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. 2007 Feb 8;580(Pt 3):907–923. doi: 10.1113/jphysiol.2006.127118

Alterations in endothelial control of the pulmonary circulation in exercising swine with secondary pulmonary hypertension after myocardial infarction

Daphne Merkus 1, Birgit Houweling 1, Vincent J de Beer 1, Zaida Everon 1, Dirk J Duncker 1
PMCID: PMC2075461  PMID: 17289783

Abstract

Secondary pulmonary hypertension after myocardial infarction (MI) has been associated with endothelial dysfunction and activation of the endothelin (ET) system. Here, we investigated whether an increased ET-mediated pulmonary vasoconstrictor influence contributes to pulmonary hypertension after MI, and whether this increased ET vasoconstriction is caused by impaired nitric oxide (NO) and prostanoid production. For this purpose, chronically instrumented swine with and without MI ran on a treadmill at 0–4 km h−1. Mixed ETA/ETB receptor blockade (tezosentan) was performed in the absence and presence of single or combined inhibition of endothelial NO synthase (eNOS, with Nω-nitro-l-arginine) and cyclo-oxygenase (COX, with indometacin). In normal swine, mixed ETA/ETB blockade decreased pulmonary vascular resistance, but only during exercise. In MI swine, an increased ET-mediated vasoconstrictor influence was observed in the pulmonary circulation both at rest and during exercise. Inhibition of COX resulted in pulmonary vasoconstriction at rest in MI, but not in normal swine; this vasoconstriction in MI swine was normalized by ETA/ETB receptor blockade. Inhibition of eNOS enhanced the vasodilator response to ETA/ETB blockade, indicating that NO blunts the pulmonary vasoconstrictor influence of ET. However, this vasodilator response was enhanced to a similar degree in MI and normal swine. In summary, swine with a recent MI are characterized by an exaggerated pulmonary vasoconstrictor influence of ET. This increased ET-mediated pulmonary vasoconstrictor influence is not caused by a loss of NO bioavailability, and is blunted by an increased prostanoid-mediated vasodilatation. In conclusion, an increased ET-mediated vasoconstriction, which does not appear to be the result of loss of endothelial vasodilators, contributes to pulmonary hypertension after MI.

Myocardial infarction (MI) results in decreased left ventricular (LV) function, thereby increasing LV filling pressures. These increased LV filling pressures are transmitted backwards into the pulmonary circulation, leading to an increase in pulmonary arterial pressure (PAP). This so-called ‘secondary pulmonary hypertension’ is frequently associated with a ‘reactive’ increase in pulmonary vascular resistance (PVR), resulting in a further increase in PAP (Moraes et al. 2000). Thus, the pulmonary circulation after MI is characterized by elevated PAP and PVR, which increase the afterload of the right ventricle (RV) and may contribute to RV dysfunction and eventually RV failure (Guarracino et al. 2005).

Pulmonary hypertension is exacerbated during exercise, as the vasodilator capacity of the pulmonary vasculature is reduced (Mabee et al. 1994; Butler et al. 1999; McLaughlin & McGoon, 2006). Moreover, in patients with heart failure, exercise capacity is inversely correlated with PVR and the failure to decrease PVR during exercise (Franciosa et al. 1985). The mechanism underlying the pulmonary vasoconstriction after MI is still incompletely understood, but may involve alterations in serotonin, thromboxane A2 and angiotensin-II (Said, 2006) as well as endothelial dysfunction (Moraes et al. 2000; Budhiraja et al. 2004). Under normal, physiological conditions, the endothelium plays a key role in maintaining vascular homeostasis by carefully balancing the production of vasodilators such as nitric oxide (NO) and prostacyclin, and vasoconstrictors such as endothelin (ET). Moreover, NO (Goligorsky et al. 1994; Richard et al. 1995; Wiley & Davenport, 2001; Kelly et al. 2004) and prostacyclin (Prins et al. 1994; Wort et al. 2002) can limit the production of and/or reduce receptor sensitivity to ET, while ET, by binding to the ETB receptor on the endothelium, stimulates the production of NO and prostanoids (Goligorsky et al. 1994; Ahlborg & Lundberg, 1997; Schiffrin & Touyz, 1998; Lavallee et al. 2001; Alonso & Radomski, 2003). Indeed, we have recently shown that in the pulmonary circulation of normal swine, endogenous NO blunts the vasoconstrictor influence of ET, thereby maintaining a low PAP and PVR particularly during exercise (Houweling et al. 2005).

In general, an increase in shear stress results in an increased production of NO and prostanoids (Osanai et al. 2000), while an increase in intravascular pressure may increase the production of ET (Falcone & Meininger, 1999; Petersen et al. 2002). Hence, following MI, a lower cardiac output may contribute to a reduced production of NO and prostanoids (Ontkean et al. 1991; Baggia et al. 1997; Cooper et al. 1998), while the increased PAP may contribute to an increased production of ET (von Lueder et al. 2004) by the pulmonary endothelial cells. This imbalance between production of vasodilators and vasoconstrictors probably contributes to the sustained state of pulmonary vasoconstriction after MI. In support of this concept, we recently observed an increased ET-mediated vasoconstrictor influence in the pulmonary circulation after MI (Houweling et al. 2006). Interestingly, the vasodilator influence of endogenous NO appeared to be maintained (Haitsma et al. 2002), while perturbations in the vasomotor influence of prostanoids have not been investigated to date.

In light of these considerations, the aim of the present study was to investigate alterations in integrated endo-thelial control of pulmonary vascular tone in swine with secondary pulmonary hypertension following a recent MI. Specifically, we investigated whether the increased pulmonary vasoconstrictor influence of endogenous ET after MI is due to a decreased influence of NO and/or prostanoids, or whether the increased ET vasoconstriction occurs independently of these endothelial vasodilators. For this purpose we investigated the effects of ET receptor blockade in the absence and presence of single or combined NO synthase (NOS) and cyclo-oxygenase (COX) inhibition. In view of the increased contribution of ET to pulmonary vasomotor tone during exercise (Houweling et al. 2006), and the close correlation between plasma ET levels and exercise capacity in patients with heart failure (Krum et al. 1995), we investigated the interaction between NO, prostanoids and ET at rest and during graded treadmill exercise.

Methods

Animals

Studies were performed in accordance with the Council of Europe Convention (ETS123)/Directive (86/609/EEC) for the protection of vertebrate animals used for experimental and other scientific purposes, and with approval of the Animal Care Committee of the Erasmus Medical Center. Thirty-four 2- to 3-month-old Yorkshire × Landrace swine (22 ± 1 kg at the time of surgery) of either sex entered the study. After completing all experimental protocols, animals were killed by an intravenous overdose of pentobarbitone sodium.

Surgery

Swine were sedated with ketamine (30 mg kg−1 i.m.), anaesthetized with thiopental (10 mg kg−1 i.v.), intubated and ventilated with a mixture of O2 and N2O (1: 2) to which 0.2–1% (v/v) isoflurane was added (Duncker et al. 2000). Anaesthesia was maintained with midazolam (2 mg kg −1 + 1 mg kg−1 h−1 i.v.) and fentanyl (10 micrograms kg−1 h−1 i.v.); depth of anaesthesia was checked regularly using a pain stimulus (toe-pinch). Under sterile conditions, the chest was opened via the fourth left intercostal space and a fluid-filled polyvinylchloride catheter was inserted into the aortic arch for aortic blood pressure measurement (Combitrans pressure transducers, Braun) and blood sampling. A calibrated electromagnetic flow probe (14–15 mm, Skalar) was positioned around the ascending aorta for measurement of cardiac output. Polyvinylchloride catheters were inserted into the left atrium to measure pressure, and into the pulmonary artery to measure pressure, administer drugs and collect mixed venous blood samples. In 14 swine the circumflex artery was permanently occluded with a suture to induce MI of the lateral left ventricular wall (infarct size ∼20% of the left ventricle (Haitsma et al. 2001, 2002)). Catheters were tunnelled to the back and animals were allowed to recover, receiving analgesia (0.3 mg buprenorphine i.m.) for 2 days and antibiotic prophylaxis (25 mg kg−1 amoxicillin and 5 mg kg−1 gentamycin i.v.) for 5 days. Three MI swine died in the first week after surgery.

