The NO donor molsidomine reduces endothelin-1 gene expression in chronic hypoxic rat lungs

Friedrich C. Blumberg1, Konrad Wolf2, Peter Sandner2, Cornelia Lorenz1, Günter A. J. Riegger1, and Michael Pfeifer1

1 Department of Internal Medicine II and 2 Institute of Physiology, University of Regensburg, 93042 Regensburg, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We investigated the effects of the nitric oxide (NO) donor molsidomine and the nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) on pulmonary endothelin (ET)-1 gene expression and ET-1 plasma levels in chronic hypoxic rats. Two and four weeks of hypoxia (10% O2) significantly increased right ventricular systolic pressure, the medial cross-sectional vascular wall area of the pulmonary arteries, and pulmonary ET-1 mRNA expression (2-fold and 3.2-fold, respectively). ET-1 plasma levels were elevated after 4 wk of hypoxia. In rats exposed to 4 wk of hypoxia, molsidomine (15 mg · kg-1 · day-1) given either from the beginning or after 2 wk of hypoxia significantly reduced pulmonary hypertension, pulmonary vascular remodeling, pulmonary ET-1 gene expression, and ET-1 plasma levels. L-NAME administration (45 mg · kg-1 · day-1) in rats subjected to 2 wk of hypoxia did not modify these parameters. Our findings suggest that in chronic hypoxic rats, exogenously administered NO acts in part by suppressing the formation of ET-1. In contrast, inhibition of endogenous NO production exerts only minor effects on the pulmonary circulation and pulmonary ET-1 synthesis in these animals.

pulmonary hypertension; chronic hypoxia; nitric oxide


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE ENDOTHELIUM PLAYS an important role in the regulation of pulmonary vascular tone and structure. Several lines of evidence suggest that an imbalance between the release of endothelium-derived vasodilators such as the endothelium-derived relaxing factor nitric oxide (NO) and vasoconstrictors such as endothelin (ET)-1 contributes significantly to the hemodynamic and structural alterations of the pulmonary circulation in both patients and experimental animals with pulmonary hypertension.

In the vascular endothelium, NO is formed from L-arginine by the enzyme nitric oxide synthase (NOS) (18). In addition to its role as a vasodilator, NO inhibits the adherence of platelets and leukocytes to the endothelium (21, 26) and suppresses the proliferation of vascular smooth muscle cells (5). Inhalation of NO (19) as well as the short-term intravenous administration of L-arginine, thereby increasing the endogenous production of NO (16), has been shown to reduce pulmonary vascular tone in patients with primary or secondary pulmonary hypertension. Moreover, prolonged inhalation of NO (13, 23) and long-term administration of L-arginine attenuate pulmonary hypertension and pulmonary vascular remodeling in chronic hypoxic and monocrotaline (MCT)-treated rats (17). Recently, it has also been demonstrated that a continuous subcutaneous infusion of the NO donor molsidomine (25) ameliorates the development of pulmonary hypertension in MCT-treated rats (15).

In vitro experiments have shown that NO inhibits the hypoxia-induced expression of ET-1 (12), a potent endothelium-derived vasoconstrictor (31) and vascular smooth muscle cell growth factor (30), whereas NOS inhibition augments the release of ET-1 in cultured endothelial cells (1). ET-1 plasma levels and lung ET-1 mRNA expression are increased in both patients (6) and experimental animals (14) with pulmonary hypertension. Moreover, ET receptor antagonists have been found to attenuate pulmonary vascular tone and pulmonary vascular remodeling in hypoxic and MCT-treated rats (3, 4, 9), underscoring the crucial role of ET-1 in the pathogenesis of pulmonary hypertension.

Furthermore, it has been reported that L-arginine reduced, whereas the NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME) increased, the amount of ET-1 mRNA in the pulmonary arteries of rats exposed to intermittent hypoxia for 6 h/day for a period of 1 or 2 wk as determined by in situ hybridization (10). However, ET-1 plasma levels as well as hemodynamic or structural effects of the intermittent hypoxic exposure and pharmacological interventions were not determined in this study (10). Nevertheless, based on these observations, we hypothesized that the vasodilatory and antiproliferative effects of therapeutic intervention with exogenously administered NO in pulmonary hypertension are, in part, promoted by suppressing the formation of ET-1. Furthermore, we speculated that inhibition of endogenous NO synthesis would increase pulmonary ET-1 production.

