Introduction

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ET-1 is a potent vasoconstrictor peptide with strong mitogenic activity for smooth muscle cells. Circulating ET-1 levels are increased in humans who have primary and secondary pulmonary hypertension (Stewart et al., 1991; Yoshibayashi et al., 1991; Cody et al., 1992; Tsutamoto et al., 1994) with an increase in local pulmonary ET-1 expression (Giaid et al., 1993), which suggests that this peptide may contribute to the pathogenic process. The responses to ET-1 are mediated via the activation of two distinct receptor subtypes (Sakurai et al., 1992). The ET_A receptors are localized on vascular smooth muscle cells and mediate the constrictive (Arai et al., 1990) and proliferative effects (Zamora et al., 1993) whereas the ET_B receptors are localized on the vascular endothelium and mediate vasorelaxation (Vane, 1990) by increasing the formation of prostacyclin and nitric oxide (De Nucci et al., 1988; Dohi and Luscher, 1991) as well as the clearance of circulating ET-1 (Dupuis et al., 1994, 1996). The ET_B is present on the smooth muscle cells as well, where it also mediates vasoconstriction (Sumner et al., 1992). MCT injected into rats produces alterations in endothelial morphology and function with subsequent pulmonary medial hypertrophy, PH and right ventricular hypertrophy (Roth and Reindel, 1990). In this model, treatment with specific ET_A receptor antagonists has yielded controversial results: chronic i.v. infusion of a specific ET_A antagonist (BQ 123) has been shown to be effective in reducing PH (Miyauchi et al., 1993), whereas s.c. administration of another such antagonist (FR 139317) prevented right ventricular hypertrophy without affecting PH (Ichikawa et al., 1996). Different potencies as well as different pharmacokinetic profiles that depend on the dose and mode of administration may explain these various findings and indicate the need for additional experiments to find an optimal orally active therapy for the treatment of PH with an ET_A antagonist. LU is a novel nonpeptidic selective ET_A antagonist with high oral bioavailability and a long half-life (Münter et al., 1996).

In the present study, we evaluated the effect of chronic treatment (21 days) with LU, administered as a once-daily p.o. dose of 50 mg/kg, on cardiac hemodynamics and hypertrophy, on pulmonary vascular morphology and on endothelial metabolic function in rats with MCT-induced PH.

	Materials and Methods

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The protocol for this study complies with the guidelines for the Care and Use of Laboratory Animals published by the Canadian Council for Animal Care and was approved by the Animal Research Committee of the Montreal Heart Institute. Male Sprague-Dawley rats weighing between 275 and 375 g (Charles River, Saint-Constant, Qc, Canada) were randomly assigned to one of four groups. The animals received an i.p. injection of either 0.5 ml 0.9% saline or 0.5 ml MCT (60 mg/kg). They were gavaged once daily with either 1 ml 0.9% saline or 1 ml LU 135252 (50 mg/kg) starting 48 h before the i.p. injection and subsequently for 3 weeks. This resulted in four groups: control + saline (n = 8), MCT + saline (n = 7), control + LU (n = 6) and MCT + LU (n = 6). The MCT was dissolved in 1.0 N HCl, and the pH was adjusted to 7.4 with 0.5 N NaOH. The compound LU 135252 was dissolved in 1.0 N NaOH, and the pH was adjusted to 7.4 with 0.5 N HCl.

Experimental protocol. Twenty-four hours after the last gavage, rats were anesthetized with sodium pentobarbital (50 mg/kg i.p.), followed by 2000 U of i.p. heparin (Sigma Chemical, St. Louis, MO). The left carotid was isolated and incised, and a polyethylene catheter (PE 50, 0.97 mm OD, 0.58 mm ID) was inserted to record the systemic arterial pressure. A second catheter (0.97 mm OD, 0.58 mm ID) was advanced into the RV through the right jugular vein for the measurement of right ventricular pressures and the first derivative of right ventricular pressures (+dP/dt, which represents the positive rate of rise of right ventricular pressure) (Gould 11-G4123-01 differential amplifier). The position of the catheter was guided by the shape of the pressure tracing displayed on an oscilloscope. The arterial and the right ventricular pressures were measured by a polygraph and recorded (Gould TA4000). Blood samples (1.5 ml) were then collected from the RV for determination of the plasma immunoreactive ET-1 concentrations. The blood was replaced by an equal volume of heparin-treated saline solution. Samples were centrifuged at 1800 × g and 4°C for 10 min, and the plasma was stored at 80°C. Immunoreactive ET-1 levels were determined as previously described (Dupuis et al., 1994).

