Department of Pediatrics, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157
Submitted 26 September 2003 ; accepted in final form 23 January 2004
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ABSTRACT |
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nitric oxide synthase isoforms; L-arginine; cyclic GMP; heat shock protein 90; caveolin-1
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 N-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).
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METHODS |
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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 100400 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 3060 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 (107 M). To check for a functional endothelium in control arteries, responses to ACh (105 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 (106 to 103 M), a NOS-2-selective antagonist, aminoguanidine (106 to 103 M), or a NOS-1-selective antagonist, 7-nitroindazole (7-NINA; 106 to 104 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 (108 to 105 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 (108 to 105 M) in the absence and presence of either the nonspecific NOS antagonist L-NAME (103 M for 20 min) or the HSP90 antagonist geldanamycin (106 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 x 108 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 (105 M) on ACh (108 to 105 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; 109 to 105 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 3050% 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 (108 to 104 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 3050% 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 (108 to 105 M) before and 30 min after adding L-arginine (102 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).
Materials. 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.
Statistics. 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.
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RESULTS |
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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.
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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).
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DISCUSSION |
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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.
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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.
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GRANTS |
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FOOTNOTES |
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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.
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REFERENCES |
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