点击显示 收起
Divisions of Pulmonary and Critical Care Medicine and Cardiology, Departments of Medicine and Anesthesiology, Johns Hopkins University, School of Medicine, Baltimore, Maryland
ABSTRACT
Rationale: Decreased nitric oxide (NO) is considered an important pathogenetic mechanism in pulmonary arterial hypertension (PAH), but clear evidence is lacking. Objectives: We used multiple techniques to assess endogenous NO in 10 patients with untreated PAH (8 idiopathic and 2 anorexigen-associated PAH) and 12 control subjects. Methods: After a nitrite/nitrate-restricted diet, NO metabolites (NOx) were assayed in 24-hour urine collections and exhaled NO (FENO) determined at multiple expiratory flows. Analysis of the relation between FENO and flow allowed derivation of three flow-independent parameters: airway wall concentration (CW), diffusing capacity (DNO), and alveolar concentration (CA). Seven patients underwent follow-up testing after 3 months of bosentan treatment. Results: At baseline, FENO was markedly decreased at the two lowest expiratory flows in PAH: 21 ± 4 versus 36 ± 4 ppb at 18 ml/second and 11 ± 2 versus 17 ± 2 ppb at 50 ml/second, for subjects with PAH and control subjects, respectively (p < 0.05). CW was 33 ± 11 ppb in subjects with PAH versus 104 ± 34 in control subjects (p = 0.04). Urinary NOx was also reduced in PAH (42 ± 6 e NOx/mM creatinine versus 62 ± 7 in control subjects; p = 0.04). After bosentan, FENO, CW, and urine NOx increased to control values (p < 0.05). Exclusion of the two anorexigen cases did not alter these results. Conclusions: FENO at low expiratory flows was decreased in PAH due to reduced CW. Bosentan reversed these abnormalities, suggesting that suppression of NO in PAH may have been caused by endothelin.
Key Words: airway; endothelin; urine nitric oxide metabolites
Impaired nitric oxide (NO) generation has long been proposed as a potential mechanism in the pathogenesis of idiopathic pulmonary arterial hypertension (IPAH); however, assessment of endogenous NO in patients with IPAH has yielded conflicting results. Reduced expression of pulmonary vascular endothelial NO synthase (eNOS) was initially described (1), but this finding has not been confirmed by others (2). NO in exhaled breath has been investigated as an indicator of pulmonary NO production in IPAH, but decreased (3, 4), normal (5), and increased (6) values have been reported. These studies used variable methodology and included subjects receiving therapy that could affect exhaled NO (FENO) concentrations (7). Circulating nitrite and nitrate metabolites (NOx) are largely derived from oxidation of NO (8) but are substantially affected by dietary factors (9) and glomerular filtration (10). Reports of plasma NOx levels in IPAH have been highly variable (4, 6, 11). Urinary NOx excretion is considered a valid indicator of whole-body NO production when dietary factors are controlled for (12).
This study used complementary techniques to assess endogenous NO in untreated patients with IPAH and anorexigen-associated PAH. FENO was measured at multiple expiratory flow rates to allow partitioning into airway and alveolar components (13). Nasal NO release was determined as a potential regulator of the pulmonary vasculature (14). Whole-body NO production was assessed by measuring NOx in 24-hour urine collections and plasma levels of NOx, L-arginine, L-citrulline, and the methylated arginines, asymmetric and symmetric dimethyl arginine (15), during a nitrate/nitrite-restricted diet. Moreover, serial testing was performed after therapy with the endothelin (ET) receptor antagonist bosentan. Some of the results of these studies have been previously reported in the form of an abstract (16).
METHODS
Subjects
This study was approved by the local institutional review board, and all participants provided written, informed consent. Ten adult patients with newly diagnosed IPAH or anorexigen-induced PAH (17) on no specific therapy were enrolled. Exclusion criteria included any factor that is known or suspected to alter respiratory or systemic NO and are listed in the online supplement. Twelve healthy nonsmoking volunteers served as control subjects. World Health Organization functional class (18) and 6-minute walk distance (19) were obtained at baseline and follow-up examinations.
