglets (shorter exposure group) and to piglets exposed to 10 days of hypoxia and their comparable age-control piglets (longer exposure group).
Newborn pigs (2–3 days old) were placed in a hypoxic normobaric chamber for 3 (n = 16) or 10 (n = 16) days. Normobaric hypoxia was produced by delivering medical-grade breathing air and N2 to an incubator (Thermocare). Oxygen content was regulated to achieve 10–11% O2 (Po2 of 60–72 Torr), and Pco2 was maintained at 3–6 Torr by absorption with soda lime. The chamber was opened two times per day for cleaning and to weigh the piglets. The animals were fed ad libitum with an artificial sow milk replacer from a feeding device attached to the chamber. Piglets raised in chambers for 3 days were 5 or 6 days old on the day of study. Piglets raised in chambers for 10 days were 12 or 13 days old on the day of study. Comparable-age piglets that had been raised on a farm were used as controls. We have previously found no differences in vascular responses between piglets raised in a room-air chamber and piglets raised on a farm. Therefore, for this study, most of the control piglets were studied on the day of arrival from the farm and were 5–7 days of age (n = 19 for the shorter exposure control group) or 12–15 days (n = 16 for the longer exposure control group).
Cannulated artery preparation.
On the day of study, the piglets were preanesthetized with ketamine (30 mg/kg im) and then anesthetized with pentobarbital (10 mg/kg iv). All animals were given heparin (1,000 IU/kg iv) and then were exsanguinated. The thorax was opened, and the lungs were removed and placed in cold (4°C) physiological saline solution (PSS) until use. The PSS had the following composition (in mM): 141 Na+, 4.7 K+, 125 Cl−, 2.5 Ca2+, 0.72 Mg2+, 1.7 H2PO4−, 25 HCO3−, and 11 glucose. Pulmonary artery segments measuring 100–400 μm in diameter were dissected from a lung lobe immediately before use.
The system used to study cannulated arteries has been described in detail previously (15, 18). Briefly, it consists of a water-jacketed plastic chamber in which proximal (inflow) and distal (outflow) cannulas were mounted. An arterial segment was threaded onto the proximal cannula and tied in place with a nylon thread. The distal end of the artery was then tied onto the distal cannula, the artery was filled with PSS, and all side branches were tied off. The distance between the cannula tips was adjusted with a micrometer connected to the proximal cannula to remove the slack from the artery. The exterior of the artery was suffused from a reservoir with PSS (37°C) aerated with a gas mixture containing O2, CO2, and N2, giving a Po2 of 140 ± 0.5 Torr, a Pco2 of 35 ± 0.3 Torr, and a pH of 7.37 ± 0.01. The arterial lumen was filled from a syringe containing similarly aerated PSS. Gas concentrations and pH were monitored in all solutions (reservoir, vessel chamber, and infusion syringes) using a Ciba-Corning 278 blood gas analyzer (Bayer, Norwood, MA).
Inflow pressure was adjusted by changing the height of the infusion syringe. Pressure transducers were placed both on the inflow side between the syringe and the artery and at the outflow end of the system. Both inflow and outflow pressures were monitored continuously on a recorder, and the artery was discarded if the pressures were not equal (indicating a leak in the vessel). The external diameter of the artery was observed continuously with a video system containing a color camera (Hitachi VCC-51) and a television monitor. Vessel diameters were measured with a video scaler (FORA IV).
Cannulated artery protocols.
Each artery was allowed to equilibrate for 30–60 min to establish basal tone. The control arteries were equilibrated at a transmural pressure of 15 cmH2O; the hypoxic arteries were equilibrated at a transmural pressure of 25 cmH2O. These pressures were chosen because they represent in vivo pressures (13, 14). We have previously found no effect of transmural pressure on ACh responses in the size of arteries used in this study (18). After establishment of basal tone, all arteries were tested for viability by contraction to U-46619 (10−7 M). To check for a functional endothelium in control arteries, responses to ACh (10−5 M) were evaluated. We previously found that hypoxic arteries constricted to ACh but dilated in response to the calcium ionophore A-23187, another endothelium-dependent vasodilator (18). Therefore, responses to A-23187 were used to verify a functional endothelium in hypoxic arteries. Thereafter, all arteries were washed with PSS and allowed to return to basal tone before proceeding with one of the following study protocols.