Pulmonary metabolic functions. After 10 min of stabilization, indicator-dilution experiments were performed. This technique has been used to measure the activity of the endothelium-bound ectoenzyme ACE by measuring fractional single-pass pulmonary hydrolysis of the hemodynamically inactive synthetic ACE substrate ³H-BPGP as previously described (Dupuis et al., 1992). The experiments were performed in duplicate by injecting 0.1 ml of ³H-BPGP (0.33 µCi, 10 pmol). Effluent from the lungs was collected from the left atrial catheter into a fraction collector equipped with tubes advancing at a rate of 1/s (50-60 µl/tube). The injection of ³H-BPGP had no effect on the base-line perfusion flow rate. The first-order rate constant (A_max/K_m) for ACE hydrolysis was calculated from the integrated Michaelis-Menten equation (Toivonen and Catavas, 1986): where F is the isolated lung perfusion flow rate, and M, the cumulative fraction of ³H-BPGP metabolized by the lungs during passage of the bolus, is calculated as where [³H]BP is the concentration of the labeled hydrolysis product of [³H]BPGP, and the denominator represents total ³H concentration in each of the dilution curve samples. Using this technique, it has previously been shown that A_max/K_m varies with the metabolically active pulmonary vascular surface area (Toivonen and Catravas, 1986; Moalli et al., 1987; McCormick et al., 1987; Pitt et al., 1987). In this set of experiments, where F is kept constant in all experimental conditions, A_max/K_m and fractional hydrolysis of ³H-BPGP vary proportionately, so M also reflects endothelial ACE activity. The experiments were performed in duplicate for each animal, and the mean value is reported. The interassay coefficient of variation for all the experiments combined (n = 27) was of 4.6 ± 2.9%. After the indicator-dilution experiments, the RV and LV + S were dissected and weighted to obtain the ratio of RV weight to LV + S weight).

Lung vascular morphometry. The pulmonary artery was injected at 50 cm H₂O pressure with a hot barium-gelatin mixture (60°C) for 3 min, and the airways were distended with 10% formaldehyde solution at a pressure of 36 cm H₂O. The lungs were immersed in an inflated state in 10% formaldehyde solution for at least 2 days. For morphometric analysis, three sections were obtained from each rat lung (one section each from the upper, median and lower parts). These were stained with hematoxylin and eosin and were microscopically assessed for medial wall thickness of pulmonary arteries.

Drugs. MCT was obtained from Sigma Chemical Co. (St. Louis, MO; the ET_A receptor antagonist LU 135252 was kindly provided by Dr. M. Kirchengast (Knoll AG, BASF Pharma, Ludwigshafen, Germany).

Statistical analysis. All values were expressed as means ± S.D. Results were compared by using an analysis of variance, followed by a Bonferroni correction for multiple groups' comparisons. Statistical significance was assumed at P < .05.

	Results

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Right Ventricular hemodynamics and circulating ET-1 levels. In controls, right ventricular systolic pressure was not affected by LU therapy (fig. 1A). In MCT-treated rats, right ventricular pressure was markedly increased to 72.5 ± 15.9 mmHg (P < .01) and was lowered by concomitant administration of LU to 42.8 ± 8.5 mmHg (P < .01). Right ventricular + dP/dt increased from 1661 ± 604 to 3446 ± 952 mmHg/s in MCT-treated rats (P < .01, fig. 1B). PH was associated with an increase in right ventricular + dP/dt that was nonsignificantly reduced by LU therapy (fig. 1B). Plasma immunoreactive ET-1 concentrations doubled in MCT-treated rats and were not modified by LU therapy (fig. 2).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Effect of LU 135252 on the right ventricular systolic pressure (panel A) and on the right ventricular rate of pressure rise (panel B) from controls and MCT-treated rats. Measurements were obtained at 3 weeks after MCT injection. Oral administration of LU 135252 was started 2 days before the injection of MCT and continued until 24 h before the experiment. Values are mean ± S.D. *P < .05, **P < .01 vs. control + NaCl; P < .01 vs. MCT + NaCl.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Effect of LU 135252 on venous ET-1 in control and MCT-treated rats after 3 weeks of therapy. Values are mean ± S.D. *P < .05, **P < .01 vs. control + NaCl.