To test these hypotheses, we examined the effects of the orally active NO donor molsidomine on the development of pulmonary hypertension, pulmonary vascular remodeling, pulmonary ET-1 mRNA gene expression, and circulating ET-1 levels in chronic hypoxic rats in vivo. In addition, we investigated whether inhibition of the endogenous NO production by the NOS inhibitor L-NAME would modify pulmonary hypertension and ET-1 formation.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental groups and chronic hypoxia. Adult male Wistar rats (250-300 g; Charles River, Sulzfeld, Germany) were randomly assigned to one of the following groups: normoxia and plain water from week 1 through week 4 (n = 5); chronic hypoxia and plain water from week 1 through week 2 (n = 5); chronic hypoxia and plain water from week 1 through week 4 (n = 5); chronic hypoxia and molsidomine (15 mg · kg-1 · day-1; Hoechst Marion Roussel, Bad Soden, Germany) in the drinking water from week 1 through week 4 (n = 5); chronic hypoxia from week 1 through week 4 and molsidomine from week 3 through week 4 of hypoxia (n = 5); and normoxia or chronic hypoxia and L-NAME (45 mg · kg-1 · day-1; Sigma-Aldrich, Steinheim, Germany) from week 1 through week 2 (n = 5 per group).

Rats were exposed to normobaric hypoxia (10% O2-90% N2) in transparent plastic chambers as previously described (20). Relative humidity within the chamber was kept at <70% with anhydrous CaSO2. Boric acid was used to keep NH3 levels within in the chamber at a minimum. The percentage of CO2 within the chamber was measured daily and did not exceed 0.3%. Normoxic rats were housed in identical cages adjacent to the chambers in the same room and breathed room air. Animal experiments were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and German laws on the protection of animals.

Hemodynamic measurements. Systolic blood pressure was measured in all rats the day before the end of the study period under prewarmed and normoxic conditions by the tail cuff method (BP monitor TSE 210 000-2T, Technical and Scientific Equipment, Kronberg, Germany).

At the end of the study period, right ventricular systolic pressure (RVSP) was measured in all rats with a closed-chest technique as previously described (20). For this purpose, each rat was taken from its cage and immediately anesthetized with thiopental sodium (50 mg/kg ip). Via a nose mask, the lungs were artificially ventilated with room air with a rodent respirator (Technical and Scientific Equipment). The right jugular vein was cannulated, and a catheter was introduced into the right ventricle. The system was filled and flushed with <2 ml of heparin solution (1,000 IU/ml). After a stable hemodynamic condition was reached, RVSP was measured with a pressure transducer (P23Db, Statham Laboratories, Hato Rey, Puerto Rico).

Organ sampling. Immediately after the hemodynamic measurements were made, the animals were killed by decapitation. Blood was collected from the carotid arteries. The heart and lungs were removed, and the pulmonary artery was perfused with 0.9% saline until the solution obtained was clear. The right lung was dissected and frozen in liquid nitrogen until RNA extraction. For morphological measurements, the left lung was fixed in the distended state by the infusion of 10% buffered formalin into the pulmonary artery and trachea at 10 and 25 cmH2O perfusion pressure, respectively, for 3 min and kept in 10% buffered formalin for at least 24 h. The lungs were then embedded in paraffin, and 5-µm sections were stained with Van Gieson's stain.

Light-microscopic analysis of pulmonary arteries. Microscopic slices were analyzed with a computerized morphometric system (AnalySIS, Soft-Imaging Software, Muenster, Germany). Total vessel area was defined as the area within the elastica externa. Medial area was defined as the area between the lamina elastica externa and the lamina elastica interna. Pulmonary arteries with an external diameter ranging between 30 and 100 µm were examined. Thirty arteries per animal were measured. The average of three measurements obtained from each artery was used for calculations. Slides were analyzed by two observers who were blinded for the modality of treatment. Variability was assessed by performing repeated analyses and was calculated as 5 (intraobserver) and 7% (interobserver).