In one series of studies, we measured responses of control and hypoxic arteries to NOS inhibition with either a nonselective NOS antagonist, l-NAME (10−6 to 10−3 M), a NOS-2-selective antagonist, aminoguanidine (10−6 to 10−3 M), or a NOS-1-selective antagonist, 7-nitroindazole (7-NINA; 10−6 to 10−4 M) by continuously monitoring the vessel diameter while cumulative doses of one of the NOS antagonists were added to the reservoir.
In another series of studies, we measured responses of control and hypoxic arteries to ACh (10−8 to 10−5 M), an agonist known to stimulate NO release. These studies were performed with vessels at basal tone. To evaluate the effects of NOS and HSP90 antagonism on ACh responses, we measured the change in diameter of control and hypoxic arteries to ACh (10−8 to 10−5 M) in the absence and presence of either the nonspecific NOS antagonist l-NAME (10−3 M for 20 min) or the HSP90 antagonist geldanamycin (10−6 M for 20 min). To further evaluate the impact of HSP90 inhibition on ACh-mediated responses and confirm our findings using the HSP90 antagonist geldanamycin, additional studies were performed in the longer exposure group with radicicol (2 × 10−8 M for 20 min), an HSP90 antagonist that is structurally unrelated to geldanamycin. Furthermore, we also performed studies in the longer exposure group to determine the influence of the cyclooxygenase (COX) synthase inhibitor indomethacin (10−5 M) on ACh (10−8 to 10−5 M) responses. We previously found that COX products are involved in regulation of ACh responses in small pulmonary arteries from piglets raised in hypoxia for 3 days and comparable age controls (18).
We also performed studies to determine whether responses to the NO donor, S-nitroso-N-acetyl-penicillamine (SNAP; 10−9 to 10−5 M) were altered by chronic hypoxia. In preliminary studies performed with vessels at basal tone, we found minimal to no responses to SNAP in arteries from either control or hypoxic piglets. Therefore, to augment responses, these studies were performed in vessels with elevated tone accomplished by the addition of endothelin to the reservoir to achieve a 30–50% decrease in vessel diameter. We subsequently monitored vessel diameter continuously while cumulative doses of SNAP were added to the reservoir. Evaluation of SNAP responses were limited to pulmonary arteries of the longer exposure group, since we have previously found no differences in SNAP responses in small pulmonary arteries from piglets exposed to 3 days of hypoxia and comparable age-control piglets (18).
Additional studies upstream and downstream of the NOS-NO signaling pathway were performed to further investigate the alterations in responses to SNAP and l-NAME that were observed only in pulmonary arteries from piglets in the longer hypoxic exposure group. Studies were performed to determine if impairments in smooth muscle cell relaxation to cyclic GMP (cGMP) were involved in the altered NO-dependent responses by measuring changes in vessel diameter to 8-bromo-cGMP (10−8 to 10−4 M) in vessels from control and hypoxic piglets of the longer exposure period. Similar to SNAP, in preliminary studies, we found minimal responses to 8-bromo-cGMP in vessels at basal tone. Therefore, these studies were performed in vessels with tone elevated 30–50% with endothelin. To help assess whether altered availability to l-arginine is involved in the loss of the vasodilatory response to ACh in hypoxic vessels of the longer exposure period, we continually measured the diameter of hypoxic arteries to ACh (10−8 to 10−5 M) before and 30 min after adding l-arginine (10−2 M) to the perfusate. These latter studies were performed in vessels at basal tone. The concentration of l-arginine was chosen based on our laboratory's previous studies with isolated lungs (16).
For all studies, vessel responses to the vehicle used for solubilization of each agent were evaluated.
Small pulmonary artery preparation for immunoblot analyses.
