© 2000 American Heart Association, Inc.
Integrative Physiology |
Downregulation of Endothelial Nitric Oxide Synthase in Rat Aorta After Prolonged Hypoxia In Vivo
From the Meakins-Christie Laboratories (M.T., K.G., M.N., D.E.), McGill University, Montréal, Québec; Université de Montréal (G.T.), Montréal, Québec; and St Michael’s Hospital (M.E.W.), University of Toronto, Toronto, Ontario, Canada.
Correspondence to M.E. Ward, St Michael’s Hospital, Room 6042 Bond Wing, 30 Bond St, Toronto, Ontario, M5B 1W8 Canada. E-mail wardm{at}smh.toronto.on.ca
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Abstract |
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Abstract—The goal of this study was to determine whether hypoxia alters expression of endothelial nitric oxide synthase (eNOS) in the systemic circulation. Rats breathed either air or 10% oxygen for 12 hours, 48 hours, or 7 days. Thoracic aortas were excised and either mounted in organ bath myographs or frozen in liquid nitrogen for later extraction of protein and RNA. eNOS protein (Western blotting) was decreased (20% of normoxic control) after 12 hours, 48 hours, and 7 days of hypoxia. eNOS mRNA (ribonuclease protection assay) was similarly reduced. Acetylcholine (10-4 mol/L) reversed phenylephrine (10-5 mol/L) preconstriction by 53.3±5.6% in aortic rings from normoxic rats and 26.1±4.8% in rings from rats exposed to hypoxia for 48 hours (P<0.05), with comparable impairment of relaxation by the calcium ionophore A23187 (10-5 mol/L). Responses to diethylamine nitric oxide and 8-bromo-cGMP were unaffected. Aortic cGMP levels after incubation with acetylcholine (10-6 mol/L) averaged 14.0±1.8 fmol/mg in rings from normoxic rats compared with 8.7±1.0 fmol/mg in rings from hypoxic rats (P<0.05). Similarly, nitrate concentration (by capillary electrophoresis) in the media in which the rings were incubated was reduced in the hypoxic group (5.6±0.23 µmol/L for hypoxic rats and 7.8±0.7 µmol/L for normoxic rats). Impaired endothelial NO release may handicap the vascular responses that defend vital organ function during hypoxia.
Key Words: endothelium • systemic vasculature • hypoxic vasodilation • autoregulation
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hypoxia occurs commonly in patients with cardiopulmonary diseases and in normal individuals at high altitude. Survival under these conditions requires adaptive responses from the systemic circulation that redistribute the available oxygen supply toward vital organs1 and enhance oxygen extraction.2 Many of the mechanisms that mediate these responses are localized to the vascular endothelium. In particular, endothelial release of nitric oxide (NO) has been shown to modulate myogenic autoregulatory responses3 and to mediate flow dilation of resistance arterioles4 ; active hyperemia5 ; reactive hyperemia6 ; and, in some vascular beds, hypoxic vasodilation.7 Thus, a central role is emerging for endothelial production of NO in maintaining the balance between tissue oxygen supply and metabolic demand. If expression of endothelial nitric oxide synthase (eNOS), the enzyme that catalyzes NO synthesis, is regulated by oxygen tension, changes in the capacity for endothelial NO release may either be an important adaptive response or else contribute to the pathogenesis of vital organ failure, depending on whether it is enhanced or impaired, respectively.
The effect of hypoxic incubation on eNOS expression has been investigated previously in endothelial cells in culture.8 9 10 Unfortunately, the results of these studies have varied in both direction and magnitude. Despite its fundamental clinical and physiological relevance, therefore, the question of whether a change in eNOS availability alters vasoregulatory responses during hypoxia remains unanswered. Accordingly, the current study was undertaken to determine whether exposure to hypoxia in vivo alters expression of eNOS protein and mRNA in the systemic vasculature and to evaluate the effect of this change on endothelium-dependent vasorelaxation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male Sprague-Dawley rats were exposed to either normoxia or hypoxia (10% O2) for 12 hours, 48 hours, or 7 days.