RNA extraction. After homogenization of the tissue in solution D (4 M guanidine thiocyanate containing 0.5% N-laurylsarcosinate, 10 mmol/l of EDTA, 25 mmol/l of sodium citrate, and 700 mmol/l of beta -mercaptoethanol), a one-tenth volume of 2 M sodium acetate (pH 4), 1 volume of phenol (water saturated), and a one-fifth volume of chloroform were added sequentially to the homogenate. After being cooled on ice for 15 min, the samples were centrifuged at 10,000 g for 15 min at 4°C. RNA in the supernatant was precipitated with an equal volume of isopropanol at -20°C for at least 1 h. The resulting RNA pellets were resuspended in 0.5 ml of solution D and again precipitated with an equal volume of isopropanol at -20°C. The pellets were finally dissolved in diethyl pyrocarbonate-treated water and stored at -80°C until further processing (2).

Quantification of ET-1 and glyceraldehyde-3-phosphate dehydrogenase mRNAs. ET-1 mRNA was measured by RNase protection assay as previously described (22). The plasmid containing the antisense sequence of ET-1 was a friendly gift from Peter Ratcliffe (Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK). It yields a 154-bp-long protected fragment in the RNase protection assay. Twenty micrograms of total lung RNA were used for the ET assays. For normalization of the ET values, we constructed a transcription vector producing a 341-bp antisense RNA fragment of rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (27). To minimize discrepancies due to RNA quantity and quality, each assayed probe was coanalyzed for GAPDH mRNA. The vector producing the GAPDH antisense fragment used in RNase protection assay was cloned as follows. The PCR-derived fragment resulting from amplification with the upstream (5'-acctgaagggtggtgcca-3'; binding at bp 356-373) and downstream primers (5'-tcagctctgggatgacct-3'; binding at bp 680-697) was cloned in the transcription vector pGEM-4Z (Pharmacia, Heidelberg, Germany). One microgram of total lung RNA was used for the GAPDH assays. After autoradiography of the dried gel, counts per minute of the protected fragments were detected and counted with a phosphorimager (Instant Imager 2024, Canberra-Packard, Dreieich, Germany).

Measurement of plasma ET-1 levels. Plasma concentrations of ET-1 were measured with a commercially available radioimmunoassay (Amersham International, Amersham, UK). Extraction of plasma was performed with the standard technique as described elsewhere (7).

Statistical analysis. ET-1 mRNA values are means ± SE. All other values are means ± SD. ANOVA followed by Student's unpaired t-test was used for interindividual comparisons. A value of P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hematocrit and hemodynamic measurements. The hematocrit was significantly elevated in hypoxic animals, indicating the effectiveness of the hypoxic model used in the present study. Molsidomine and L-NAME treatment had no effect on the hematocrit compared with that in the respective hypoxic control group (Table 1).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Hematocrit, hemodynamic measurements, ET-1 plasma levels, and medial vessel wall area

As shown in Table 1, hypoxia did not significantly affect systemic arterial blood pressure. Molsidomine did not significantly reduce arterial blood pressure. L-NAME significantly increased systolic blood pressure in both normoxic and hypoxic animals (Table 1).

Two and four weeks of hypoxia significantly increased RVSP compared with that in normoxic control animals (Table 1). Molsidomine significantly attenuated the hypoxia-induced pulmonary hypertension in both treatment groups, whereas L-NAME did not modify RVSP (Table 1).

Morphological studies. Pulmonary arteries from chronically hypoxic rats showed a significant increase in the medial cross-sectional vascular wall area compared with that in normoxic control rats (Table 1). Molsidomine treatment significantly reduced medial vascular wall hypertrophy (Table 1). L-NAME had no effect on the structure of the pulmonary arteries of rats exposed to normoxia or chronic hypoxia (Table 1).

ET-1 plasma levels. ET-1 plasma levels of all study groups are presented in Table 1. ET-1 plasma levels were significantly increased only in rats exposed to 4 wk of hypoxia (Table 1). Molsidomine significantly reduced circulating ET-1 levels in both treatment groups compared with the group exposed solely to 4 wk of hypoxia (Table 1). L-NAME had no significant influence on ET-1 plasma levels (Table 1).

Pulmonary GAPDH and ET-1 gene expression. Pulmonary GAPDH mRNA expression did not significantly change in the different study groups. Figure 1 shows a representative autoradiograph of the GAPDH mRNA expression in normoxic, chronic hypoxic, and molsidomine-treated rats.