Control and hypoxic piglets from both exposure times were preanesthetized with ketamine (30 mg/kg im), anesthetized with pentobarbital (10 mg/kg iv), given heparin (1,000 IU/kg iv), and then exsanguinated. Next, the lungs of the piglets were excised, and small pulmonary arteries (20- to 600-μm diameter) were dissected. Some small pulmonary arteries were frozen in liquid nitrogen and stored at −80°C for immunoblot analysis.
Frozen samples of small pulmonary arteries from control and hypoxic piglets of both exposure times were homogenized in 10 mM HEPES buffer containing (in mM) 250 sucrose, 3 EDTA, and 1 phenylmethylsulfonyl fluoride, pH 7.4, on ice using three 15-s pulses of a Polytron blender, taking care to avoid foaming of the homogenate. Protein concentration of the vessel homogenate was determined by the Bio-Rad protein assay. Each vessel homogenate was diluted with phosphate-buffered saline (PBS) to obtain a protein concentration of 1 mg/ml. Aliquots of the protein concentrations were solubilized in equal volumes of denaturing, reducing sample buffer [Novex; 0.25 M Tris·HCl, 5% (wt/vol) SDS, 2.5% (vol/vol) 2-mercaptoethanol, 10% glycerol, 0.05% bromphenol blue, pH 6.8], heated to 80°C for 15 min, and centrifuged for 3 min at 5,600 g in a microfuge. Equal volumes and, therefore, equal protein amounts of the supernatants were applied to Tris-glycine precast 8% polyacrylamide gels (Novex). Electrophoresis was carried out in 25 mM Tris, 192 mM glycine, and 0.1% SDS (pH 8.3) at 125 V for 1.7 h. The proteins were transferred from the gel to a nitrocellulose membrane (Novex) at 100 V for 1 h in 25 mM Tris, 192 mM glycine, and 20% methanol (pH 8.3). The membrane was incubated overnight at 4°C in PBS containing 10% nonfat dried milk and 0.1% Tween 20 to block nonspecific protein binding. To detect the protein of interest, the nitrocellulose membrane was incubated for 1 h at room temperature with the primary antibody diluted in PBS containing 0.1% Tween 20 and 1% nonfat dried milk (carrier buffer), followed by incubation for either 60 min with an appropriate horseradish peroxidase-conjugated (Zymed) secondary antibody diluted in the carrier buffer or for 30 min at room temperature with an appropriate biotinylated (Vector Elite, ABC Kit, Vector Laboratories) secondary antibody diluted in the carrier buffer, followed by incubation for 30 min at room temperature with streptavidin-horseradish peroxidase conjugate (Amersham) diluted in PBS containing 0.1% Tween 20. The nitrocellulose membrane was washed three times between the first two incubations with the carrier buffer and three times with the carrier buffer plus one time with PBS containing 0.1% Tween 20 after the final incubation. To visualize the biotinylated antibody, the membranes were developed using enhanced chemiluminescence (ECL) reagents (Amersham), and the chemiluminescent signal was captured on X-ray film (ECL hyperfilm, Kodak). The bands for each protein were quantified using densitometry.
Immunoblot analysis for NOS-3, NOS-1, caveolin-1, and HSP90.
We assessed whether a change in protein abundance of NOS-3, NOS-1, caveolin-1, or HSP90 might contribute to altered NO-dependent responses in hypoxic arteries. We were unable to detect NOS-2 protein in lungs of newborn piglets by immunoblot. Note that we have previously published immunoblot analyses for NOS-3 and NOS-1 in small pulmonary arteries from piglets exposed to 3 days of hypoxia and comparable age-control piglets (47). Therefore, in this study, evaluation of NOS-3 and NOS-1 expression was limited to pulmonary arteries from piglets of the longer exposure group.
For each protein, preliminary studies with various amounts of total protein were performed to determine the dynamic range of each antibody for detection by immunoblot analysis. We chose an amount of protein that was within the dynamic range to compare protein abundance between homogenates of small pulmonary arteries from control and hypoxic piglets as follows: NOS-3 (BD Biosciences Pharmingen; 1:500, 10 μg of protein per lane), NOS-1 (BD Biosciences Pharmingen; 1:500; 10 μg of protein per lane), caveolin-1 (BD Biosciences Pharmingen; 1:1,000; 2.5 μg of protein per lane for the shorter exposure period and 1 μg of protein per lane for the longer exposure period), HSP90 (BD Biosciences Pharmingen; 1:2,000; 2.5 μg of protein per lane for the shorter exposure period and 10 μg of protein per lane for the longer exposure period), and appropriate horseradish peroxidase-conjugated secondary antibodies (Sigma or Zymed).