Urine and Plasma Measurements
Subjects were asked to follow a nitrite/nitrate-restricted diet (20) for 48 hours. During the second day of the diet, a 24-hour urine collection was obtained. On the third morning, participants underwent FENO and nasal NO measurements (see below). Blood was collected into heparinized tubes and plasma separated immediately. Urine NOx was determined with a chemiluminescent analyzer (Sievers 280 NOA; Sievers Instruments, Boulder, CO) (21) and corrected for urine creatinine. Plasma NOx was similarly assayed after deproteinization with cold ethanol (21). L-arginine, L-citrulline, and asymmetric and symmetric dimethyl arginine were assayed by HPLCeCmass spectrometry (22). One patient with IPAH did not provide urine or blood samples but underwent FENO measurements.
FENO Measurements
Online recording of FENO was performed according to the recommendations of the American Thoracic Society (23) at expiratory flow rates of 18, 50, 100, and 250 ml/minute. Using the method described by Silkoff and coworkers (13), the curvilinear relationship between FENO and flow rate was analyzed by nonlinear least squares regression to derive the airway wall NO concentration (CW), the diffusing capacity of NO from airway wall to lumen (DNO), and the alveolar NO concentration (CA) according to the following equation:
where V = expiratory flow rate.
Nasal NO
Ambient air (always < 20 ppb NO) was suctioned through an olive snuggly fitted into one nasal cavity at a rate of 3 L/minute and sampled through the NO analyzer (13).
Follow-up Evaluation
Of the 10 patients with PAH, 7 (including both of the anorexigen-associated cases) were restudied after 3 months of therapy with bosentan (Tracleer; Actelion Pharmaceuticals, South San Francisco, CA) administered according to the package insert.
Statistics
Data are expressed as means ± SEM. Comparison of FENO values of the patients with PAH at baseline with the control group and between the patients at baseline with patients after bosentan was performed with analysis of variance. The mean FENO values at the different flow rates for each group were used to derive CW, DNO, and CA by nonlinear regression as described above. Individual FENO data were also used to calculate these parameters, which were converted to natural logarithms (13) and compared between groups with a paired or unpaired t test, as appropriate. Nasal NO output and the urine and plasma variables were compared between the control and PAH baseline groups with an unpaired t test or the nonparametric Mann-Whitney test as appropriate. A Wilcoxon matched pairs test was used to compare the seven bosentan-treated patients with PAH at baseline and at follow-up.
RESULTS
Subjects
Table 1 shows the demographic and clinical characteristics of the subjects with PAH and control subjects. All had moderateeCsevere disease with a mean pulmonary arterial pressure of greater than 40 mm Hg, and all but one had World Health Organization class IIIeCIV symptoms. Two had a history of fenfluramine use. Although anorexigen-associated PAH is classified separately from IPAH, there is no evidence that these groups differ clinically (24) or pathologically (25). Moreover, the various NO parameters obtained from these two individuals were comparable to the remainder of the IPAH group and excluding them from the analyses did not significantly alter the results. The control group was well matched in terms of sex, age, and height—factors that may affect FENO (26).
FENO
FENO concentrations were markedly reduced in the PAH group at baseline compared with control subjects (Figure 1). This was particularly evident at the lower expiratory flow rates (e.g., at 18 ml/second, mean FENO in the patients with PAH was 21 ppb compared with 36 ppb in control subjects). Application of the model described above to the mean group FENO data revealed that CW was reduced to less than half the normal value in the patients at baseline (Figure 2A). When individual FENO data were used, the fit of two patients with PAH at baseline did not converge. The CW of the other eight patients was 33.1 ± 11 ppb compared with 104 ± 34 in the control group (p = 0.04; Figure 2B). No significant differences in DNO or CA were observed.