Experimental protocols

Studies were performed ∼2 weeks after surgery, with animals exercising on a motor driven treadmill. We previously demonstrated excellent reproducibility of the cardiovascular response to consecutive exercise trials with 90 min of rest in between in both normal swine (Duncker et al. 2000) and MI swine (Haitsma et al. 2002; Duncker et al. 2005). In the present study, four exercise protocols were performed on different days and in random order. Most swine participated in several protocols. Overlap of animals between protocols is shown in Table 1. We have previously published part of our data on endothelial control of pulmonary vasomotor tone in normal swine (Houweling et al. 2005) and on the role of endothelin in control of pulmonary vasomotor tone in MI swine (Houweling et al. 2006).

Table 1.

Schematic representation of the overlap of animals used in the various protocols

Protocol Con–Tezo Indo–Tezo NLA–Tezo NLA–Indo–Tezo Total
Con–Tezo 17N/9MI 8N 9N 7N
Indo–Tezo 8MI 10N/9MI 3 N 3N
NLA–Tezo 6MI 7MI 10 N/7MI 6N
NLA–Indo–Tezo 5MI 6MI 6MI 7N/7MI
Total 20N/11MI
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Con, control; Tezo, tezosentan; Indo, indometacin; N normal.

Endothelin

With swine (normal, n = 17 and MI, n = 9) lying quietly on the treadmill, resting haemodynamic measurements, consisting of heart rate, cardiac output, mean aortic pressure (MAP), mean pulmonary arterial pressure (PAP), and mean left atrial pressure (LAP) were obtained and blood samples collected. Haemodynamic measurements were repeated and rectal temperature was measured with animals standing on the treadmill. Subsequently, a four-stage (1–4 km h−1) treadmill exercise protocol was started; each exercise stage lasted 2–3 min. This exercise protocol results in heart rates of up to 230–240 beats min−1, which equals approximately 85% MI of the estimated maximal heart rate of these animals (Haitsma et al. 2001; Duncker et al. 2005). Haemodynamic variables were continuously recorded and blood samples collected during the last 45 s of each stage. The slope of the electromagnetic flow probe was calibrated using an electric signal, and the zero-value was set during diastole. Fluid-filled pressure transducers were positioned on the back of the animals and calibrated at mid-chest level. After completing the exercise protocol, animals were allowed to rest on the treadmill for 90 min, after which the mixed ETA/ETB receptor antagonist tezosentan (a gift from Dr Clozel, Actelion Pharmaceuticals Ltd) was intravenously administered over 10 min in a dose of 3 mg kg−1, followed by a continuous infusion of 6 mg kg−1 h−1i.v. (Merkus et al. 2003), and the exercise protocol was repeated. We have previously shown that this dose of tezosentan abolished the ∼35 mmHg increase in arterial pressure in response to administration of exogenous endothelin in a dose of 50 ng kg −1min−1 (Merkus et al. 2003).

Prostanoids and endothelin

Ninety minutes after 10 normal and 9 MI swine had undergone a control exercise trial (as described above), animals received the COX inhibitor indometacin ((Sigma) 10 mg kg−1i.v. over 10 min (Merkus et al. 2004)) and 5 min later underwent a second exercise trial. Ninety minutes later, animals received indometacin in a dose of 5 mg kg−1i.v., which resulted in haemodynamic conditions that were identical to those following administration of 10 mg kg−1 prior to the second exercise trial (Houweling et al. 2005). Subsequently, animals received tezosentan (3 mg kg−1i.v. + 6 mg kg−1 h−1i.v.) and underwent a third exercise trial.

NO and endothelin

Ninety minutes after 10 normal and 7 MI swine had undergone a control exercise trial, animals received the NOS inhibitor Nω-nitro-l-arginine (NLA (Sigma), 20 mg kg−1i.v.; (Duncker et al. 2000; Merkus et al. 2004)), and underwent a second exercise trial. Ninety minutes later, animals received tezosentan (3 mg kg−1i.v. + 6 mg kg−1 h−1i.v.) and underwent a third exercise trial.

NO, prostanoids and endothelin

Ninety minutes after 7 normal and 7 MI swine underwent an exercise trial in the presence of NLA (20 mg kg−1i.v.) and indo-metacin (10 mg kg−1i.v.), animals received indometacin (5 mg kg−1i.v.) and tezosentan (3 mg kg−1i.v. and 6 mg kg−1 h−1i.v.) and underwent a second exercise trial (Houweling et al. 2005).

Blood gas measurements

Blood samples were kept in iced syringes until the conclusion of each exercise trial. Measurements of Inline graphic (mmHg), Inline graphic (mmHg) and pH were then immediately performed with a blood gas analyser (Acid-Base Laboratory Model 505, Radiometer, Copenhagen, Denmark), and corrected for body temperature. O2 saturation (%) and haemoglobin (g (100 ml)−1) were measured with a haemoximeter (OSM3, Radiometer). Blood O2 content (μmol ml−1) was computed as (Hb 0.621 O2-saturation) + (0.00131 Inline graphic). Body O2 consumption (B Inline graphic) was calculated as the product of cardiac output and the difference in O2 content between arterial and mixed venous blood (Stubenitsky et al. 1998; Duncker et al. 2000).

Data analysis

Digital recording and off-line analysis of haemodynamics have been previously described (Stubenitsky et al. 1998; Duncker et al. 2000). Systemic vascular resistance (SVR) was computed as mean aortic blood pressure divided by cardiac output. Pulmonary vascular resistance (PVR) was computed as mean pulmonary arterial pressure minus mean left atrial pressure divided by cardiac output (Merkus et al. 2004).

Statistical analysis

Haemodynamic data were digitally recorded and analysed off-line. Haemodynamic variables were analysed using analysis of variance (ANOVA) for repeated measures. Post hoc testing was done using Dunnett's test. The relationships between B Inline graphic and SVR and PVR were analysed using multiple regression analysis with B Inline graphic, tezosentan, NLA, indometacin and their interactions as independent variables and with each animal as a dummy variable. Normal animals and animals with MI were first analysed separately (Statview). Subsequently, interaction between the drugs and MI was included in the regression analysis. Statistical significance was accepted when P ≤ 0.05. Data are presented as mean ± s.e.m. Since no significant differences were found between male and female swine, data from both sexes were pooled.

Results

Haemodynamic responses to exercise in normal and MI swine

Exercise up to 4 km h−1 in normal swine resulted in a tripling of B Inline graphic which was met by a doubling of cardiac output and an increase in O2 extraction from 47 ± 1% to 67 ± 1% (Fig. 1). The increase in cardiac output was principally due to an increase in heart rate, as stroke volume increased by only 7%. Mean aortic blood pressure was minimally affected, implying that the increase in cardiac output was balanced by a similar decrease in systemic vascular resistance (Table 2). In contrast, mean pulmonary arterial pressure almost doubled in normal swine during exercise (Fig. 1). The transpulmonary pressure gradient (pulmonary arterial pressure minus left atrial pressure) increased commensurate with cardiac output, so that PVR did not change during exercise.