Concentrations for each drug listed in cannulated artery protocols were expressed as final molar concentrations in the vessel bath. ACh, l-NAME, aminoguanidine, 7-NINA, radicicol, l-arginine, indomethacin, and 8-bromo-cGMP were obtained from Sigma Chemical. Geldanamycin was from Alexis. SNAP was from Biomol. SNAP and aminoguanidine were solubilized in distilled water. ACh, l-NAME, l-arginine, and 8-bromo-cGMP were solubilized in normal saline. Geldanamycin and 7-NINA were solubilized in DMSO. Radicicol was solubilized in ethanol. Indomethacin was solubilized in a mixture of equal parts saline and 8% NaHCO3.
Data are presented as means ± SD. Unpaired t-tests were used to compare data between control and hypoxic animals; a paired t-test was used to compare changes in vessel diameters before and after treatment with either l-NAME, geldanamycin, radicicol, indomethacin, or l-arginine for each dose of ACh. When more than one vessel was used from a given animal for a particular experimental protocol, the results were averaged and used as an n of one such that all comparisons were done on the number of animals. P < 0.05 was considered significant.
For the cannulated artery studies, the mean diameter of vessels used for all studies were similar: 228 ± 55 and 244 ± 76 μm, respectively, for control and hypoxic arteries from the shorter exposure group and 241 ± 58 and 245 ± 55 μm, respectively, for control and hypoxic arteries from the longer exposure group. In the concentrations used for drug solubilization, none of the vehicles significantly changed arterial diameter.
The nonspecific NOS antagonist l-NAME caused a similar decrease in diameter of arteries from both control and hypoxic piglets in the shorter exposure group (Fig. 1A). In contrast, responses to the NOS-1-selective antagonist (Fig. 1B) and to the NOS-2-selective antagonist (Fig. 1C) were minimal and did not differ between arteries from control and hypoxic piglets.
For the longer exposure group (Fig. 1, D–F), the diameter of arteries from both control and hypoxic piglets decreased with the nonspecific NOS antagonist l-NAME (Fig. 1D). In contrast to findings from the shorter exposure group (Fig. 1A), the magnitude of decrease in diameter to l-NAME was markedly less for arteries from hypoxic compared with control piglets of the longer exposure group (Fig. 1D). Similar to our findings in the shorter exposure group, neither arteries from control nor hypoxic piglets of the longer exposure group responded significantly to either NOS-1 (Fig. 1E)- or NOS-2 (Fig. 1F)-selective antagonists.
The influence of the nonspecific NOS antagonist l-NAME on responses to ACh for vessels from the shorter and longer exposure groups are shown in Fig. 2. Arteries from control piglets of both exposure periods dilated to ACh (Fig. 2, A and C, for the shorter exposure and longer exposure groups, respectively), whereas arteries from hypoxic piglets of both exposure periods constricted to ACh (Fig. 2, B and D, for the shorter exposure and longer exposure groups, respectively). Treatment with l-NAME reversed ACh responses from dilation to constriction in control arteries from both the shorter exposure group (Fig. 2A) and the longer exposure group (Fig. 2C). Treatment with l-NAME augmented the constriction to ACh in hypoxic arteries from the shorter exposure group (Fig. 2B), whereas the magnitude of constriction to ACh was similar in the presence and absence of l-NAME in arteries from the longer hypoxic exposure group (Fig. 2D).
