1 Department of Internal Medicine II and 2 Institute of Physiology, University of Regensburg, 93042 Regensburg, Germany
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ABSTRACT |
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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 · kg1 · 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
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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 · kg1 · 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).
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
-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.
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RESULTS |
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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).
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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.
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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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