Figure 1. Effect of MI on metabolic and haemodynamic responses to treadmill exercise.

Figure 1

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Shown are the responses to exercise of various parameters in relation to the body oxygen consumption (B Inline graphic) under control conditions in 16 normal swine (○) and 9 MI swine (▪). *P ≤ 0.05 MI versus normal (change in intercept and/or slope).

Table 2.

Haemodynamic variables in normal swine at rest and during exercise before and after ETA/ETB receptor blockade in the absence or presence of single or combined blockade of COX and eNOS

Rest Exercise level (km h−1)
Lying Standing 1 2 3 4
HR (beats min−1)
 Control 131 ± 4 148 ± 4* 170 ± 4* 185 ± 4* 202 ± 5* 233 ± 6*
 Tezo 150 ± 4‡ 162 ± 4*‡ 185 ± 5*‡ 185 ± 7*‡ 215 ± 6*‡ 244 ± 7*‡
 Indo 86 ± 7† 97 ± 5*† 121 ± 6*† 134 ± 6*† 150 ± 6*† 174 ± 5*†
 Indo + Tezo 97 ± 7†‡ 112 ± 5*†‡ 128 ± 6*† 141 ± 6*† 157 ± 7*† 178 ± 8*†
 NLA 103 ± 6† 120 ± 4*† 132 ± 5*† 142 ± 5*† 159 ± 5*† 191 ± 6*†
 NLA + Tezo 118 ± 5‡ 141 ± 6*‡ 152 ± 6*†‡ 160 ± 6*†‡ 179 ± 6*†‡ 204 ± 6*†‡
 NLA + Indo 82 ± 4† 95 ± 6*† 112 ± 6*† 121 ± 8*† 134 ± 7*† 164 ± 9*†
 NLA + Indo + Tezo 101 ± 8 116 ± 7*‡ 128 ± 6*‡ 141 ± 8* 155 ± 10*†‡ 179 ± 9*†
CO (l min−1)
 Control 3.8 ± 0.2 4.6 ± 0.2* 5.3 ± 0.3* 5.9 ± 0.3* 6.5 ± 0.3* 7.3 ± 0.3*
 Tezo 4.2 ± 0.2‡ 4.8 ± 0.3* 5.7 ± 0.3* 6.2 ± 0.3* 6.9 ± 0.4*‡ 7.7 ± 0.4*‡
 Indo 2.6 ± 0.3† 3.2 ± 0.3*† 4.1 ± 0.3*† 4.5 ± 0.3*† 5.1 ± 0.3*† 6.2 ± 0.3*†
 Indo + Tezo 3.0 ± 0.2† 3.8 ± 0.3*†‡ 4.4 ± 0.3*† 5.2 ± 0.4*‡ 5.6 ± 0.4*†‡ 6.4 ± 0.4*†
 NLA 3.1 ± 0.3† 3.8 ± 0.2*† 4.5 ± 0.3*† 4.9 ± 0.4*† 5.5 ± 0.4*† 6.4 ± 0.4*†
 NLA + Tezo 3.8 ± 0.3‡ 4.8 ± 0.4*‡ 5.3 ± 0.4*†‡ 5.6 ± 0.4*†‡ 6.4 ± 0.4*‡ 7.3 ± 0.5*†‡
 NLA + Indo 1.9 ± 0.2 2.4 ± 0.2* 3.1 ± 0.2*† 3.5 ± 0.3* 4.0 ± 0.2*† 4.9 ± 0.4*
 NLA + Indo + Tezo 2.8 ± 0.3‡ 3.5 ± 0.3*‡ 4.0 ± 0.2*‡ 4.6 ± 0.3*‡ 5.0 ± 0.3*‡ 5.7 ± 0.2*‡
SV (ml)
 Control 29 ± 2 31 ± 2* 32 ± 2* 32 ± 2* 32 ± 2* 32 ± 2*
 Tezo 28 ± 2 30 ± 2* 31 ± 2* 31 ± 2* 33 ± 2* 32 ± 2*
 Indo 30 ± 2 32 ± 2* 34 ± 2* 34 ± 2* 35 ± 2*† 36 ± 1*†
 Indo + Tezo 31 ± 2† 34 ± 2* 34 ± 1* 37 ± 1*† 36 ± 2*† 36 ± 1*†
 NLA 30 ± 3 32 ± 2* 34 ± 2* 34 ± 2* 35 ± 2* 34 ± 2
 NLA + Tezo 32 ± 3 34 ± 3* 35 ± 3* 35 ± 3* 36 ± 2‡ 36 ± 3*‡
 NLA + Indo 23 ± 1 25 ± 1 27 ± 1* 28 ± 1* 29 ± 1* 30 ± 1*†
 NLA + Indo + Tezo 27 ± 1 30 ± 1‡ 31 ± 1*‡ 32 ± 2* 32 ± 1*†‡ 31 ± 1*†
MAP (mmHg)
 Control 95 ± 2 89 ± 3* 87 ± 2* 88 ± 2* 88 ± 2* 90 ± 2*
 Tezo 89 ± 2‡ 79 ± 2*‡ 79 ± 2*‡ 79 ± 2*‡ 81 ± 2*‡ 82 ± 1*‡
 Indo 124 ± 7† 120 ± 7† 107 ± 6*† 106 ± 6*† 107 ± 5*† 104 ± 5*†
 Indo + Tezo 111 ± 3† 93 ± 3*‡ 89 ± 2*‡ 89 ± 3*‡ 89 ± 3*‡ 88 ± 3*‡
 NLA 122 ± 2† 120 ± 3† 118 ± 3† 118 ± 2† 121 ± 2† 123 ± 2†
 NLA + Tezo 111 ± 4†‡ 104 ± 2†‡ 98 ± 3*†‡ 99 ± 2*†‡ 102 ± 3†‡ 101 ± 2*†‡
 NLA + Indo 161 ± 7† 156 ± 8† 146 ± 6*† 142 ± 5*† 137 ± 4*† 132 ± 4*†
 NLA + Indo + Tezo 131 ± 7‡ 121 ± 7*‡ 114 ± 5*‡ 114 ± 5*‡ 115 ± 5*‡ 113 ± 5*‡
PAP (mmHg)
 Control 15 ± 1 15 ± 1 18 ± 1* 21 ± 1* 23 ± 1* 28 ± 1*
 Tezo 15 ± 1 13 ± 1 17 ± 1‡ 17 ± 1*‡ 21 ± 1*‡ 25 ± 1*‡
 Indo 19 ± 1† 18 ± 2 18 ± 2 20 ± 2 22 ± 2* 26 ± 2*
 Indo + Tezo 17 ± 2 14 ± 2*‡ 15 ± 2† 17 ± 2† 20 ± 2† 23 ± 2*†
 NLA 23 ± 2† 23 ± 2† 27 ± 3† 29 ± 3† 34 ± 3*† 41 ± 2*†
 NLA + Tezo 16 ± 2‡ 17 ± 2 17 ± 2†‡ 19 ± 2‡ 25 ± 2*†‡ 29 ± 3*‡
 NLA + Indo 24 ± 2† 26 ± 3 27 ± 3 29 ± 3* 32 ± 3* 37 ± 3*
 NLA + Indo + Tezo 21 ± 2 18 ± 3 20 ± 2‡ 24 ± 3 26 ± 3* 31 ± 3*†‡
LAP (mmHg)
 Control 4 ± 1 2 ± 1 3 ± 1 5 ± 1 6 ± 1* 8 ± 1*
 Tezo 4 ± 1 0 ± 1* 3 ± 1 3 ± 1 6 ± 1* 8 ± 1*
 Indo 10 ± 2† 7 ± 2 3 ± 2* 6 ± 1* 6 ± 1* 7 ± 1
 Indo + Tezo 8 ± 1† 2 ± 1*‡ 3 ± 1* 4 ± 1* 6 ± 1* 7 ± 1
 NLA 10 ± 1† 5 ± 1 7 ± 1 8 ± 1 9 ± 1 10 ± 1
 NLA + Tezo 2 ± 2 3 ± 2 4 ± 1 6 ± 1 9 ± 1* 10 ± 1*
 NLA + Indo 15 ± 2† 13 ± 3 12 ± 3† 12 ± 2*† 13 ± 2* 12 ± 2*
 NLA + Indo + Tezo 8 ± 2‡ 4 ± 3 5 ± 2‡ 8 ± 2‡ 9 ± 1 11 ± 2
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HR, heart rate; CO, cardiac output; MAP, mean arterial pressure; PAP, pulmonary arterial pressure; LAP, left atrial pressure; Tezo, tezosentan; Indo, indometacin. Data are mean ± s.e.m.