The effects of the HSP90 antagonist geldanamycin on ACh responses are shown in Fig. 3. Dilation to ACh was abolished by treatment with geldanamycin in control arteries from both exposure groups (Fig. 3, A and C, for the shorter and longer exposure periods, respectively). Unexpectedly, constriction to ACh was diminished by geldanamycin treatment in hypoxic arteries from both the shorter exposure period (Fig. 3B) and the longer exposure period (Fig. 3D). Treatment of longer exposure arteries with radicicol (Fig. 4) yielded results similar to those for geldanamycin (Fig. 3, C and D). Specifically, after radicicol treatment, dilation to ACh was abolished in control arteries (Fig. 4A), and hypoxic arteries from the longer exposure group no longer constricted to ACh (Fig. 4B). Notably, treatment with the NOS precursor l-arginine also abolished constriction to ACh in hypoxic arteries from the longer exposure group (Fig. 5). Moreover, treatment with the COX inhibitor indomethacin nearly abolished constriction to ACh in hypoxic arteries from the longer exposure group (Fig. 6B) and reversed the ACh responses from dilation to constriction in comparable age-control arteries (Fig. 6A).
Responses of the longer exposure group to the NO donor SNAP are shown in Fig. 7. Both control and hypoxic arteries of the longer exposure group dilated to all doses of SNAP, but the dilation to SNAP was blunted in hypoxic compared with control arteries (Fig. 7A). Interestingly, there were no differences between responses of control and hypoxic arteries of the longer exposure group to 8-bromo-cGMP (Fig. 7B).
Immunoblot analyses for NOS-3, NOS-1, caveolin-1, and HSP90 in small pulmonary artery homogenates from control and hypoxic piglets are shown in Figs. 8 and 9. We were unable to detect NOS-2 protein by the immunoblot technique applied to small pulmonary artery homogenates from control and hypoxic lungs. In the shorter exposure group, there were no differences in the mean values for the absorbance of HSP90 (Fig. 8A) and caveolin-1 (Fig. 8B) as determined by densitometry between small pulmonary artery homogenates from hypoxic and control piglets. Our laboratory has previously reported no difference between the intensity of bands for either NOS-3 or NOS-1 in small pulmonary artery homogenates from lungs of the shorter exposure group (47).
In the longer exposure group, the mean data for the absorbance of NOS-3 (Fig. 9A), HSP90 (Fig. 9C), and caveolin-1 (Fig. 9D) bands did not differ between homogenates of small pulmonary arteries from hypoxic compared with control piglets. The mean data for the absorbance of NOS-1 bands (Fig. 9B) were significantly greater for homogenates of small pulmonary arteries from hypoxic compared with control piglets of the longer exposure group.
An important finding in this study is that the nonselective NOS antagonist l-NAME had a greater constrictor effect on small pulmonary arteries from control piglets than those from hypoxic piglets of the longer exposure group. This finding suggests that after 10 days of in vivo hypoxia, the contribution of basal NO production to resting tone in resistance level pulmonary arteries is reduced. In contrast to these findings, l-NAME caused a similar degree of constriction in small pulmonary arteries from piglets exposed to 3 days of hypoxia and their age-matched controls. Thus, consistent with our laboratory's previous studies (13, 47), findings in this study indicate that basal NO production by small pulmonary arteries of newborn piglets remains unaltered after 3 days of in vivo hypoxia. Moreover, the effect of chronic hypoxia on the NOS-NO pathway in resistance-level pulmonary arteries is dependent on the length of exposure to hypoxia.
Another important finding in this study is that neither the NOS-1-selective antagonist 7-NINA nor the NOS-2-selective antagonist aminoguanidine altered basal diameter of small pulmonary arteries from either control or hypoxic piglets of either exposure group. These findings combined with the finding that arteries of all types constricted with nonselective NOS antagonism indicate that NOS-3, but neither NOS-1 nor NOS-2, underlies basal NO production in resistance-level pulmonary arteries of newborn piglets and that NOS-3, but neither NOS-2 nor NOS-1, is involved with the diminished basal NO production that occurs in resistance-level pulmonary arteries with the longer exposure to chronic hypoxia.