Nasal NO
Nasal NO output was similar in patients with PAH at baseline (512 ± 48 nl/minute) and control subjects (760 ± 125 nl/minute; p = 0.2).
Urine and Plasma NOx
Urinary excretion of NOx was significantly reduced in patients with PAH at baseline compared with control subjects (Table 2). In contrast, plasma NOx was similar in the two groups. Creatinine clearance, as expected, was mildly decreased in the PAH group.
Plasma L-arginine, L-citrulline, and Asymmetric and Symmetric Dimethyl Arginine
Table 2 summarizes the HPLC data for control subjects and patients with PAH at baseline. Although L-arginine levels were identical between the two groups, plasma L-citrulline was markedly decreased in the patients with PAH, resulting in a significantly lower L-citrulline/L-arginine ratio. The patients with PAH also demonstrated small but significant increases in asymmetric and symmetric dimethyl arginine plasma levels.
Changes in Response to Bosentan Therapy
All but one of the seven patients restudied after 3 months of bosentan therapy reported subjective improvement in symptoms. Six-minute walk distance increased from 296 ± 34 to 341 ± 41 m (p = 0.02). FENO at the lower expiratory flow rates increased dramatically, becoming similar to those observed in the control group. Figure 1 displays the comparison between all patients with PAH at baseline and the seven treated with bosentan at follow-up. Similar results were obtained when the comparison was limited to the seven bosentan-treated patients (data not shown).
Using mean group FENO data for the partitioning analysis, CW similarly normalized, whereas DNO and CA did not appear to change (Figure 2A). We also performed this analysis for each individual patient at baseline and follow-up. For two of the bosentan-treated patients at baseline, the model fit did not converge. Comparison of individual CW values among eight patients at baseline with seven patients after bosentan treatment revealed a marked increase from 33 ± 11 to 105 ± 44 ppb (p = 0.01; Figure 2B). Similar results were obtained when the comparison was limited to the five bosentan-treated patients whose data were available for partitioning analysis at both baseline and follow-up (p < 0.05; data not shown). No significant changes in DNO or CA occurred with either comparison.
Nasal NO output at follow-up (659 ± 146 nl/minute) was similar to baseline (498 ± 46 nl/minute; p = 0.7). Urinary excretion of NOx significantly increased after 3 months of bosentan therapy (Table 2). No significant changes were detected in any of the plasma variables, whereas a trend was noted for a reduction in creatinine clearance at follow-up.
DISCUSSION
The status of endogenous NO in PAH is unclear and highly controversial. This study demonstrated that patients with PAH have markedly reduced FENO at lower expiratory flow rates, which may be the result of decreased CW. We also show that whole-body NO production, as assessed by 24-hour urinary NOx excretion, is decreased. In response to treatment with the nonselective ET receptor antagonist bosentan, these abnormalities reverted to normal after 3 months.
Shortly after the discovery that NO was detectable in exhaled breath, investigators began to explore this critical vasoactive mediator in IPAH, yielding highly variable results (3, 5, 6). These studies were conducted before the realization that FENO varies widely with the expiratory flow rate and that the nasal passages need to be reliably excluded. Researchers at the Cleveland Clinic demonstrated significantly reduced lower respiratory tract NO concentrations in IPAH using a bronchoscopic technique (4). The current study uses the standardized procedure for online recording of FENO recommended by the American Thoracic Society (23). Also, unlike previous studies, only untreated patients were included. It is well recognized that epoprostenol administration can dramatically increase FENO (7, 27). Moreover, care was taken to exclude subjects with concomitant factors that could alter respiratory or systemic NO. The most important finding of our study is the profound reduction in FENO at lower expiratory flow rates in patients with PAH at baseline and the normalization of these abnormalities with bosentan therapy.