Western Blot
Proteins from thoracic aorta and lung were analyzed
using an eNOS-specific monoclonal antibody (Transduction
Laboratories).
cDNA cloning
Aortic RNA was extracted using TRIzol.11 One
microgram of RNA was reverse transcribed, and an 871-bp eNOS cDNA was
amplified by polymerase chain reaction (PCR) using sense
(5'-AGCTGGCATGGGCAACTTGAA-3') and antisense
(5'-CAGCACATCAAAGCGGCCATT-3') primers and subcloned into the PCRII
vector (Invitrogen) behind the T7 promoter (GenBank accession No.
AF085195). ß-Actin cDNA (292 bp) was also amplified by PCR (sense,
5'- AAGTACCCCATTGAACACGGCA-3'; antisense, 5'-TAGATGGGCACAGTGTGGGTGA-3')
and subcloned into the PCRII vector (Invitrogen) behind the Sp6
promoter (GenBank accession No. AF122903).
32P–Labeled Riboprobe Synthesis and Ribonuclease
Protection Assay
eNOS and ß-actin constructs were linearized with
BamHI and Bsu36I, respectively. Antisense RNA for
eNOS and ß-actin were synthesized using T7 and Sp6 RNA polymerases,
respectively, from 1 µg of linearized construct and
-32P–labeled CTP (Amersham). The eNOS cRNA
spanned regions homologous to exons 4 to 7 of the human eNOS
sequence.12 The ß-actin probe spanned exons 3 and 4
of the rat ß-actin gene. Ribonuclease protection assays for eNOS and
ß-actin were carried out simultaneously on aortic RNA
from each of the rats in each group. Protected mRNA was quantified by
densitometry.
Endothelium-Dependent and -Independent
Relaxation
Aortic segments (4 mm) from normoxic rats and rats exposed
to hypoxia for 48 hours were treated with
10-5 mol/L phenylephrine.
Concentration-response curves for acetylcholine (Ach;
10-9 to 10-4 mol/L) were
generated, and maximum relaxations in response to A23187
(10-5 mol/L), diethylamine nitric oxide (DEA/NO,
10-4 mol/L), and 8-bromo-cGMP (8-Br-cGMP,
10-4 mol/L) were assessed.
cGMP Radioimmunoassay
Aortic segments (2 mm) from normoxic and 48-hour hypoxic
rats were incubated at 37°C for 1 hour in Krebs solution, for 10
minutes in isobutylmethylxanthine (0.5
mmol/L), and then with Ach (10-6 mol/L) for 1 or
4 minutes or for 1 minute in the absence of Ach. The reaction was
stopped with trichloroacetic acid (10% wt/vol). Trichloroacetic acid
was removed with water-saturated ether, and the samples were
acetylated with a 2:1 mixture of trifluoroethane and glacial
acetic acid. The samples were incubated with a rabbit anti-rat cGMP
antibody for 48 hours before overnight incubation with
125I-labeled cGMP. Rabbit serum and IgG were
added, the samples were precipitated with 12.5% polyethylene glycol,
and radioactivity was measured with a gamma counter.
Capillary Electrophoresis (CE)
Plasma nitrate (NO3-)
concentration was measured by CE.13 14 Plasma was
collected from normoxic and 48-hour hypoxic rats, filtered (0.2
µm), and analyzed in a 50 mmol/L phosphate buffer (pH
2.5) with 0.5 mmol/L spermine using an ABI270 CE instrument
with a 50-µm fused silica capillary (Polymicro Technologies). Samples
were separated at -347 Vcm-1 (45 to 50
µA) at 30°C, and the NO3-
peak was detected by absorption at 214 nm. Nitrate concentration was
also measured in the reaction media from unstimulated and
Ach-stimulated (4 minutes) aortic segments. Calibration curves were
constructed with plasma or reaction media spiked with standard nitrate
solutions.