Only a limited number of previous studies have examined the functional contribution of NOS-1 and NOS-2 to basal pulmonary vascular pressure in lungs of either normal animals or those with pulmonary hypertension. Different from our findings, there is evidence that, in addition to NOS-3, both NOS-1 and NOS-2 contribute to pulmonary vascular resistance in the normal fetal sheep lung under basal conditions (36, 37). Also different from our findings, other investigators reported that NOS-3 and NOS-2, but not NOS-1, play a role in modulating basal tone in the normal adult murine pulmonary circulation (12). Differences in methodology could contribute to the discrepant results. Regardless of the specific NOS isoform involved or methodology used, several studies indicate that the NO-dependent contribution to basal pulmonary vascular pressure varies with maturation (22, 35, 42) and between species (9, 23).
In addition to interspecies and maturational differences, there is speculation that the development of pulmonary hypertension may alter the role of one or more NOS isoforms in the regulation of pulmonary vascular pressure. For example, it has been proposed that upregulation of NOS-1 and/or NOS-2 may modulate contraction and oppose acute or chronic increases in pulmonary vascular pressure in various models of pulmonary hypertension. If this were the case, one might anticipate greater constriction to NOS-2- and/or NOS-1-selective antagonists as well as enhanced expression of these NOS isoforms in the pulmonary circulation of animals with pulmonary hypertension compared with controls. Indeed, a number of studies have revealed increased NOS-2 in lungs of chronically hypoxic adult rats (28, 34, 39, 43). Yet, selective NOS-2 antagonists have not elicited greater pulmonary vasoconstriction in hypertensive compared with control adult rat lungs (39, 48). Moreover, there is a recent report that NOS-1 is reduced rather than increased in the pulmonary circulation of fetal lambs with pulmonary hypertension induced by in utero ligation of the ductus arteriosus (49). In addition, others have reported that NOS-2 activity was decreased in homogenates containing both conduit- and resistance-level pulmonary arteries of piglets with pulmonary hypertension after both 3 and 14 days of exposure to hypoxia (4). Although we found that NOS-1 levels were increased, the functional effects of NOS-1 and NOS-2 antagonists were unaltered in resistance-level pulmonary arteries of newborn piglets exposed to 10 days of chronic hypoxia compared with age-matched controls (Fig. 1, E and F). The use of different-sized arteries, species, postnatal ages, and stimuli for inducing pulmonary hypertension could explain differences between all these studies.
There is general consensus that NOS-3 plays a role in the vascular alterations that occur in the lungs of animals with pulmonary hypertension. Virtually all of the studies in adult animals are consistent with the concept that either the level or function of NOS-3 is altered in the lungs of animals with chronic hypoxia-induced pulmonary hypertension (see reviews in Refs. 23, 27). Data from studies in newborn lungs also consistently support the notion that basal pulmonary NO production is decreased with chronic hypoxia and that either decreased protein expression or activity of NOS-3 is involved with this change (4, 7, 14, 17, 24, 41).
In addition to altered basal NO production, both our findings and those of others suggest that changes in agonist-stimulated NO production occur in lungs of newborns exposed to chronic hypoxia (14, 33, 46). Regardless of the length of hypoxic exposure, pulmonary artery responses to ACh were blunted in studies with either whole lungs (14), conduit-level pulmonary arteries (33, 46), or resistance-level pulmonary arteries (18) from chronically hypoxic newborn animals. Responses to ACh are often used to reflect stimulated NO production. Indeed, findings in this study support the contention that ACh-induced pulmonary arterial dilation is at least in part mediated by stimulation of NO release in normal lungs in that we found that l-NAME abolished ACh-mediated dilation in resistance-level pulmonary arteries from the control piglets of both exposure periods (Fig. 2, A and C). It is possible that a diminished ability to stimulate NO release in response to ACh or other perturbations in the NO-dilation pathway contribute to the constrictor response to ACh that developed in pulmonary arteries from hypoxic piglets of one or both exposure groups. In this study, we found that l-NAME augmented the constrictor response to ACh in resistance pulmonary arteries from piglets exposed to shorter (Fig. 2B) but not to longer (Fig. 2D) hypoxia. One possible explanation for the latter finding is that after the longer but not the shorter exposure to hypoxia, pulmonary resistance arteries can no longer respond to ACh with enhanced NO release. Thus consistent with our findings regarding basal NO production, these latter findings suggest that perturbations in the NOS-3-NO pathway depend on length of hypoxic exposure, with greater dysfunction after the longer exposure period.