Right Ventricular hypertrophy and pulmonary arteries remodeling. At 21 days after MCT, the RV to LV + S weight ratio (0.58 ± 0.08) was greater than the control value (0.33 ± 0.03; P < .01, fig. 3). In MCT-treated rats only, LU induced a significant reduction in right ventricular hypertrophy to a RV/(LV + S) weight ratio of 0.42 ± 0.05 (P < .05). A linear-regression analysis revealed a good correlation between increasing right ventricular pressure and hypertrophy (r = 0.83; P < .001) (fig. 4); the MCT-treated rats that received LU occupied an intermediate position between the controls and the MCT-treated rats gavaged with saline.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Effect of LU 135252 on the heart weight ratio [RV/(LV + S)] from controls and MCT-treated rats. Measurements were obtained at 3 weeks after MCT injection. Values are mean ± S.D. *P < .05, **P < .01 vs. control + NaCl; P < .01 vs. MCT + NaCl.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Correlation between the right ventricular systolic pressure (RVP) and the weight ratio of the right ventricle to the left ventricle plus septum [RV/(LV + S)].

Pulmonary ACE activity. The isolated lungs were perfused at a flow rate of 10 ml/min. To achieve this, perfusion pressure was set at 11.5 ± 1.2 mmHg in controls + NaCl and at 11.9 ± 1.8 mmHg in controls + LU (P = N.S.). Because of higher pulmonary vascular resistance, perfusion pressure in MCT + NaCl was increased to 28.2 ± 2.9 mmHg (P < .01 vs. control), and it was intermediate in the MCT + LU rats at 19.5 ± 4.0 mmHg (P < .01 vs. control; P < .01 vs. MCT + LU).

	Discussion

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The present study demonstrates that the ET_A receptor antagonist LU 135252, administered p.o. and once daily, significantly reduced the development of MCT-induced PH and prevented the reduction in pulmonary ACE activity. The compound LU 135252 is the (+) enantiomer of LU 127043, and it has been investigated as a selective ET_A receptor antagonist with good oral bioavailability and a long duration of action (Raschack et al., 1995; Riechers et al., 1996). A p.o. dose of LU of 50 mg/kg/day effectively reduces neointimal proliferation in a vascular injury model (Münter et al., 1996). LU 135252 binds to the ET_A receptor with a higher affinity (k_i = 1.5 nM) (Münter et al., 1996) than LU 127043 (k_i = 6.0 nM), BQ 123 (k_i = 19 nM) or BMS 182874 (k_i = 57 nM) and with affinity similar to that of FR 139317 (k_i = 1.0 nM). LU reduced the increase in right ventricular systolic pressure and hypertrophy while having no effect on mean systemic arterial pressure. Continuous infusion of BQ 123, another selective ET_A antagonist, has been shown to be effective in reducing PH, right ventricular hypertrophy and pulmonary artery remodeling in hypoxia (DiCarlo et al., 1995), as well as MCT-induced (Miyauchi et al., 1993) PH. Endogenous myocardial ET-1 synthesis may directly contribute to hypertrophy, because BQ 123 can attenuate left ventricular hypertrophy induced by aortic banding (Ito et al., 1994). Accordingly, the growth regulatory properties of endothelin may play a role in myocardial hypertrophy (Chua et al., 1992). In MCT-treated rats, the present study with LU and that of Miyauchi et al. (1993) with BQ 123 suggest that the inhibition of right ventricular hypertrophy is due not only to blockade of excessive stimulation of the heart by endogenous ET-1 but also to the prevention of PH.

Our histological studies did not demonstrate a significant reduction of pulmonary artery medial wall hypertrophy, and because the lung also contains ET_B receptors, this raises the possibility that activity against the ET_B may be necessary to affect pulmonary remodeling. This possibility seems unlikely, however, because another specific ET_A antagonist, BQ 123, was able to affect remodeling with a similar reduction in pulmonary pressures and right ventricular hypertrophy (Miyauchi et al., 1993). It is possible that the dose of LU may not have been high enough to reduce remodeling of the pulmonary arteries, and arteries of a smaller diameter than those studied may have been affected. Endothelin is considered to function as a cofactor in the hypertensive process by acting in synergy with various mitogens, such as transforming growth factor, epidermal growth factor, platelet-derived growth factor and insulin, to potentiate cellular transformation and replication (Battistini et al., 1993). Platelet-activating factor (Ono et al., 1992) and angiotensin II (Abraham et al., 1995) may also contribute to the lung vascular remodeling in hypoxic pulmonary hypertensive rats. The involvement of other factors in addition to ET-1 may also explain our finding that LU did not reduce vascular remodeling in pulmonary arteries of MCT-treated rats. It has been demonstrated that in addition to the morphological changes, changes in pulmonary arterial smooth muscle contractility also appear to play a role in the development and/or maintenance of PH (Griffith et al., 1994). The prevention of PH by the p.o. administration of LU 135252 was consistent with a major role of ET-1 as a potent vasoconstrictor that contributes to increasing the basal pulmonary tone in the development of PH.