Partitioning and Anatomic Source of FENO
Detailed analyses of pulmonary NO exchange dynamics have led to a two-compartment model (airways and alveolar region) describing three flow-independent NO exchange parameters during exhalation: mean NO concentration in the airway wall, total airway compartment NO diffusing capacity from the airway wall to lumen, and NO concentration in alveolar gas (28). Derivation of these parameters requires measurements of FENO at multiple expiratory flow rates, as described by Silkoff and coworkers (13). For all but two measurements (two patients with PAH at baseline), the data fit the model well, and the flow-independent parameters obtained in the control subjects were comparable to previously published results (28). On the basis of this model, we have shown that the reduced FENO concentrations observed in the PAH group were due to decreased CW. The normal nasal NO output in our patients with PAH indicates that this abnormality is localized to the lower respiratory tract.
Since the discovery of NO in exhaled breath, there has been considerable controversy regarding the extent to which the pulmonary vasculature contributes to FENO. The term "airway wall" in the model used here encompasses all of the tissue between the lumens of the airways and blood vessels, including pulmonary and bronchial endothelium, vascular and bronchial smooth muscle, and airway epithelium. Because of its extremely high avidity for hemoglobin, most NO produced in the blood vessels is expected to be consumed within the vascular lumen. Although several lines of evidence support the contention that the majority of FENO originates from cells lining the airways and alveoli (29, 30), pulmonary vascular factors may also influence FENO (31). Thus, the precise origin of airway wall tissue NO remains to be fully elucidated.
Significance of Reduced FENO in PAH
Irrespective of the exact source of FENO, our findings have potential clinical and mechanistic importance. The striking reductions at baseline and significant increases in FENO in response to therapy observed here and by others (4, 7), together with the recent suggestion that serial measurements may correlate with clinical outcomes (32), point to the potential utility of this noninvasive, easily obtained assessment as a clinical biomarker pending further validation. Although decreased FENO in IPAH may reflect reduced NO release from the pulmonary vasculature as a consequence of endothelial dysfunction, reduced lower airway NO may also have pathogenetic importance as a regulator of the adjacent pulmonary arteries (33).
Mechanism(s) for Decreased FENO in PAH
The main determinant of CW is the production of NO in the airway wall tissue relative to its catabolism (13). Decreased pulmonary expression and/or activity of NOS could account for the low CW observed in the patients with PAH. Recent studies in humans suggests that the type II, inducible (iNOS) isoform is the major determinant of FENO (34). Although the abundance of vascular endothelial NOS in PAH is debatable, there is no evidence for altered expression of any of the NOS isoforms in PAH airways (1, 35). However, available data are quite limited in this regard.
A major route of NO consumption is reaction with superoxide, forming peroxynitrite. There is considerable evidence for oxidative stress in PAH (36) and intense nitrotyrosine (a marker of peroxynitrite formation) expression in the lung (37). Thus, enhanced oxidative consumption of NO could explain reduced exhaled and airway wall concentrations. Oxidants can also directly suppress NOS activity (15). Enhanced binding of NO to metalloproteins is another potential mechanism for reduced airway NO (38).
NO is generated by the NOS-induced oxidation of L-arginine to L-citrulline. The reduction in plasma L-citrulline/L-arginine ratio observed in the patients with PAH suggests decreased NOS activity. We detected elevated asymmetric dimethyl arginine, a potent endogenous inhibitor of NOS (15), in PAH plasma. However, the magnitude of the observed increase is probably not biologically significant (39). Normal asymmetric dimethyl arginine levels in IPAH have been recently reported (35), whereas another, preliminary, study found significantly increased values (40). Similarly, the significance of our finding of high circulating symmetric dimethyl arginine in the patients with PAH is unclear. Although not a direct inhibitor of NOS, symmetric dimethyl arginine may interfere with L-arginine uptake into cells by competing with the cationic amino acid transporter (41), but considerably higher concentrations than detected here in the plasma would be required for this effect.