*

P ≤ 0.05 versus rest (lying)

†

P ≤ 0.05 versus corresponding control

‡

P ≤ 0.05 effect of Tezo.

The 18% lower stroke volume in MI compared to normal swine was accompanied by an increase in heart rate. Yet, the increase in heart rate did not fully compensate for the decrease in stroke volume. Hence, cardiac output was slightly lower at all levels of treadmill exercise, which necessitated a small increase in O2 extraction that resulted in a further decrease in mixed venous O2 saturation during exercise in MI compared to normal swine (Fig. 1, Table 3). However, aerobic metabolism was still maintained as arterial (Tables 4 and 5) and mixed venous (not shown) pH and HCO3− were not different between normal swine and swine with MI. Pulmonary arterial pressure was significantly elevated in MI swine both at rest and during exercise, which was principally due to a marked increase in left atrial pressure, but also due to a ∼20% increase in PVR (Fig. 1).

Table 3.

Haemodynamic variables in swine with MI at rest and during exercise before and after ETA/ETB receptor blockade in the absence and presence of single or combined blockade of COX and eNOS

Rest Exercise level (km h−1)
Lying Standing 1 2 3 4
HR (beats min−1)
 Control 147 ± 4§ 158 ± 5* 187 ± 2*§ 203 ± 4*§ 219 ± 6*§ 244 ± 8*
 Tezo 157 ± 4‡ 169 ± 4*‡ 190 ± 4* 205 ± 4* 222 ± 7* 243 ± 6*
 Indo 104 ± 3†§ 124 ± 3*†§ 139 ± 3*†§ 143 ± 4*† 150 ± 6*† 181 ± 5*†
 Indo + Tezo 112 ± 7† 125 ± 2†§ 135 ± 7† 148 ± 5*† 159 ± 4*† 175 ± 7*†
 NLA 111 ± 4† 139 ± 6 155 ± 7*†§ 167 ± 6*†§ 180 ± 6*†§ 209 ± 8*
 NLA + Tezo 131 ± 5‡ 152 ± 5 165 ± 5* 176 ± 8† 190 ± 9† 216 ± 10†
 NLA + Indo 91 ± 6 99 ± 6* 115 ± 6* 125 ± 6* 142 ± 5* 165 ± 5*
 NLA + Indo + Tezo 86 ± 5 100 ± 4* 122 ± 4* 133 ± 4* 148 ± 4* 173 ± 3*‡
CO (l min−1)
 Control 3.5 ± 0.2 4.0 ± 0.3* 4.7 ± 0.3* 5.1 ± 0.3* 5.6 ± 0.4* 6.0 ± 0.4*§
 Tezo 3.6 ± 0.3 4.6 ± 0.4*‡ 5.2 ± 0.4*‡ 5.6 ± 0.4*‡ 6.0 ± 0.4*‡ 6.4 ± 0.4*‡
 Indo 2.8 ± 0.2† 3.6 ± 0.3*† 4.2 ± 0.3*† 4.6 ± 0.3*† 4.9 ± 0.3*† 5.8 ± 0.3*†
 Indo + Tezo 3.4 ± 0.3‡ 4.2 ± 0.3*‡ 4.7 ± 0.3*† 5.2 ± 0.3*†‡ 5.6 ± 0.3*†‡ 6.2 ± 0.3*†
 NLA 3.0 ± 0.3† 3.9 ± 0.3*‡ 4.4 ± 0.3*† 4.6 ± 0.3*† 5.1 ± 0.3*† 5.8 ± 0.4*†
 NLA + Tezo 3.7 ± 0.2‡ 4.8 ± 0.3*‡ 5.2 ± 0.3*‡ 5.8 ± 0.4*‡ 6.1 ± 0.5*‡ 6.8 ± 0.4*‡
 NLA + Indo 2.6 ± 0.3† 3.3 ± 0.3† 4.2 ± 0.3*† 4.6 ± 0.3*† 5.2 ± 0.3*† 6.2 ± 0.4*†
 NLA + Indo + Tezo 3.4 ± 0.3‡ 4.5 ± 0.3*‡ 5.1 ± 0.2*‡ 5.6 ± 0.3*‡ 6.2 ± 0.3*‡ 7.1 ± 0.4*‡
MAP (mmHg)
 Control 90 ± 2 83 ± 2* 81 ± 2*§ 81 ± 2*§ 82 ± 3* 83 ± 3
 Tezo 80 ± 2‡§ 76 ± 2‡ 75 ± 2‡ 76 ± 2‡ 76 ± 2‡ 77 ± 2‡
 Indo 126 ± 5† 114 ± 4† 104 ± 4*† 101 ± 4*† 99 ± 4*† 94 ± 5*†
 Indo + Tezo 103 ± 4†‡ 97 ± 5†‡ 94 ± 3*†‡ 90 ± 4*‡ 87 ± 4*‡ 85 ± 4*‡
 NLA 125 ± 5† 112 ± 5† 113 ± 4*† 115 ± 4*† 115 ± 3*† 111 ± 3*†
 NLA + Tezo 115 ± 5†‡ 97 ± 4*†‡ 94 ± 5*†‡ 94 ± 5*†‡ 94 ± 5*†‡ 93 ± 4*†‡
 NLA + Indo 147 ± 5† 141 ± 8† 133 ± 7*† 132 ± 7*† 127 ± 7*† 118 ± 5*†
 NLA + Indo + Tezo 129 ± 4‡ 117 ± 4*‡ 112 ± 5*‡ 109 ± 5*‡ 108 ± 5* 105 ± 5*
SV (ml)
 Control 24 ± 2 26 ± 2* 24 ± 1 25 ± 2 25 ± 2* 25 ± 1
 Tezo 23 ± 1 27 ± 2* 27 ± 2* 27 ± 2*‡ 27 ± 2*‡ 27 ± 2*‡
 Indo 30 ± 2 32 ± 2* 34 ± 2* 34 ± 2* 35 ± 2*† 36 ± 1*†
 Indo + Tezo 31 ± 2† 34 ± 2* 34 ± 1* 37 ± 1*† 36 ± 2*† 36 ± 1*†
 NLA 26 ± 2 28 ± 2 29 ± 2*† 28 ± 2†§ 29 ± 2 28 ± 2
 NLA + Tezo 28 ± 1 32 ± 2 32 ± 2*‡ 33 ± 3‡ 33 ± 3 32 ± 3
 NLA + Indo 27 ± 1 33 ± 1† 36 ± 1 36 ± 1 36 ± 1 37 ± 1
 NLA + Indo + Tezo 39 ± 2†‡ 43 ± 2*†‡ 41 ± 1†‡ 42 ± 1†‡ 42 ± 1†‡ 41 ± 2†‡
PAP (mmHg)
 Control 25 ± 3§ 24 ± 2§ 29 ± 2*§ 31 ± 2*§ 34 ± 2*§ 38 ± 2*§
 Tezo 21 ± 2§ 21 ± 2‡§ 25 ± 2‡§ 28 ± 2*‡§ 31 ± 2*§ 33 ± 2*‡§
 Indo 28 ± 3†§ 29 ± 3†§ 32 ± 3§ 33 ± 3§ 35 ± 3*§ 37 ± 3*§
 Indo + Tezo 21 ± 2‡ 22 ± 3‡§ 28 ± 4*‡§ 29 ± 3*§ 29 ± 3*†‡§ 33 ± 3*†‡§
 NLA 29 ± 2† 35 ± 2 38 ± 3*†§ 41 ± 3*†§ 44 ± 3*†§ 48 ± 2*†§
 NLA + Tezo 25 ± 2 24 ± 3 26 ± 2‡§ 30 ± 2‡ 33 ± 3*‡ 37 ± 3*‡
 NLA + Indo 35 ± 4†§ 34 ± 4 37 ± 5 39 ± 5 43 ± 5* 45 ± 4*
 NLA + Indo + Tezo 29 ± 3‡ 27 ± 4*‡ 31 ± 3*‡§ 34 ± 4*‡ 38 ± 4*‡§ 42 ± 4*§
LAP (mmHg)
 Control 12 ± 2§ 10 ± 2§ 13 ± 2§ 14 ± 2§ 16 ± 2*§ 18 ± 2*§
 Tezo 11 ± 2§ 10 ± 2§ 12 ± 2§ 15 ± 2*§ 17 ± 2*§ 18 ± 2*§
 Indo 17 ± 3† 15 ± 3† 18 ± 3†§ 20 ± 3§ 20 ± 3§ 20 ± 2§
 Indo + Tezo 11 ± 2‡ 11 ± 3§ 15 ± 2*†§ 16 ± 2*†§ 17 ± 2*§ 19 ± 2*§
 NLA 17 ± 1†§ 14 ± 2§ 14 ± 1§ 16 ± 1§ 17 ± 1§ 18 ± 1§
 NLA + Tezo 13 ± 1‡§ 9 ± 2* 10 ± 1*†‡§ 13 ± 1 15 ± 1 17 ± 1
 NLA + Indo 14 ± 3† 14 ± 3 15 ± 3 16 ± 3 17 ± 3 18 ± 3
 NLA + Indo + Tezo 15 ± 3†‡ 13 ± 2 14 ± 2§ 15 ± 3 18 ± 3 18 ± 3
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HR, heart rate; CO, cardiac output; MAP, mean arterial pressure; PAP, pulmonary arterial pressure; LAP, left atrial pressure; Tezo, tezosentan; Indo, indometacin. Data are mean ± s.e.m.