An expanded Materials and Methods section is available online at http://www.circresaha.org.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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eNOS Protein
Representative Western blots conducted on proteins from thoracic aortas and lungs from a normoxic rat and rats exposed to hypoxia for 12 hours, 48 hours, and 7 days are illustrated in Figures 1A
and 1B, respectively.
The accompanying histograms illustrate the mean aortic and lung eNOS
protein levels (arbitrary units of optical density) for each group (n=4
per group). Aortic eNOS protein was decreased (P<0.01
versus normoxic controls) after 12 hours, 48 hours, and 7 days of
hypoxia. Lung eNOS protein levels were unchanged after 12 and
48 hours of hypoxia but were increased (P<0.05
versus normoxic controls) after 7 days of hypoxia, as
previously reported.19 20 21
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eNOS mRNA
A representative autoradiogram of
an RNase protection assay carried out on aortic RNA from a normoxic rat
and from rats exposed to hypoxia for 12 hours, 48 hours, and 7
days is illustrated in the top panel of Figure 2. The histogram in the lower panel
illustrates the mean aortic eNOS mRNA levels in each group (n=5 per
group) expressed as a percentage of the ß-actin mRNA level. Aortic
eNOS mRNA levels were decreased (P<0.01) after exposure to
hypoxia for 12 hours, 48 hours, and 7 days compared with
normoxic controls.
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Endothelium-Dependent and -Independent
Relaxation
The concentration-response relationships for Ach-induced
relaxation in aortic rings from normoxic rats and from rats exposed to
hypoxia for 48 hours are illustrated in the top panel of Figure 3. The tension generated during
contraction with 10-5 mol/L
phenylephrine was 1.67 ± 0.08 g/mg dry weight in
rings from normoxic rats. As has been reported
previously,24 tension was lower (0.88±0.06 g/mg dry
weight) in rings from hypoxic rats. Maximal relaxation by Ach was
53.3±5.6% of the phenylephrine-induced contraction in
rings from normoxic rats, compared with 26.1±4.8% in rings from rats
exposed to hypoxia (P<0.05 for difference). The
pEC50 for Ach-induced relaxation was 6.90±0.18
in rings from normoxic rats and 7.21±0.14 in rings from hypoxic rats
(P>0.05).
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The responses to A23187, DEA/NO, and 8-Br-cGMP in aortic rings from
normoxic rats and from rats exposed to hypoxia for 48 hours are
compared in the bottom panel of Figure 3
. Relaxation of
phenylephrine contraction by A23187 was reduced in the
hypoxic group. The responses to DEA/NO and 8-Br-cGMP in aortic rings
from hypoxic rats did not differ from those in rings from normoxic
rats. In this group, Ach reversed 48.5±3.8% of the
phenylephrine-induced contraction in rings from
normoxic rats and 20.1±2.8% in rings from rats exposed to
hypoxia (P<0.05 for difference).
cGMP Generation
The effect of hypoxia on aortic cGMP levels during in
vitro stimulation with Ach is illustrated in Figure 4. Unstimulated cGMP levels did not
differ between normoxic rats and those exposed to hypoxia for
48 hours. The mean aortic cGMP level after 4 minutes of incubation with
Ach was lower (P<0.05) in the group exposed to
hypoxia for 48 hours than in the normoxic group.
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Plasma Nitrate Concentration and Aortic Nitrate Production
Concentrations of NO3- in
plasma from normoxic rats (n=7) and from rats exposed to
hypoxia for 48 hours (n=6) averaged 70.3±2.6 and
72.1±5.2 µmol/L, respectively (P>0.05 for
difference). The concentrations of nitrate in the reaction buffer from
aortic rings from normoxic rats and from rats exposed to
hypoxia for 48 hours that were not treated with Ach (control)
and that were incubated with 10-6 mol/L Ach for
4 minutes are illustrated in Figure 5.