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 1.   Autoradiograph of a representative RNase protection assay showing pulmonary glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in normoxic control rats (lane a), in rats exposed to 2 (lane b) and 4 (lane c) wk of hypoxia, and in rats exposed to 4 wk of hypoxia and receiving molsidomine from either week 1 through week 4 (lane d) or week 3 through week 4 (lane e) of hypoxia. One microgram of total lung RNA was used for the assay. For each group, 2 samples are shown.

After 2 and 4 wk of hypoxia, pulmonary ET-1 mRNA increased significantly (Figs. 2 and 3). Molsidomine treatment from week 1 through week 4 of hypoxia as well as from week 3 through week 4 of hypoxia significantly reduced ET-1 mRNA expression compared with rats exposed to 4 wk of hypoxia (Figs. 2 and 3). In contrast, L-NAME treatment had no effect on pulmonary ET-1 expression in normoxic (data not shown) and chronic hypoxic animals (Figs. 4 and 5).


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 2.   Autoradiograph of a representative RNase protection assay showing pulmonary endothelin (ET)-1 mRNA expression in normoxic control rats (lane a), in rats exposed to 2 (lane b) and 4 (lane c) wk of hypoxia, and in rats exposed to 4 wk of hypoxia and receiving molsidomine from either week 1 through week 4 (lane d) or week 3 through week 4 (lane e) of hypoxia. Twenty micrograms of total lung RNA were used for the assay.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 3.   Changes in pulmonary ET-1 mRNA expression. Norm, normoxic control rats (=100%); 2wHyp, rats exposed to 2 wk of hypoxia; 4wHyp, rats exposed to 4 wk of hypoxia; 4wHyp+MDw1-4, rats exposed to 4 wk of hypoxia and receiving molsidomine from week 1 through week 4; 4wHyp+MDw3-4, rats exposed to 4 wk of hypoxia and receiving molsidomine from week 3 through week 4. Values are means ± SE; n = 5 rats/group. * P < 0.05 vs. Norm. # P < 0.05 vs. 4wHyp.



View larger version (58K):
[in this window]
[in a new window]
 
Fig. 4.   Autoradiograph of a representative RNase protection assay showing pulmonary ET-1 mRNA expression in normoxic control rats (lane a), in rats exposed to 2 wk of hypoxia (lane b), and in rats exposed to 2 wk of hypoxia and receiving N-nitro-L-arginine methyl ester (LN; lane c). Twenty micrograms of total lung RNA were used for the assay.



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 5.   Changes in pulmonary ET-1 mRNA expression. Values are means ± SE; n = 5 rats/group. * P < 0.05 vs. Norm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study describes for the first time that in rats exposed to chronic hypoxia, the orally active NO donor molsidomine ameliorates the development of both pulmonary hypertension as indicated by the reduction in RVSP and pulmonary vascular remodeling as indicated by a decrease in the medial cross-sectional vascular wall area of pulmonary arteries. These beneficial effects are comparable to those observed during chronic NO inhalation (13, 23) or prolonged administration of L-arginine (17), a precursor of endogenous NO production.

The effects of long-term NO administration in rats with already established pulmonary hypertension have not yet been investigated. In the present study, we were able to show that molsidomine prevented a further increase in RVSP and pulmonary vascular remodeling in animals that were first exposed to 2 wk of hypoxia and then treated with molsidomine during an additional 2 wk of hypoxia. Although molsidomine did not reverse the hemodynamic and structural alterations induced by the first 2 wk of hypoxia, our data indicate that prolonged NO administration is effective in preventing further progression of chronic hypoxic pulmonary hypertension. On the other hand, confirming the findings of Hampl et al. (8), NOS inhibition did not alter RVSP or the structure of the pulmonary arteries in our experiments.

Endothelial cells play a key role in the local regulation of vascular resistance via the release of vasoactive factors, enhancing or reducing smooth muscle cell proliferation and contractility. Consistent with a previous report (14), we found a significant increase in pulmonary ET-1 mRNA expression in hypertensive rat lungs compared with normoxic control lungs. Despite the increase in pulmonary ET-1 gene expression after 2 wk of hypoxia, ET-1 plasma levels significantly increased only in the group of rats exposed to 4 wk of hypoxia. However, because ET-1 acts predominantly in a paracrine fashion and endothelial ET-1 secretion is mainly directed to the smooth muscle cells, only part of the ET-1 produced is secreted into the circulation (29). Therefore, enhancement of ET-1 gene expression may not inevitably be followed by a measurable increase in plasma ET-1 levels.