Our study also provides important information regarding potential mechanisms contributing to the impairments in NO function that are manifest in resistance-level pulmonary arteries after 10 days of in vivo hypoxia. Figure 10 is a simplified schematic of the NOS-NO vascular signaling pathway, indicating several targets that were explored in this study. Target 1 in Fig. 10 explored the hypothesis that impaired NO-mediated vascular function in resistance arteries from the longer exposure group was associated with limited availability of the NOS substrate l-arginine. Our results demonstrate that treatment with the NO precursor l-arginine abolished the constrictor response to ACh that develops in resistance arteries after 10 days of exposure to hypoxia (Fig. 5). Consistent with both our laboratory's previous findings (16) and those of others (6, 11), this finding suggests that l-arginine bioavailability to NOS-3 is compromised during chronic hypoxia. Although the mechanism remains uncertain (5, 16, 30), this study lends further support to the notion that l-arginine supplementation might restore impairments in NO function that occur upstream of NOS (Fig. 10) with chronic hypoxia.
Our studies also addressed whether changes in levels of NOS isoforms were involved in the pulmonary vascular dysfunction observed in the prolonged hypoxic-exposure group. Our inability to detect NOS-2 in homogenates of pulmonary arteries from control and hypoxic piglets precludes us from making definitive conclusions regarding the role of the NOS-2 isoform. Studies of NOS-3 (target 2 of Fig. 10) reveal preserved protein expression with chronic hypoxia. Together with responses to the NOS antagonists, these findings imply that changes in the function, but not the level, of NOS-3 underlies impaired basal NO production in resistance-level pulmonary arteries of piglets exposed to the longer period of hypoxia.
We also examined the impact of chronic in vivo hypoxia on other proteins that are known to be important in the regulation of NOS activity. Direct interaction of NOS-3 with caveolin-1 (target 3 in Fig. 10) inhibits enzyme activity; agonists that stimulate NOS activity reduce the association of NOS-3 with caveolin-1 (2). NOS-3 function might be impaired by increased levels of caveolin-1. However, our results reveal no alterations in caveolin-1 expression by immunoblot (Fig. 9D). Despite this result, we cannot rule out that interactions between NOS-3 and caveolin-1 might be altered by chronic hypoxia. In this regard, a recent study (32) with intrapulmonary arteries (∼1-mm diameter) from adult rats showed that exposure to 1 wk of chronic hypoxia failed to alter levels of either NOS-3 or caveolin-1 but did alter the normal coupling between these two proteins. Further investigation will be required to pursue the possibility that altered protein-protein interactions contribute to the impaired NO signaling in resistance pulmonary arteries of chronically hypoxic newborn piglets.
The chaperone protein HSP90 has also been shown to regulate NOS-3 activity. NOS-3 binding to HSP90 (target 4 in Fig. 10) increases enzyme activity (20). Therefore, NOS-3 function might be impaired by decreased levels of HSP90 or altered colocalization of HSP90 with its client protein, NOS-3. We found similar levels of HSP90 in resistance arteries from control piglets and those exposed to chronic hypoxia. Thus our study does not support the idea that altered amounts of this NOS regulatory protein underlies impaired NO function.
Nonetheless, our functional studies with cannulated resistance arteries lend support to the idea that recruitment of HSP90 and its association with NOS-3 is involved with ACh-mediated responses in resistance-level pulmonary arteries. In control piglets, pretreatment with the HSP90 antagonist geldanamycin abolished ACh-induced dilation (Fig. 3, A and C). Geldanamycin binds to the ATP binding site on HSP90, interfering with normal chaperone function, and thus normal coupling of NADPH oxidation and NO production by NOS (2). Our results in control resistance-level pulmonary arteries pretreated with geldanamycin are consistent with reduced NO production and/or bioavailability by inhibition of HSP90 chaperone function and subsequent NOS uncoupling.