Unstimulated values in the two groups did not differ. Nitrate
concentrations were higher (P<0.05) after incubation with
Ach in the normoxic than in the hypoxic group.
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Discussion |
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The results of this study indicate that in rats, prolonged exposure to hypoxia results in (1) a decrease in aortic eNOS protein and mRNA, (2) impaired endothelium-dependent relaxation of phenylephrine-precontracted aortic rings, and (3) impaired capacity of aortic segments to generate cGMP and NO3- in response to stimulation by Ach. This is the first demonstration that physiologically relevant levels of hypoxia in vivo alter eNOS expression in the systemic vasculature and, consequently, impair endothelium-dependent vascular responses. Our finding that, after exposure to hypoxia, relaxation in response to A23187 is decreased to a similar extent as relaxation to Ach points to an abnormality distal to events occurring at the level of the endothelial cell plasma membrane (ie, Ach receptor activation and Ca2+ entry). The alteration must also be upstream of soluble guanylate cyclase activation or cGMP target sites, because the relaxant effects of DEA/NO, and 8-br-cGMP were not affected. The abnormality in endothelium-dependent relaxation after in vivo hypoxia, therefore, is not due to the inability to activate eNOS or to respond to its product (NO), but rather to a reduction in the availability of this enzyme.
Previous studies of the effects of hypoxia on eNOS protein and mRNA expression in cultured endothelial cells have yielded conflicting results. Hypoxic incubation (0% O2 for 24 hours) decreased eNOS protein and mRNA in endothelial cells from human umbilical8 and saphenous15 16 veins and from bovine pulmonary artery9 and aorta.9 15 In contrast, Arnet et al10 reported that eNOS protein and mRNA were increased in bovine aortic endothelial cells after 24 hours of incubation at 1% O2. Moreover, a luciferase reporter construct consisting of the eNOS 5' regulatory region could be activated in these cells by hypoxia. Upregulation of eNOS protein and activity has also been demonstrated in early-passage porcine coronary arteriolar endothelial cells after hypoxic exposures of 30 to 240 minutes’ duration.17 This variability reflects differences in the species and vascular bed from which the endothelial cells were derived, the methods used to maintain the cells, and the duration and severity of the hypoxic exposures. Even if the previous data were consistent, however, cell culture experiments may not accurately reproduce the microenvironment to which these cells are normally exposed nor the chemical and mechanical stimuli that interact with the effects of hypoxia under physiological conditions. Convincing evidence that hypoxic regulation of eNOS protein expression is a physiologically relevant mechanism, therefore, requires its demonstration in vivo, and the present results complement and extend the findings of the previous studies.
The effect of in vivo exposure to hypoxia on eNOS protein expression has previously been investigated in the rat pulmonary circulation.18 19 20 In those studies, breathing 10% oxygen for 7 days and for 3 weeks increased pulmonary eNOS levels. This could not be attributed to the known stimulatory effect of increased flow (shear stress) on eNOS expression,21 because inhibiting the increase in pulmonary blood flow by surgical stenosis of the pulmonary artery failed to prevent the increases in eNOS. Our present results confirm upregulation of lung eNOS protein after 7 days of hypoxia and demonstrate that hypoxia has the opposite effect on eNOS expression in the aorta.
Aortic blood flow is also increased in rats during hypoxia.1 Although shear stress at the endothelial-luminal interface may not necessarily increase in tandem with flow, it is highly unlikely to change in the opposite direction. Consequently, the decrease in aortic eNOS that we describe in this report is not likely to be attributable to changes in flow. This study does not include experiments designed to dissociate the influence of other hemodynamic or neurohumoral stimuli from the direct effects of hypoxia. Nonetheless, these factors compose part of the response to systemic hypoxia, and the hypoxic exposures as presented in this study simulate a clinically and physiologically relevant condition. Taken together, our current results and those of previous in vivo studies indicate marked regional variability in eNOS protein expression during systemic hypoxia.