The interactions between the different vasoactive factors are not well understood. An in vitro study by Boulanger and Luscher (1) showed that the thrombin-stimulated release of ET-1 from the intact porcine aorta is inhibited by cGMP, the second messenger of NO, and potentiated by NOS inhibition. Saijonmaa et al. (24) reported that NO delivered exogenously by nitroprusside or nitroglycerin reduced the basal and thrombin-stimulated production of ET-1 by cultured human endothelial cells. Moreover, Kourembanas et al. (12) demonstrated in these same cells that nitroprusside suppresses the hypoxia-induced production of ET-1 at the level of gene expression, whereas, conversely, NOS inhibition increased ET-1 gene expression. Recently, it has also been shown (10) that L-arginine treatment reduced, whereas L-NAME administration enhanced, ET-1 mRNA expression in the pulmonary vasculature of rats subjected to intermittent hypoxia.

We were now able to demonstrate that in chronic hypoxic rats in vivo, exogenously administered NO suppresses both pulmonary ET-1 gene expression and circulating ET-1 plasma levels and that the reduction in ET-1 formation is associated with hemodynamic and structural improvement. These effects could be observed in rats treated from the beginning of hypoxia as well as in rats with already established hypoxic pulmonary hypertension.

In contrast to the above-mentioned reports, inhibition of endogenous NO production by L-NAME did not modify pulmonary ET-1 gene expression in our experiments. Whether these contradictory results are due to methodological differences, particularly intermittent versus chronic hypoxia, needs to be elucidated by further study. Moreover, although systolic blood pressure was elevated in L-NAME-treated animals, indicating the effectiveness of NOS inhibition in the systemic circulation, we cannot rule out that the pulmonary circulation is less sensitive to L-NAME, and thus pulmonary NOS inhibition might have been partially ineffective in our series. Another possible limitation is that L-NAME treatment lasted only 2 wk. We selected this time period because after 2 wk of hypoxia, rats already exhibit pulmonary hypertension and increased pulmonary ET-1 gene expression. However, the hemodynamic changes and alterations in gene expression have not yet reached their maximum, and thus an L-NAME-induced increase in pulmonary hypertension or ET-1 gene expression should be easier to detect after 2 wk than after 4 wk of hypoxia. Moreover, in the study by Hampl et al. (8), animals were treated for 3 wk with L-NAME and no effects on pulmonary circulation could be observed. Therefore, it seems unlikely that a prolongation of treatment would have changed the results in our study.

Although the pulmonary circulation seems to be less sensitive against inhibition of the endogenous NO synthesis, it is well established that in pulmonary hypertension, exogenously administered NO causes pulmonary vasodilatation and a reduction in pulmonary vascular remodeling (13, 19, 23). The mechanisms underlying these potentially contradictory findings remain unclear, and further study is needed to elucidate this point. Furthermore, NO or NO donors probably act not solely by reducing ET-1 production. They may also promote some of their effects by altering the redox state of the cell (11) or by stimulating other mediators such as prostaglandins (28). However, we did not investigate these possibilities in our study.

Furthermore, a limitation of the present study is that we did not measure cardiac output because Hampl et al. (8) described a slightly reduced cardiac index during L-NAME treatment. However, in the study by Hampl et al. as well as in our experiments, pulmonary hypertension and pulmonary vascular remodeling did not change significantly during L-NAME treatment compared with those in hypoxic control animals. Thus the determination of cardiac output would not have made any difference in the interpretation of our results.

In conclusion, our findings suggest that exogenously administered NO attenuates chronic hypoxic pulmonary hypertension in adult rats in part via a reduction of pulmonary ET-1 production. In contrast, inhibition of the endogenous NO formation by L-NAME induces systemic arterial hypertension but exerts only minor effects on the pulmonary circulation and pulmonary ET-1 synthesis, indicating that in these animals under in vivo conditions, the pulmonary circulation is less sensitive to NOS inhibition than the systemic circulation.