Twenty-foureChour urinary NOx excretion can serve as a qualitative indicator of whole-body NO production, once dietary intake and renal function are controlled for, and this has been shown to be reduced in systemic vascular diseases (42). A recent study in four patients with IPAH has shown results consistent with ours (43). Although reduced pulmonary NO production and/or consumption may be the predominant source of the lower urine NOx, a systemic or renal (12) contribution is not excluded. Plasma NOx has been assessed previously in IPAH with variable results (4, 11). Archer and coworkers (6) reported normal plasma NOx in IPAH, but increased values in fenfluramine-related PAH. The latter had a lower cardiac index and, consequently, lower glomerular filtration rate. There is evidence for diurnal variation in urinary NOx excretion (44). Hence, a 24-hour urine collection provides a more reliable global assessment of NO production than a single random plasma sample.
Changes in Endogenous NO in Response to Bosentan Therapy
We were surprised by the significant increase in FENO (and CW) and urinary NOx in response to bosentan. These changes suggest increased airway and possibly pulmonary and/or bronchial vascular NO production and/or reduced consumption. There is a well-recognized reciprocal relationship between ET and NO (45). Stimulation of ET-B receptors on vascular endothelial cells enhances NO release, whereas activation of ET-A receptors located predominantly on vascular smooth muscle inhibits NO production. Both receptors are abundantly expressed on airway epithelial and smooth muscle cells, mediating a host of biological effects (46). Analogous to the actions of ET in arteries, there is some evidence for the presence of bronchodilatory ET-B receptors on bronchial epithelial cells that release NO (47). ET-1 infusion acutely increased FENO in guinea pigs (48), and bosentan acutely reduced FENO in anesthetized dogs (49). Consistent with our results, however, is the normalization of a reduced FENO in chronically hypoxic pulmonary hypertensive piglets by chronic administration of an ET receptor-A selective antagonist (50). Both in vitro (51) and in vivo (52, 53) studies have demonstrated enhanced NOS activity in response to ET-A or combined ET-A+B receptor blockade. Several mechanisms may be involved, including enhanced transcription of eNOS (52), post-transcriptional regulation (53), and attenuation of the prooxidant effects of ET (54).
Given the small number of patients studied here, our results must be considered preliminary, and more research is required to further characterize the mechanisms accounting for our observations. An important limitation of our study is the lack of data regarding the level of NOx, oxidants, and nitroso-compounds within the epithelial lining fluid, which could be assessed in bronchoalveolar lavage fluid or exhaled breath condensate. Such information would help clarify the basis for our FENO findings. Previous studies have reported reduced NOx in bronchoalveolar lavage fluid (4) and exhaled breath condensate in patients with IPAH (32).
In summary, we have demonstrated that FENO at low expiratory flow rates is markedly reduced in untreated patients with PAH. Partitioning analysis reveals that this reduction is due to decreased airway wall tissue concentrations of NO. This is accompanied by a reduction in whole-body NO production as indicated by 24-hour urinary NOx excretion. Moreover, the ET receptor antagonist bosentan normalized these abnormalities after 3 months of treatment. Decreased production and/or increased consumption of pulmonary NO may contribute to the pathogenesis of PAH, and FENO may serve as a useful biomarker in this challenging disease.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
REFERENCES
Giaid A, Saleh D. Reduced expression of endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension. N Engl J Med 1995;333:214eC221.
Mason NA, Springall DR, Burke M, Pollock J, Mikhail G, Yacoub MH, Polak JM. High expression of endothelial nitric oxide synthase in plexiform lesions of pulmonary hypertension. J Pathol 1998;185:313eC318.
Cremona G, Higenbottam T, Borland C, Mist B. Mixed expired nitric oxide in primary pulmonary hypertension in relation to lung diffusion capacity. QJM 1994;87:547eC551.
Kaneko FT, Arroliga AC, Dweik RA, Comhair SA, Laskowski D, Oppedisano R, Thomassen MJ, Erzurum SC. Biochemical reaction products of nitric oxide as quantitative markers of primary pulmonary hypertension. Am J Respir Crit Care Med 1998;158:917eC923.
Riley MS, Porszasz J, Miranda J, Engelen MP, Brundage B, Wasserman K. Exhaled nitric oxide during exercise in primary pulmonary hypertension and pulmonary fibrosis. Chest 1997;111:44eC50.