The endothelium normally exerts an inhibitory effect on vascular reactivity, and agonist-induced contraction is greater in arterial segments from which the endothelium has been removed compared with those in which it is intact.22 23 In previous studies, we have noted that, after prolonged hypoxic exposure, endothelial ablation resulted in a decrease rather than an increase in the contractile response of rat aortic segments to phenylephrine.23 After hypoxia, therefore, the endothelium serves as a source of substances that enhance rather than inhibit contraction. Our current results provide a partial mechanistic explanation for this finding, because the inhibitory influence of the endothelium on vascular reactivity has been attributed to endothelial NO release.22 A decreased capacity for vasodilator synthesis, however, cannot account for endothelial enhancement of contractility, and a concomitant increase in endothelium-derived constricting factor release must be proposed. Synthesis and receptor binding of both endothelin-124 25 and thromboxane A226 27 have been reported to be under the negative regulatory influence of NO. Accordingly, hypoxic inhibition of eNOS expression may play an additional role in the alteration in endothelial function through the removal of an inhibitor of vasoconstrictor production and activity.
Plasma nitrate levels did not differ between normoxic rats and rats exposed to hypoxia for 48 hours in the current study, indicating that factors other than aortic eNOS levels determine the circulating NO3- concentration. During hypoxia, increased flow1 will stimulate NO synthesis by the remaining enzyme,28 the duration of eNOS activation may be increased as a result of increased pH of the endothelial intracellular space,29 30 and increased eNOS expression in the pulmonary circulation18 may counterbalance the effect of decreased NO synthesis in the systemic vasculature. Our inability to demonstrate a decrease in circulating NO3-, therefore, does not diminish the pathophysiological significance of hypoxic inhibition of eNOS expression in the systemic circulation. The dilatory response to flow needed to maximize perfusion4 and the decrease in transvascular resistance necessary to accommodate increased metabolic activity5 and to preserve vital organ perfusion during superimposed hypotensive stresses3 require that local NO production be intact. A decrease in the capacity of the endothelium to maximally respond to dilatory stimuli would undermine these responses.
Inhibition of NO synthesis by infusion of L-arginine analogues has been shown to inhibit hypoxic vasodilation in the guinea pig heart7 and canine diaphragm.31 In at least some essential vascular beds, therefore, this pathway must be intact to optimize perfusion and maintain tissue oxygenation in the face of decreased systemic oxygen delivery. These responses will be impaired by a reduction in the local capacity to produce NO. Because the decrease in eNOS protein expression occurs relatively quickly (hours to days), impaired endothelial NO release is relevant to cardiopulmonary diseases associated with hypoxia (eg, pneumonia, congestive heart failure, and exacerbations of chronic obstructive lung disease) of which the natural histories evolve over this time frame. Hypoxic inhibition of eNOS expression in the systemic circulation may, therefore, represent an important mechanism in the pathogenesis of organ dysfunction in critically ill patients.
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Acknowledgments |
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This study was funded by a grant from the Medical Research Council of Canada. M.T. is a recipient of a doctoral fellowship from the Fonds pour la Formation de Chercheurs et l’Aide a la Recherche de Québec.
Received August 2, 1999; accepted December 1, 1999.