    ACKNOWLEDGEMENTS

We thank Prof. A. Kurtz (Institute of Physiology, University of Regensburg, Regensburg, Germany) for critical reading of the manuscript and helpful suggestions. The expert technical assistance provided by Dr. F. Muders, M. Hamann and K. H. Götz is gratefully acknowledged.


    FOOTNOTES

Address for reprint requests and other correspondence: F. C. Blumberg, Dept. of Internal Medicine II, Univ. of Regensburg, 93042 Regensburg, Germany (E-mail friedrich.blumberg{at}klinik.uni-regensburg.de).

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.

Received 11 May 2000; accepted in final form 25 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Boulanger, C, and Luscher TF. Release of endothelin from the porcine aorta: inhibition by endothelium-derived nitric oxide. J Clin Invest 85: 587-590, 1990[ISI][Medline].

2.   Chomczynski, P, and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159, 1987[ISI][Medline].

3.   DiCarlo, VS, Chen SJ, Meng QC, Durand J, Yano M, Chen YF, and Oparil S. ETA-receptor antagonist prevents and reverses chronic hypoxia-induced pulmonary hypertension in rat. Am J Physiol Lung Cell Mol Physiol 269: L690-L697, 1995[Abstract/Free Full Text].

4.   Eddahibi, S, Raffestin B, Clozel M, Levame M, and Adnot S. Protection from pulmonary hypertension with an orally active endothelin receptor antagonist in hypoxic rats. Am J Physiol Heart Circ Physiol 268: H828-H835, 1995[Abstract/Free Full Text].

5.   Garg, UC, and Hassid A. Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest 83: 1774-1777, 1989[ISI][Medline].

6.   Giaid, A, Yanagisawa M, Langleben D, Michel RP, Levy R, Shennib H, Kimura S, Masaki T, Duguid WP, and Stewart DJ. Expression of endothelin-1 in the lungs of patients with pulmonary hypertension. N Engl J Med 328: 1732-1739, 1993[Abstract/Free Full Text].

7.   Gulberg, V, Gerbes AL, Vollmar AM, and Paumgartner G. Endothelin-3 like immunoreactivity in plasma of patients with cirrhosis of the liver. Life Sci 51: 1165-1169, 1992[ISI][Medline].

8.   Hampl, V, Archer SL, Nelson DP, and Weir EK. Chronic EDRF inhibition and hypoxia: effects on pulmonary circulation and systemic blood pressure. J Appl Physiol 75: 1748-1757, 1993[Abstract].

9.   Hill, NS, Warburton RR, Pietras L, and Klinger JR. Nonspecific endothelin-receptor antagonist blunts monocrotaline-induced pulmonary hypertension in rats. J Appl Physiol 83: 1209-1215, 1997[Abstract/Free Full Text].

10.   Junbao, D, Jianfeng J, Wanzhen L, Bin Z, and Heping Z. Nitric oxide impacts endothelin-1 gene expression in intrapulmonary arteries of chronically hypoxic rats. Angiology 50: 479-485, 1999[ISI][Medline].

11.   Kirsch, M, and de Groot H. Reaction of peroxynitrite with reduced nicotinamide nucleotides, the formation of hydrogen peroxide. J Biol Chem 274: 24664-24670, 1999[Abstract/Free Full Text].

12.   Kourembanas, S, McQuillan LP, Leung GK, and Faller DV. Nitric oxide regulates the expression of vasoconstrictors and growth factors by vascular endothelium under both normoxia and hypoxia. J Clin Invest 92: 99-104, 1993[ISI][Medline].

13.   Kouyoumdjian, C, Adnot S, Levame M, Eddahibi S, Bousbaa H, and Raffestin B. Continuous inhalation of nitric oxide protects against development of pulmonary hypertension in chronically hypoxic rats. J Clin Invest 94: 578-584, 1994[ISI][Medline].

14.   Li, H, Chen SJ, Chen YF, Meng QC, Durand J, Oparil S, and Elton TS. Enhanced endothelin-1 and endothelin receptor gene expression in chronic hypoxia. J Appl Physiol 77: 1451-1459, 1994[Abstract/Free Full Text].

15.   Mathew, R, Gloster ES, Sundararajan T, Thompson CI, Zeballos GA, and Gewitz MH. Role of inhibition of nitric oxide production in monocrotaline-induced pulmonary hypertension. J Appl Physiol 82: 1493-1498, 1997[Abstract/Free Full Text].