Archer SL, Djaballah K, Humbert M, Weir KE, Fartoukh M, Dall'ava-Santucci J, Mercier JC, Simonneau G, Dinh-Xuan AT. Nitric oxide deficiency in fenfluramine- and dexfenfluramine-induced pulmonary hypertension. Am J Respir Crit Care Med 1998;158:1061eC1067.
Ozkan M, Dweik RA, Laskowski D, Arroliga AC, Erzurum SC. High levels of nitric oxide in individuals with pulmonary hypertension receiving epoprostenol therapy. Lung 2001;179:233eC243.
Rhodes P, Leone AM, Francis PL, Struthers AD, Moncada S, Rhodes PM. The L-arginine:nitric oxide pathway is the major source of plasma nitrite in fasted humans. Biochem Biophys Res Commun 1995;209:590eC596.
Wang J, Brown MA, Tam SH, Chan MC, Whitworth JA. Effects of diet on measurement of nitric oxide metabolites. Clin Exp Pharmacol Physiol 1997;24:418eC420.
Wennmalm A, Benthin G, Edlund A, Kieler-Jensen N, Lundin S, Petersson AS, Waagstein F. Nitric oxide synthesis and metabolism in man. Ann N Y Acad Sci 1994;714:158eC164.
Mikhail G, Chester AH, Gibbs JS, Borland JA, Banner NR, Yacoub MH. Role of vasoactive mediators in primary and secondary pulmonary hypertension. Am J Cardiol 1998;82:254eC255.
Baylis C, Vallance P. Measurement of nitrite and nitrate levels in plasma and urine: what does this measure tell us about the activity of the endogenous nitric oxide system Curr Opin Nephrol Hypertens 1998;7:59eC62.
Silkoff PE, Sylvester JT, Zamel N, Permutt S. Airway nitric oxide diffusion in asthma: role in pulmonary function and bronchial responsiveness. Am J Respir Crit Care Med 2000;161:1218eC1228.
Lundberg JO, Weitzberg E. Nasal nitric oxide in man. Thorax 1999;54:947eC952.
Cooke JP. Asymmetrical dimethylarginine: the uber marker Circulation 2004;109:1813eC1818.
Girgis RE, Mozamell S, Hassoun PM, Permutt S, Sylvester JT. Endogenous nitric oxide in primary pulmonary hypertension: response to bosentan therapy . Am J Respir Crit Care Med 2004;169:A393.
Rich S, Dantzker DR, Ayres SM, Bergofsky EH, Brundage BH, Detre KM, Fishman AP, Goldring RM, Groves BM, Koerner SK. Primary pulmonary hypertension: a national prospective study. Ann Intern Med 1987;107:216eC223.
McGoon M, Gutterman D, Steen V, Barst R, McCrory DC, Fortin TA, Loyd JE. Screening, early detection, and diagnosis of pulmonary arterial hypertension: ACCP evidence-based clinical practice guidelines. Chest 2004;126:14SeC34S.
American Thoracic Society. ATS statement: guidelines for the six-minute walk test. Am J Respir Crit Care Med 2002;166:111eC117.
Evans TG, Rasmussen K, Wiebke G, Hibbs JB Jr. Nitric oxide synthesis in patients with advanced HIV infection. Clin Exp Immunol 1994;97:83eC86.
Yang BK, Vivas EX, Reiter CD, Gladwin MT. Methodologies for the sensitive and specific measurement of S-nitrosothiols, iron-nitrosyls, and nitrite in biological samples. Free Radic Res 2003;37:1eC10.
Martens-Lobenhoffer J, Bode-Boger SM. Simultaneous detection of arginine, asymmetric dimethylarginine, symmetric dimethylarginine and citrulline in human plasma and urine applying liquid chromatography-mass spectrometry with very straightforward sample preparation. J Chromatogr B Analyt Technol Biomed Life Sci 2003;798:231eC239.