*

P ≤ 0.05 versus rest (lying)

†

P ≤ 0.05 versus corresponding control

‡

P ≤ 0.05 effect of Tezo

§

P ≤ 0.05 MI versus normal.

Table 4.

Arterial blood gas values in normal swine at rest and during exercise before and after ETA/ETB receptor blockade in the absence and presence of single or combined blockade of COX and eNOS

Rest Exercise level (km h−1)
(Lying) 1 2 3 4
Inline graphic (mmHg)
 Control 97 ± 2 96 ± 2 95 ± 2 95 ± 2 91 ± 2*
 Tezo 98 ± 2 95 ± 4 95 ± 2 93 ± 2* 92 ± 2*
 Indo 115 ± 5† 110 ± 5† 116 ± 4† 109 ± 7 107 ± 5†
 Indo + Tezo 115 ± 4† 104 ± 4* 107 ± 5† 105 ± 5 100 ± 5*†
 NLA 98 ± 2  91 ± 3 94 ± 3 92 ± 4 93 ± 5
 NLA + Tezo 95 ± 3 92 ± 3 92 ± 4 92 ± 4 88 ± 2
 NLA + Indo 125 ± 6† 128 ± 8† 120 ± 3† 112 ± 3† 106 ± 3*†
 NLA + Indo + Tezo 111 ± 8 114 ± 11 109 ± 4‡ 106 ± 3 103 ± 5
Inline graphic(%)
 Control 93 ± 1 92 ± 1 93 ± 1 93 ± 1 93 ± 1
 Tezo 93 ± 1 92 ± 1* 93 ± 1 92 ± 1 92 ± 1
 Indo 93 ± 1 93 ± 1 94 ± 1 93 ± 1 93 ± 1
 Indo + Tezo 93 ± 1 92 ± 1 93 ± 1 93 ± 1 93 ± 1
 NLA 93 ± 1 92 ± 1 93 ± 1 93 ± 1 93 ± 1
 NLA + Tezo 93 ± 1 92 ± 1 93 ± 1 93 ± 1 93 ± 1
 NLA + Indo 94 ± 1 95 ± 1 95 ± 1 95 ± 1 94 ± 1
 NLA + Indo + Tezo 94 ± 1 93 ± 1 94 ± 1 94 ± 1 94 ± 1
pH
 Control 7.439 ± 0.019 7.467 ± 0.008 7.474 ± 0.007 7.484 ± 0.007 7.490 ± 0.007*
 Tezo 7.454 ± 0.006 7.455 ± 0.006 7.473 ± 0.008* 7.481 ± 0.008* 7.490 ± 0.007*
 Indo 7.500 ± 0.008† 7.501 ± 0.007† 7.509 ± 0.008† 7.505 ± 0.009† 7.511 ± 0.007†
 Indo + Tezo 7.486 ± 0.007† 7.490 ± 0.008† 7.494 ± 0.007 7.502 ± 0.007*† 7.503 ± 0.007
 NLA 7.495 ± 0.011 7.502 ± 0.014 7.512 ± 0.013† 7.513 ± 0.014 7.527 ± 0.009
 NLA + Tezo 7.478 ± 0.009‡ 7.488 ± 0.013 7.503 ± 0.010* 7.501 ± 0.007 7.516 ± 0.011*
 NLA + Indo 7.494 ± 0.011 7.493 ± 0.013 7.495 ± 0.010 7.489 ± 0.012 7.476 ± 0.017
 NLA + Indo + Tezo 7.477 ± 0.006 7.484 ± 0.009 7.494 ± 0.008 7.507 ± 0.009 7.502 ± 0.009
Inline graphic (mmHg)
 Control 44 ± 1 44 ± 1 43 ± 1 42 ± 1* 41 ± 1*
 Tezo 46 ± 1 46 ± 1 45 ± 1 42 ± 1* 42 ± 1*
 Indo 39 ± 4 39 ± 4 39 ± 4 39 ± 4 38 ± 4
 Indo + Tezo 37 ± 1† 37 ± 1† 36 ± 1† 35 ± 1*† 35 ± 1†
 NLA 43 ± 1 42 ± 1 41 ± 1 41 ± 1 37 ± 1
 NLA + Tezo 44 ± 1 42 ± 1 41 ± 1* 41 ± 1* 38 ± 1*
 NLA + Indo 31 ± 1† 33 ± 2† 32 ± 2† 34 ± 2† 34 ± 1
 NLA + Indo + Tezo 37 ± 2†‡ 37 ± 1† 37 ± 1† 35 ± 1† 35 ± 1
HCO3− (mmol l−1)
 Control 30 ± 1 31 ± 1 31 ± 1 30 ± 1 30 ± 1
 Tezo 36 ± 5 36 ± 5 36 ± 5 35 ± 5 35 ± 5
 Indo 29 ± 3 30 ± 3 30 ± 4 30 ± 3 30 ± 3
 Indo + Tezo 27 ± 1† 27 ± 1* 27 ± 1* 26 ± 1 26 ± 1
 NLA 33 ± 1 32 ± 1 32 ± 1 32 ± 1 30 ± 1
 NLA + Tezo 32 ± 1 32 ± 1 31 ± 1 31 ± 1 30 ± 1
 NLA + Indo 23 ± 1† 24 ± 2† 24 ± 2† 25 ± 2† 25 ± 2†
 NLA + Indo + Tezo 27 ± 1† 27 ± 1† 28 ± 1† 27 ± 1† 27 ± 1†
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Inline graphic, oxygen saturation; Tezo, tezosentan; Indo, indometacin. Data are mean ± s.e.m.