16.   Mehta, S, Stewart DJ, Langleben D, and Levy RD. Short-term pulmonary vasodilation with L-arginine in pulmonary hypertension. Circulation 92: 1539-1545, 1995[Abstract/Free Full Text].

17.   Mitani, Y, Maruyama K, and Sakurai M. Prolonged administration of L-arginine ameliorates chronic pulmonary hypertension and pulmonary vascular remodeling in rats. Circulation 96: 689-697, 1997[Abstract/Free Full Text].

18.   Moncada, S, and Higgs A. The L-arginine-nitric oxide pathway. N Engl J Med 329: 2002-2012, 1993[Free Full Text].

19.   Pepke-Zaba, J, Higenbottam TW, Dinh-Xuan AT, Stone D, and Wallwork J. Inhaled nitric oxide as a cause of selective pulmonary vasodilatation in pulmonary hypertension. Lancet 338: 1173-1174, 1991[ISI][Medline].

20.   Pfeifer, M, Wolf K, Blumberg FC, Elsner D, Muders F, Holmer SR, Riegger GA, and Kurtz A. ANP gene expression in rat hearts during hypoxia. Pflügers Arch 434: 63-69, 1997[ISI][Medline].

21.   Radomski, MW, Palmer RM, and Moncada S. The role of nitric oxide and cGMP in platelet adhesion to vascular endothelium. Biochem Biophys Res Commun 148: 1482-1489, 1987[ISI][Medline].

22.   Ritthaler, T, Gopfert T, Firth JD, Ratcliffe PJ, Kramer BK, and Kurtz A. Influence of hypoxia on hepatic and renal endothelin gene expression. Pflügers Arch 431: 587-593, 1996[ISI][Medline].

23.   Roberts, JD, Jr, Roberts CT, Jones RC, Zapol WM, and Bloch KD. Continuous nitric oxide inhalation reduces pulmonary arterial structural changes, right ventricular hypertrophy, and growth retardation in the hypoxic newborn rat. Circ Res 76: 215-222, 1995[Abstract/Free Full Text].

24.   Saijonmaa, O, Ristimaki A, and Fyhrquist F. Atrial natriuretic peptide, nitroglycerine, and nitroprusside reduce basal and stimulated endothelin production from cultured endothelial cells. Biochem Biophys Res Commun 173: 514-520, 1990[ISI][Medline].

25.   Schutte, H, Grimminger F, Otterbein J, Spriestersbach R, Mayer K, Walmrath D, and Seeger W. Efficiency of aerosolized nitric oxide donor drugs to achieve sustained pulmonary vasodilation. J Pharmacol Exp Ther 282: 985-994, 1997[Abstract/Free Full Text].

26.   Tsao, PS, Lewis NP, Alpert S, and Cooke JP. Exposure to shear stress alters endothelial adhesiveness. Role of nitric oxide. Circulation 92: 3513-3519, 1995[Abstract/Free Full Text].

27.   Tso, JY, Sun XH, Kao TH, Reece KS, and Wu R. Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene. Nucleic Acids Res 13: 2485-2502, 1985[Abstract].

28.   Upmacis, RK, Deeb RS, and Hajjar DP. Regulation of prostaglandin H2 synthase activity by nitrogen oxides. Biochemistry 38: 12505-12513, 1999[ISI][Medline].

29.   Wagner, OF, Christ G, Wojta J, Vierhapper H, Parzer S, Nowotny PJ, Schneider B, Waldhausl W, and Binder BR. Polar secretion of endothelin-1 by cultured endothelial cells. J Biol Chem 267: 16066-16068, 1992[Abstract/Free Full Text].

30.   Weissberg, PL, Witchell C, Davenport AP, Hesketh TR, and Metcalfe JC. The endothelin peptides ET-1, ET-2, ET-3 and sarafotoxin S6b are co-mitogenic with platelet-derived growth factor for vascular smooth muscle cells. Atherosclerosis 85: 257-262, 1990[ISI][Medline].

31.   Yanagisawa, M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, and Masaki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332: 411-415, 1988[ISI][Medline].


Am J Physiol Lung Cell Mol Physiol 280(2):L258-L263
1040-0605/01 $5.00 Copyright © 2001 the American Physiological Society