Based on this explanation, the predicted effect of geldanamycin treatment would be to enhance ACh-mediated constriction in hypoxic arteries from the shorter exposure group and to either enhance or have no effect on ACh-mediated constriction in the longer exposure group, similar to our results with l-NAME (Fig. 2, B and D). Unexpectedly, we found that geldanamycin had the opposite effect, i.e., diminished ACh-mediated constriction in hypoxic arteries in both exposure groups (Fig. 3, B and D). Moreover, radicicol, a macrocyclic antifungal that is structurally unrelated to geldanamycin but similarly binds to the HSP90 ATP binding site (40), also reduced constriction to ACh in hypoxic arteries from the longer exposure group (Fig. 4B). Although it is likely that NO production, at least in resistance arteries from the shorter hypoxic exposure period is diminished by HSP90 antagonism, this effect cannot fully explain our results. Instead, our findings suggest that in hypoxic arteries the effects of geldanamycin and radicicol are to enhance production of a vasodilator other than NO and/or to inhibit production of a vasoconstrictor. The hypothesis that HSP90 is involved in the signal transduction of a vasoconstrictor that may be interfering with or counteracting optimal NO function in hypoxic arteries is intriguing and currently under investigation in our laboratories.
Our study also examined two other potential sites for NO pathway dysfunction downstream of NOS-3. Specifically, we found that responses to the NO donor SNAP (target 5 in Fig. 10) were impaired, whereas responses to the stable cGMP analog 8-bromo-cGMP (target 6 in Fig. 10) were preserved in resistance pulmonary arteries of hypoxic piglets from the longer exposure group. These findings respectively indicate that diminished responses to NO develop after 10 days of in vivo hypoxia but that mechanisms regulating NO-induced dilation downstream of guanylate cyclase (target 5 in Fig. 10) remain intact. This latter finding is of particular interest because it indicates that cGMP analogs might be therapeutically useful in clinical conditions associated with chronic hypoxia.
Last, we extended our previous findings in the shorter exposure period (18) to show that COX-dependent dilators are involved with ACh-induced dilation responses in control arteries (Fig. 6A). Notably also consistent with our laboratory's previous findings (18), a COX-dependent contracting factor is at least partly responsible for the abnormal pulmonary vascular response to ACh that develops when newborn piglets are exposed to hypoxia for 10 days (Fig. 6B).
The limitations of our methodologies employed in these studies must be considered when interpreting the results. In cannulated artery studies, it must be considered that all agonists used to elevate tone have the potential to confound results because of their potential influence on a myriad of signaling pathways. For example, the use of endothelin, which has been shown to stimulate NOS activity, may have influenced responses to cGMP. Even when tone is not elevated with an agonist, differences in basal tone could influence the results. The immunoblot technique also had limitations that must be considered when this methodology is used in a semiquantitative fashion. For example, the pulmonary artery homogenates contain a mixture of cell types, the composition of which could change with hypoxia. The ability to normalize the samples is further complicated by the present lack of knowledge as to which proteins remain unaltered during hypoxia. However, combining our findings using the immunoblot technique with functional studies in cannulated arteries lends strength to our conclusions.
In summary, our findings show that NO signaling is impaired in resistance-level pulmonary arteries of newborn piglets exposed to chronic hypoxia. This impairment is not due to diminished levels of either NOS-3 or NOS-1 proteins nor to altered levels of the regulatory proteins HSP90 and caveolin-1. Notably, the NO dysfunction appears to involve diminished bioavailability of l-arginine, an impairment upstream of NOS. Moreover, impairments downstream of NOS also appear to be involved so that, as with other models of neonatal pulmonary hypertension (45), multiple impairments in the NOS signal transduction pathway are evident with chronic hypoxia. Finally, consistent with our previous studies (14, 17, 47), NO dysfunction in resistance pulmonary arteries is dependent on the length of in vivo exposure to chronic hypoxia. Findings from this study point out important potential targets in the NO pathway that might be manipulated to prevent the progression of or enhance the recovery from chronic hypoxia-induced pulmonary hypertension in newborns.
This work was supported in part by American Heart Association Mid-Atlantic Affiliate Grant 51107U (to C. D. Fike) and by National Heart, Lung, and Blood Institute Grants HL-62489 (to J. L. Aschner) and HL-68572 (to C. D. Fike).
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- Copyright © 2004 the American Physiological Society