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D. Lopez, X. Orta, K. Casos, M. P. Saiz, P. Puig-Parellada, M. Farriol, and M. T. Mitjavila Upregulation of endothelial nitric oxide synthase in rat aorta after ingestion of fish oil-rich diet Am J Physiol Heart Circ Physiol, August 1, 2004; 287(2): H567 - H572. [Abstract] [Full Text] [PDF]
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F. Coulet, S. Nadaud, M. Agrapart, and F. Soubrier Identification of Hypoxia-response Element in the Human Endothelial Nitric-oxide Synthase Gene Promoter J. Biol. Chem., November 21, 2003; 278(47): 46230 - 46240. [Abstract] [Full Text] [PDF]
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G. M. T. Hare, C. D. Mazer, W. Mak, R. M. Gorczynski, K. M. Hum, S. Y. Kim, L. Wyard, A. Barr, R. Qu, and A. J. Baker Hemodilutional anemia is associated with increased cerebral neuronal nitric oxide synthase gene expression J Appl Physiol, May 1, 2003; 94(5): 2058 - 2067. [Abstract] [Full Text] [PDF]
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S. Earley and B. R. Walker Increased nitric oxide production following chronic hypoxia contributes to attenuated systemic vasoconstriction Am J Physiol Heart Circ Physiol, May 1, 2003; 284(5): H1655 - H1661. [Abstract] [Full Text] [PDF]
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F. C. Blumberg, K. Wolf, M. Arzt, C. Lorenz, G. A. J. Riegger, and M. Pfeifer Effects of ET-A receptor blockade on eNOS gene expression in chronic hypoxic rat lungs J Appl Physiol, February 1, 2003; 94(2): 446 - 452. [Abstract] [Full Text] [PDF]
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 I. M. Bird, L. Zhang, and R. R. Magness Possible mechanisms underlying pregnancy-induced changes in uterine artery endothelial function Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2003; 284(2): R245 - R258. [Abstract] [Full Text] [PDF]
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L. Xi, D. Tekin, E. Gursoy, F. Salloum, J. E. Levasseur, and R. C. Kukreja Evidence that NOS2 acts as a trigger and mediator of late preconditioning induced by acute systemic hypoxia Am J Physiol Heart Circ Physiol, July 1, 2002; 283(1): H5 - H12. [Abstract] [Full Text] [PDF]
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M. E. Zacour, H. Teoh, A. J. Halayko, and M. E. Ward Mechanisms of aortic smooth muscle hyporeactivity after prolonged hypoxia in rats J Appl Physiol, June 1, 2002; 92(6): 2625 - 2632. [Abstract] [Full Text] [PDF]
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P. Kinnunen, J. Piuhola, H. Ruskoaho, and I. Szokodi AM reverses pressor response to ET-1 independently of NO in rat coronary circulation Am J Physiol Heart Circ Physiol, September 1, 2001; 281(3): H1178 - H1183. [Abstract] [Full Text] [PDF]
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 J. M. Pearl, D. P. Nelson, C. J. Wagner, J. P. Lombardi, and J. Y. Duffy Endothelin receptor blockade reduces ventricular dysfunction and injury after reoxygenation Ann. Thorac. Surg., August 1, 2001; 72(2): 565 - 570. [Abstract] [Full Text] [PDF]
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R. M. Mohan, S. Golding, and D. J. Paterson Intermittent hypoxia modulates nNOS expression and heart rate response to sympathetic nerve stimulation Am J Physiol Heart Circ Physiol, July 1, 2001; 281(1): H132 - H138. [Abstract] [Full Text] [PDF]
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 C. R. Ferreiro, A. C. P. Chagas, M. H. C. Carvalho, A. P. Dantas, M. B. Jatene, L. C. Bento de Souza, and P. Lemos da Luz Influence of Hypoxia on Nitric Oxide Synthase Activity and Gene Expression in Children With Congenital Heart Disease : A Novel Pathophysiological Adaptive Mechanism Circulation, May 8, 2001; 103(18): 2272 - 2276. [Abstract] [Full Text] [PDF]
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D. Xiao, I. M. Bird, R. R. Magness, L. D. Longo, and L. Zhang Upregulation of eNOS in pregnant ovine uterine arteries by chronic hypoxia Am J Physiol Heart Circ Physiol, February 1, 2001; 280(2): H812 - H820. [Abstract] [Full Text] [PDF]
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