*

P ≤ 0.05 versus rest (lying)

†

P ≤ 0.05 versus corresponding control

‡

P ≤ 0.05 effect of Tezo.

Table 5.

Arterial blood gas values in MI swine at rest and during exercise before and after ETA/ETB receptor blockade in the absence and presence of single or combined blockade of COX and eNOS

Rest Exercise level (km h−1)
(Lying) 1 2 3 4
Inline graphic (mmHg)
 Control 101 ± 2 89 ± 5* 86 ± 4*§ 90 ± 3* 86 ± 3*
 Tezo 93 ± 2† 87 ± 3 87 ± 4* 86 ± 3* 84 ± 3*
 Indo 122 ± 7† 121 ± 7 117 ± 5† 109 ± 3† 105 ± 6
 Indo + Tezo 109 ± 4 102 ± 5* 108 ± 7‡ 106 ± 4 101 ± 3
 NLA 95 ± 3 93 ± 4 93 ± 5 93 ± 4 91 ± 5
 NLA + Tezo 92 ± 5 90 ± 5 94 ± 3 93 ± 3 85 ± 4
 NLA + Indo 117 ± 3† 113 ± 9 104 ± 6*§ 103 ± 6 102 ± 5*
 NLA + Indo + Tezo 104 ± 3‡ 104 ± 5 97 ± 5 99 ± 5 92 ± 7
Inline graphic (%)
 Control 91 ± 1 89 ± 1§ 89 ± 1*§ 90 ± 1§ 90 ± 1§
 Tezo 91 ± 1 89 ± 1§ 90 ± 1*§ 89 ± 1§ 89 ± 1§
 Indo 93 ± 1 94 ± 1† 94 ± 1† 94 ± 1† 92 ± 2
 Indo + Tezo 93 ± 1 92 ± 1 93 ± 1 94 ± 1† 93 ± 1†
 NLA 91 ± 1 92 ± 1 92 ± 1 92 ± 1 92 ± 1
 NLA + Tezo 91 ± 1 92 ± 2 94 ± 1 93 ± 2 92 ± 2
 NLA + Indo 96 ± 1 96 ± 1† 95 ± 1 95 ± 1 95 ± 1
 NLA + Indo + Tezo 95 ± 1‡ 94 ± 1‡ 94 ± 1‡ 94 ± 1 92 ± 2
pH
 Control 7.457 ± 0.006 7.457 ± 0.009 7.460 ± 0.009 7.482 ± 0.015* 7.480 ± 0.013*
 Tezo 7.455 ± 0.005 7.464 ± 0.010 7.462 ± 0.005 7.474 ± 0.009* 7.474 ± 0.011
 Indo 7.479 ± 0.012 7.502 ± 0.013† 7.514 ± 0.012*† 7.511 ± 0.015 7.488 ± 0.026
 Indo + Tezo 7.464 ± 0.007 7.487 ± 0.017 7.489 ± 0.014 7.507 ± 0.013*† 7.508 ± 0.015*
 NLA 7.467 ± 0.005 7.491 ± 0.009 7.503 ± 0.011*† 7.505 ± 0.008*† 7.516 ± 0.011*†
 NLA + Tezo 7.472 ± 0.007 7.490 ± 0.005* 7.495 ± 0.007 7.494 ± 0.011 7.494 ± 0.011
 NLA + Indo 7.508 ± 0.016 7.516 ± 0.011 7.504 ± 0.012 7.503 ± 0.013 7.505 ± 0.014
 NLA + Indo + Tezo 7.463 ± 0.008‡ 7.555 ± 0.060 7.486 ± 0.010 7.500 ± 0.010* 7.489 ± 0.011
Inline graphic (mmHg)
 Control 45 ± 1 44 ± 1 45 ± 1 42 ± 1* 41 ± 1*
 Tezo 46 ± 1 45 ± 1 44 ± 1 43 ± 1* 42 ± 1*
 Indo 33 ± 1† 33 ± 1† 32 ± 1† 34 ± 1† 36 ± 2
 Indo + Tezo 39 ± 1†‡ 39 ± 1†‡ 38 ± 1†‡ 37 ± 1†‡ 36 ± 1*†
 NLA 47 ± 3 44 ± 2* 42 ± 2* 42 ± 2* 40 ± 2*
 NLA + Tezo 50 ± 3 48 ± 4* 47 ± 4 48 ± 5 55 ± 9
 NLA + Indo 34 ± 3 35 ± 3 36 ± 3* 34 ± 1 33 ± 1
 NLA + Indo + Tezo 43 ± 4‡ 40 ± 3*‡ 41 ± 4‡ 40 ± 3* 40 ± 3
HCO3− (mmol l−1)
 Control 30 ± 1 30 ± 1 30 ± 1 30 ± 1 29 ± 1*
 Tezo 31 ± 1 31 ± 1 30 ± 1 31 ± 1 30 ± 1*
 Indo 24 ± 1† 25 ± 1* 25 ± 1 26 ± 1* 25 ± 1
 Indo + Tezo 27 ± 1‡ 28 ± 1*‡ 28 ± 1‡ 28 ± 1‡ 27 ± 1
 NLA 32 ± 2 32 ± 2 32 ± 2 32 ± 2 31 ± 2
 NLA + Tezo 35 ± 3 35 ± 3 35 ± 3 36 ± 4 41 ± 7
 NLA + Indo 26 ± 2 28 ± 3 28 ± 3 26 ± 1 26 ± 1
 NLA + Indo + Tezo 30 ± 3‡ 31 ± 4‡ 31 ± 3‡ 31 ± 3 31 ± 3
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Inline graphic, oxygen saturation; Tezo, tezosentan; Indo, indometacin. Data are mean ± s.e.m.

*

P ≤ 0.05 versus rest (lying)

†

P ≤ 0.05 versus corresponding control

‡

P ≤ 0.05 effect of Tezo

§

P ≤ 0.05 MI versus normal.

Role of NO and prostanoids in the regulation of vascular tone

Systemic circulation

Administration of the eNOS inhibitor NLA or the COX inhibitor indometacin resulted in a pressor response due to systemic vasoconstriction as evidenced by marked increases in mean arterial pressure and SVR in both normal and MI swine (Fig. 2, Tables 2 and 3). As a result heart rate decreased, while stroke volume was not altered, resulting in a decrease in cardiac output. During exercise the systemic vasoconstriction and the pressor response to indometacin were progressively blunted, whereas the pressor response and systemic vasoconstriction in response to NLA were maintained (Fig. 2, Tables 2 and 3).