American Thoracic Society. Recommendations for standardized procedures for the on-line and off-line measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide in adults and children-1999. Am J Respir Crit Care Med 1999;160:2104eC2117.
Simonneau G, Fartoukh M, Sitbon O, Humbert M, Jagot JL, Herve P. Primary pulmonary hypertension associated with the use of fenfluramine derivatives. Chest 1998;114:195SeC199S.
Tuder RM, Radisavljevic Z, Shroyer KR, Polak JM, Voelkel NF. Monoclonal endothelial cells in appetite suppressant-associated pulmonary hypertension. Am J Respir Crit Care Med 1998;158:1999eC2001.
Tsang KW, Ip SK, Leung R, Tipoe GL, Chan SL, Shum IH, Ip MS, Yan C, Fung PC, Chan-Yeung M, et al. Exhaled nitric oxide: the effects of age, gender and body size. Lung 2001;179:83eC91.
Forrest IA, Small T, Corris PA. Effect of nebulized epoprostenol (prostacyclin) on exhaled nitric oxide in patients with pulmonary hypertension due to congenital heart disease and in normal controls. Clin Sci (Lond) 1999;97:99eC102.
George SC, Hogman M, Permutt S, Silkoff PE. Modeling pulmonary nitric oxide exchange. J Appl Physiol 2004;96:831eC839.
Sartori C, Lepori M, Busch T, Duplain H, Hildebrandt W, Bartsch P, Nicod P, Falke KJ, Scherrer U. Exhaled nitric oxide does not provide a marker of vascular endothelial function in healthy humans. Am J Respir Crit Care Med 1999;160:879eC882.
Vaughan DJ, Brogan TV, Kerr ME, Deem S, Luchtel DL, Swenson ER. Contributions of nitric oxide synthase isozymes to exhaled nitric oxide and hypoxic pulmonary vasoconstriction in rabbit lungs. Am J Physiol Lung Cell Mol Physiol 2003;284:L834eCL843.
Tworetzky W, Moore P, Bekker JM, Bristow J, Black SM, Fineman JR. Pulmonary blood flow alters nitric oxide production in patients undergoing device closure of atrial septal defects. J Am Coll Cardiol 2000;35:463eC467.
Machado RF, Londhe Nerkar MV, Dweik RA, Hammel J, Janocha A, Pyle J, Laskowski D, Jennings C, Arroliga AC, Erzurum SC. Nitric oxide and pulmonary arterial pressures in pulmonary hypertension. Free Radic Biol Med 2004;37:1010eC1017.
Ide H, Nakano H, Ogasa T, Osanai S, Kikuchi K, Iwamoto J. Regulation of pulmonary circulation by alveolar oxygen tension via airway nitric oxide. J Appl Physiol 1999;87:1629eC1636.
Lane C, Knight D, Burgess S, Franklin P, Horak F, Legg J, Moeller A, Stick S. Epithelial inducible nitric oxide synthase activity is the major determinant of nitric oxide concentration in exhaled breath. Thorax 2004;59:757eC760.
Xu W, Kaneko FT, Zheng S, Comhair SA, Janocha AJ, Goggans T, Thunnissen FB, Farver C, Hazen SL, Jennings C, et al. Increased arginase II and decreased NO synthesis in endothelial cells of patients with pulmonary arterial hypertension. FASEB J 2004;18:1746eC1748.
Cracowski JL, Cracowski C, Bessard G, Pepin JL, Bessard J, Schwebel C, Stanke-Labesque F, Pison C. Increased lipid peroxidation in patients with pulmonary hypertension. Am J Respir Crit Care Med 2001;164:1038eC1042.
Bowers R, Cool C, Murphy RC, Tuder RM, Hopken MW, Flores SC, Voelkel NF. Oxidative stress in severe pulmonary hypertension. Am J Respir Crit Care Med 2004;169:764eC769.
Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Physiol 1996;271:C1424eCC1437.