As previously shown, plasma ET-1 levels were significantly increased in MCT-treated rats. This increase in plasma ET-1 has been shown to precede the development of PH and does not seem to originate from the lungs, because pulmonary production of ET-1 is decreased, whereas in the prehypertensive stage, the increase in plasma ET-1 levels may be partly ascribed to increased production by the kidneys (Miyauchi et al., 1993). The marked increase in plasma ET-1 may also be due to MCT-induced pulmonary inflammation, because the lungs, kidneys and liver are the main sites for the removal of circulating ET-1 (Shiba et al., 1989). The p.o. administration of LU did not significantly modify plasma ET-1 concentrations in either control or MCT-treated rats. It has also been shown that neither of the ET_A receptor antagonists FR 139317 and BQ 123 normalized the plasma ET-1 concentration in MCT-treated rats (Ichikawa et al., 1996) or hypoxia-exposed rats (DiCarlo et al., 1995), respectively. Our results are consistent with the previous observations suggesting that the increase in plasma ET-1 by MCT cannot be normalized despite improvement in PH.

Pulmonary vascular endothelial dysfunction is associated with primary and secondary PH and may be a reflection of, or even contribute to, the pathophysiological process. ACE is located at the luminal surface of the pulmonary vascular endothelium and represents one of the most important metabolic properties of the pulmonary vasculature. In this study, we therefore used the synthetic ACE substrate ³H-BPGP to probe the metabolic activity of the pulmonary vascular endothelium. This substrate was previously used in awake dogs to demonstrate that exercise results in an increase in the metabolically active pulmonary vascular surface area (capillary recruitment) (Dupuis et al., 1992). Another ACE substrate, BPAP, has been used in various experiments to measure the metabolic integrity of the pulmonary vascular endothelium (Catravas and White, 1984; Toivonen and Catravas, 1986; McCormick et al., 1987; Moalli et al., 1987; Pitt et al., 1987). In the present experiments, where isolated lung perfusion rate was kept identical in the four experimental groups, fractional ³H-BPGP hydrolysis and the first-order parameter A_max/K_m vary proportionately, a reduction representing a decrease in the perfused metabolically active pulmonary vascular surface area. We found that the pulmonary hydrolysis of ³H-BPGP was significantly reduced in MCT-treated rats but was normalized by daily p.o. administration of LU.

It has previously been shown that MCT induces a reduction in pulmonary ACE activity in rats (Mathew et al., 1990), but this reduction has little effect on the conversion of angiotensin I to angiotensin II or on the degradation of bradykinin (Ito et al., 1988). Others have shown that hypoxic PH is associated with a significant inhibition of the transpulmonary conversion of angiotensin I to angiotensin II (Jackson et al., 1986) and that toxic injury to the pulmonary vascular endothelium also reduces pulmonary ACE activity (McCormick et al., 1987). ACE inhibition therapy with captopril (Ishikawa et al., 1995) does not prevent the development of PH in the MCT model, which suggests that the reduction in ACE activity does not play a significant pathophysiological role in this model. MCT causes direct damage to the vascular endothelium and may adversely affect numerous endothelial cell functions; for that reason, we measured ACE activity as an index of the integrity of the endothelial metabolic functions. In human primary PH, a reduction in endothelial nitric oxide (Giaid and Saleh, 1995) and prostacyclin production and an increase in endothelin production (Giaid et al., 1993) suggest that endothelial abnormalities play a key role in the pathogenic process. Secondary PH is also associated with endothelial dysfunction, however, which suggests that PH in itself causes endothelial cell dysfunction; increased endothelin levels correlate with the severity of secondary PH in heart failure (Cody et al., 1992; Tsutamoto et al., 1994).

In the present study, pulmonary ACE activity was reduced in MCT PH but was completely restored after administration of the specific ET_A antagonist LU 135252. This restoration of an endothelial metabolic function occurred without any significant decrease in the circulating ET-1 levels but with reduction of PH and right ventricular hypertrophy. This suggests that the reduction in pulmonary ACE activity was not the direct consequence of MCT-induced endothelial injury but rather was a consequence of PH. The sustained increase in ET-1 levels, combined with the therapeutic benefit of LU 135252, however, suggests that ET-1 plays an important primary pathophysiological role in the MCT PH model.

In conclusion, administration of an orally active ET_A receptor antagonist reduces the development of MCT-induced PH and right ventricular hypertrophy and improves the metabolic properties of the pulmonary vascular endothelium. The present results strongly support the important role played by ET-1 in this model of PH and establish that other endothelial metabolic abnormalities, such as that of ACE, are secondary to PH and are markers rather than mediators of the disease. This work supports the development of orally active ET_A antagonists for the treatment of PH and the associated endothelial metabolic dysfunction.