Figure 2. Interaction between NO, prostanoids and endothelin in the regulation of systemic vascular tone in normal swine and swine with MI.

Figure 2

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From left to right: effect of endothelin receptor blockade (tezosentan (Tezo), COX inhibition and endothelin receptor blockade (Indo + Tezo), NOS inhibition and endothelin receptor blockade (NLA + Tezo) and combined inhibition of COX and NOS and endothelin receptor blockade (NLA + Indo + Tezo) on the relation between body O2 consumption (B Inline graphic) and systemic vascular resistance (SVR) in normal swine (upper panels) and swine with MI (lower panels). Significance symbols denote a change in intercept or intercept and slope. *P ≤ 0.05 effect of Tezo; †P ≤ 0.05 effect of Tezo in presence of Indo, NLA or NLA + Indo versus Tezo alone; ‡P ≤ 0.05 effect of Tezo is different after MI.

Pretreatment with NLA enhanced the vasoconstriction induced by indometacin, particularly at rest, as evidenced by an exaggerated increase in SVR in normal swine, indicating that NO and prostanoids act synergistically to maintain a low vasomotor tone. Yet, despite the enhanced response at rest, exercise-induced vasodilatation was unmitigated in the presence of both NLA and indometacin (Fig. 2). In contrast to the findings in normal swine, the vasoconstrictor response to indometacin was not altered by pretreatment with NLA in swine with MI, either at rest or during exercise.

Pulmonary circulation

Indometacin had no effect on the PVR during either rest or exercise in normal swine; however, PVR increased by 42 ± 8% in response to indometacin at rest, but not during exercise in MI swine (P ≤ 0.05). Administration of NLA produced a similar rise in PVR in normal and MI swine at rest, which persisted during exercise (Fig. 3). Subsequent inhibition of COX with indometacin resulted in a 71 ± 18% increase in PVR in MI swine at rest (P ≤ 0.05), but did not increase PVR in either normal or MI swine during exercise (Fig. 3).

Figure 3. Interaction between NO, prostanoids and endothelin in the regulation of systemic vascular tone in normal swine and swine with MI.

Figure 3

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From left to right: effect of endothelin receptor blockade (tezosentan (Tezo); 16 normal and 9 MI swine); COX inhibition and endothelin receptor blockade (Indo + Tezo; 9 normal and 8 MI swine); NOS inhibition and endothelin receptor blockade (NLA + Tezo; 10 normal and 7 MI swine) and combined inhibition of COX and NOS and endothelin receptor blockade (NLA + Indo + Tezo; 6 normal and 6 MI swine) on the relation between body O2 consumption (B Inline graphic) and pulmonary vascular resistance (PVR) in normal swine (upper panels) and swine with MI (lower panels). Significance symbols denote a change in intercept or intercept and slope. *P ≤ 0.05 effect of Tezo versus corresponding control; †P ≤ 0.05, ††P ≤ 0.10 effect of Tezo in presence of Indo, NLA or NLA + Indo versus Tezo alone; ‡P ≤ 0.05 effect of Tezo is different after MI.

The pulmonary haemodynamic effects of indometacin and NLA did not result in impaired pulmonary gas exchange in either normal (Table 4) or MI swine (Table 5). Thus, arterial Inline graphic was not altered by NLA in either group, while indometacin actually produced a small increase in arterial Inline graphic that was, however, similar in both groups of swine. In view of the small indometacin-induced decrease in arterial Inline graphic, it is likely that this was caused by mild hyperventilation.

The role of endothelin in the regulation of vascular tone

Systemic circulation

Administration of the mixed ETA/ETB antagonist tezosentan resulted in a decrease of mean arterial pressure and SVR in both normal and MI swine. The systemic vasodilatation resulted in a compensatory, probably baroreflex-mediated increase in heart rate and cardiac output (Tables 2 and 3). The tezosentan-induced vasodilator response diminished with increasing exercise levels (Fig. 2). Pretreatment with the eNOS inhibitor NLA enhanced the vasodilator response to tezosentan at rest and during exercise in both normal and MI swine (Fig. 2). Administration of COX inhibitor indometacin prior to tezosentan, increased the vasodilator response to tezosentan at rest, however, this effect waned with exercise (Fig. 2). In normal swine, combined administration of indometacin and NLA increased the vasodilator responses to tezosentan even further, particularly at rest (Fig. 2). These observations suggest that both NO and prostanoids act in concert to blunt the vasoconstrictor effects of ET in the systemic circulation.

Although plasma ET levels were increased following MI (3.9 ± 0.5 pm versus 2.3 ± 0.3 pm in normal swine, P ≤ 0.05), MI did not change the vasodilator effect of tezosentan on the systemic circulation after administration of NLA or indometacin alone (Fig. 2), indicating that after MI, NO and prostanoids still exert part of their vasodilator effect through inhibition of ET-induced vasoconstriction. However, the effect of tezosentan after combined administration of NLA and indometacin was reduced in swine with MI as compared to normal swine, and was similar to the effect of tezosentan in the presence of NLA or indometacin alone (Fig. 2). Thus, the vasoconstrictor effect of ET in the systemic circulation after MI is limited by NO and prostanoids, but loss of both NO and prostanoids does not result in further activation of the ET system as compared to loss of one of these compounds (Fig. 2).

Pulmonary circulation

Administration of tezosentan did not exert any effect on the pulmonary circulation of normal swine at rest. However, during exercise pulmonary arterial pressure and pulmonary vascular resistance decreased in response to administration of tezosentan (Fig. 3). In agreement with our previous observations (Houweling et al. 2006), the vasodilator response to tezosentan was more pronounced in MI swine as compared to normal swine (Fig. 3). Tezosentan had no effect on arterial blood gas values in either normal (Table 4) or MI swine (Table 5).

Pretreatment with NLA markedly augmented the vasodilator effect of tezosentan at rest as well as during exercise in normal swine and swine with MI (Fig. 3), indicating that NO limits the ET-induced pulmonary vasoconstriction to the same extent in both groups. Pretreatment with indometacin did not affect vasodilatation in response to tezosentan in normal swine. However, indometacin resulted in an increase in the vasodilator response to tezosentan in MI swine particularly at rest (Fig. 3), indicating that prostanoids limit ET-induced pulmonary vasoconstriction in swine with MI. In the presence of NLA, the pulmonary vasodilator effect of tezosentan in normal swine was not affected by administration of indometacin (Fig. 3). In contrast, in swine with MI, the effect of tezosentan was smaller in the presence of NLA and indometacin as compared to the presence of NLA alone (Fig. 3).

Discussion

The main findings of the present study are that in swine with pulmonary hypertension secondary to MI: (i) the contribution of endogenous ET to pulmonary vasomotor tone is enhanced as compared to normal swine; (ii) prostanoids exert a vasodilator influence on the pulmonary vasculature and attenuate the vasoconstrictor influence of endogenous ET, but only under resting conditions and not during exercise, while in normal swine prostanoids do not appear to play a role in control of pulmonary resistance vessel tone either at rest or during exercise and do not modulate the pulmonary vasoconstrictor influence of ET; (iii) the contribution of endogenous NO to control of pulmonary resistance vessel tone is not altered as compared to normal swine, while endogenous NO blunts the vasoconstrictor influence of ET to a similar extent as in normal swine.