Boger RH. The emerging role of asymmetric dimethylarginine as a novel cardiovascular risk factor. Cardiovasc Res 2003;59:824eC833.
Hoeper MM, Bode-Boger SM, Schubert B, Gatzke R, Takacs A, Fliser D, Kielstein J. Plasma levels of asymmetric dimethylarginine (ADMA) and L-arginine in patients with primary pulmonary hypertension . Am J Respir Crit Care Med 2003;167:A842.
Closs EI, Basha FZ, Habermeier A, Forstermann U. Interference of L-arginine analogues with L-arginine transport mediated by the y+ carrier hCAT-2B. Nitric Oxide 1997;1:65eC73.
Boger RH, Bode-Boger SM, Thiele W, Junker W, Alexander K, Frolich JC. Biochemical evidence for impaired nitric oxide synthesis in patients with peripheral arterial occlusive disease. Circulation 1997;95:2068eC2074.
Demoncheaux EA, Higenbottam TW, Kiely DG, Wong JM, Wharton S, Varcoe R, Siddons T, Spivey AC, Hall K, Gize AP. Decreased whole body endogenous nitric oxide production in patients with primary pulmonary hypertension. J Vasc Res 2005;42:133eC136.
Borgonio A, Witte K, Stahrenberg R, Lemmer B. Influence of circadian time, ageing, and hypertension on the urinary excretion of nitric oxide metabolites in rats. Mech Ageing Dev 1999;111:23eC37.
Rossi GP, Seccia TM, Nussdorfer GG. Reciprocal regulation of endothelin-1 and nitric oxide: relevance in the physiology and pathology of the cardiovascular system. Int Rev Cytol 2001;209:241eC272.
Henry PJ. Endothelin receptor distribution and function in the airways. Clin Exp Pharmacol Physiol 1999;26:162eC167.
Mazzoni MR, Breschi MC, Ceccarelli F, Lazzeri N, Giusti L, Nieri P, Lucacchini A. Suc-[Glu9,Ala11,15]-endothelin-1 (8eC21), IRL 1620, identifies two populations of ET(B) receptors in guinea-pig bronchus. Br J Pharmacol 1999;127:1406eC1414.
Malmstrom RE, Tornberg DC, Settergren G, Liska J, Angdin M, Lundberg JO, Weitzberg E. Endogenous nitric oxide release by vasoactive drugs monitored in exhaled air. Am J Respir Crit Care Med 2003;168:114eC120.
Hubloue I, Biarent D, Abdel KS, Bejjani G, Kerbaul F, Naeije R, Leeman M. Endogenous endothelins and nitric oxide in hypoxic pulmonary vasoconstriction. Eur Respir J 2003;21:19eC24.
Perreault T, Berkenbosch JW, Barrington KJ, Decker ER, Wu C, Brock TA, Baribeau J. TBC3711, an ET(A) receptor antagonist, reduces neonatal hypoxia-induced pulmonary hypertension in piglets. Pediatr Res 2001;50:374eC383.
Ikeda U, Yamamoto K, Maeda Y, Shimpo M, Kanbe T, Shimada K. Endothelin-1 inhibits nitric oxide synthesis in vascular smooth muscle cells. Hypertension 1997;29:65eC69.
Maeda S, Miyauchi T, Iemitsu M, Tanabe T, Goto K, Yamaguchi I, Matsuda M. Endothelin receptor antagonist reverses decreased NO system in the kidney in vivo during exercise. Am J Physiol Endocrinol Metab 2004;286:E609eCE614.
Taner CB, Severson SR, Best PJ, Lerman A, Miller VM. Treatment with endothelin-receptor antagonists increases NOS activity in hypercholesterolemia. J Appl Physiol 2001;90:816eC820.
Wedgwood S, Black SM. Endothelin-1 decreases endothelial NOS expression and activity through ETA receptor-mediated generation of hydrogen peroxide. Am J Physiol Lung Cell Mol Physiol 2005;288:L480eCL487.