Vasomotor control of the pulmonary vasculature: matching of ventilation to perfusion

For the lungs to perform their gas-exchange function efficiently, the pulmonary circulation must maintain low pulmonary arterial and capillary pressure, while accommodating the entire cardiac output. In addition, perfusion must be distributed among the ventilated units through local variations in pulmonary vascular resistance so that flow matches local ventilation (Dawson, 1984). The pulmonary vascular resistance is determined by passive distension as well as active regulation of vasomotor tone (Dawson, 1984). During exercise, the increase in cardiac output results in an increased flow through the pulmonary vascular bed, which together with the increase in left atrial pressure accounts for the majority of the increase in pulmonary arterial pressure. In the human lung with its large base and small top, the well-ventilated base is mainly perfused in the upright posture under resting conditions, while the increase in pressure during exercise recruits perfusion of the top and results in a ∼40% decrease in pulmonary vascular resistance (Bake et al. 1974; Reeves & Taylor, 1996). In contrast to humans, quadrupeds have small ventral but large dorsal lung volumes. As a result, resting pulmonary pressure is slightly higher in quadrupeds, but ventilation and perfusion are more evenly distributed across the entire lung (Reeves & Taylor, 1996). During exercise, flows in all lung areas increase proportionally to their resting flows so that flow distribution does not change, and hence pulmonary vascular resistance decreases by only 10–20% (Reeves & Taylor, 1996). Another consequence of the body posture of quadrupeds is that perfusion and ventilation are already matched under resting conditions, with little additional recruitment of lung areas during exercise (Reeves & Taylor, 1996). This may explain our observation of a small decrease in arterial Inline graphic at the highest level of exercise, which is in accordance with reports from various laboratories (Hastings et al. 1982; Armstrong et al. 1987; Merkus et al. 2006), although this is not a uniform finding (Laughlin et al. 1989; Duncker et al. 1998). It is important to note that small changes in arterial Inline graphic contribute little to the arterial oxygen content, since the arterial oxygen– haemoglobin dissociation curve operates at its upper plateau. Consequently, arterial haemoglobin oxygen saturation was maintained (Tables 4 and 5).

The changes in pulmonary vascular resistance induced by ET receptor blockade and eNOS inhibition do not affect arterial Inline graphic, suggesting that matching between ventilation and perfusion is not affected. In contrast, COX inhibition did not affect pulmonary vascular resistance, but increased arterial Inline graphic, which may have been the result of the increased pulmonary retention time of the blood due to the decrease in cardiac output, in conjunction with mild hyperventilation.

Vasomotor control of the pulmonary vasculature following MI

MI results in neurohumoral activation, i.e. activation of the sympathetic nervous system and the renin–angiotensin system. In our model, elevations of adrenaline, noradrenaline and angiotensin-II are most pronounced during exercise (Haitsma et al. 2001; Merkus et al. 2006). Additionally, MI increases LV filling pressures that are transmitted backwards into the pulmonary circulation (Moraes et al. 2000). Although the secondary increase in pulmonary arterial pressure may initially act to passively decrease PVR, it also results in a ‘reactive’ increase in PVR and inward remodelling and stiffening of the pulmonary vasculature (Moraes et al. 2000). This reactive increase in PVR may be caused by increased contribution of vasoconstrictors or a decreased contribution of vasodilators to pulmonary vasomotor tone and may limit the decrease in PVR during exercise (Moraes et al. 2000). This reduced vasodilator capacity of the pulmonary vasculature results in exacerbation of pulmonary hypertension during exercise, which increases RV afterload and contributes to RV hypertrophy and RV failure (Moraes et al. 2000). Moreover, it has been reported that exercise capacity is inversely correlated with PVR and with the failure to decrease PVR during exercise in patients with heart failure (Franciosa et al. 1985). The pathogenesis of the increased PVR in pulmonary hypertension is incompletely understood, but may involve alterations in serotonin, thromboxane A2 and/or angiotensin-II (Said, 2006). Additionally, it may be mediated in part by downregulation or desensitization of β-adrenoceptors. In the pulmonary vasculature of healthy swine, the sympathetic nervous system exerts a vasodilator influence that is mediated through β-adrenoceptors (Stubenitsky et al. 1998). Although the increased sympathetic drive following MI may initially counteract the increase in PVR, β-blockade does not affect PVR in patients with chronic heart failure (Metra et al. 1994; Nodari et al. 2003), suggesting that prolonged β-adrenergic stimulation may have caused desensitization of the β-adrenoceptors, thereby contributing to the increase in PVR following pulmonary hypertension. Additionally, endothelial dysfunction may play a role in this process (Moraes et al. 2000; Budhiraja et al. 2004; Humbert et al. 2004). With endothelial dysfunction, the production of the vasodilators NO and prostacyclin decreases, while the production of the vasoconstrictor ET increases. This imbalance between vasodilators and vasoconstrictors is likely to contribute to an increase in pulmonary vascular tone, thereby increasing PVR. In agreement with a previous study from our laboratory (Houweling et al. 2006), we observed an enhanced ET-mediated vasoconstriction in the present study.

ET and secondary pulmonary hypertension following MI

Activation of the ET system can contribute to the progression of pulmonary hypertension, and has been shown to occur in various animal models as well as patients with pulmonary hypertension. Thus, production of ET is increased in the pulmonary endothelium of subjects with pulmonary hypertension (Giaid et al. 1993), while pulmonary clearance of ET is reduced (Dupuis et al. 1998a,b; Staniloae et al. 2004; Langleben et al. 2005). The increased production and decreased clearance of ET together result in increased circulating levels of ET, which is an independent predictor of clinical prognosis and survival (Spieker et al. 2001). Moreover, the ET plasma levels correlate closely with the reduced exercise capacity of patients with pulmonary hypertension (Krum et al. 1995).

In addition to increased plasma levels of ET, which in themselves could be sufficient to explain the increased vasoconstrictor influence of ET on the pulmonary circulation, we found that the sensitivity of the pulmonary circulation to administration of exogenous ET was increased after MI (Houweling et al. 2006). Moreover, while the response to endogenous ET was exclusively ETB receptor mediated in normal swine, an ETA receptor-mediated component emerged in swine with MI (Houweling et al. 2006). In contrast, studies on isolated pulmonary resistance vessels from rats (Sauvageau et al. 2006) and rabbits (Docherty & MacLean, 1998) with and without MI show no difference in response to ET or in ETA or ETB receptor expression (Sauvageau et al. 2006). However, the response of isolated arterioles to administration of ET may be different from the response of the intact vasculature (Merkus et al. 2005). This could be due to factors such as NO (Goligorsky et al. 1994; Wiley & Davenport, 2001) and possibly prostacyclin, that are capable of modifying ET receptor sensitivity and are present in vivo, while they are reduced in the in vitro preparations.

Prostanoids and pulmonary hypertension

Prostacyclin has very potent pulmonary vasodilator properties (Albertini et al. 1996). Moreover, both COX-1 and COX-2 have been shown to be expressed in the pulmonary vasculature of rats (Ermert et al. 1998) and mice (Baber et al. 2005). Yet, in agreement with other studies (Newman et al. 1986; Albertini et al. 1996; Merkus et al. 2004), endogenous prostanoids did not contribute to regulation of basal pulmonary tone and did not influence ET-mediated pulmonary vasoconstriction (Houweling et al. 2005), suggesting that endogenous prostanoid production is absent in the normal healthy pulmonary circulation. However, after MI, inhibition of COX resulted in pulmonary vasoconstriction at rest in both the absence and presence of inhibition of NO synthesis, suggesting that after MI, prostanoids exert a basal vasodilator influence and therefore act to limit the pulmonary vasoconstriction that occurs after MI. These data are in accordance with a study on isolated pulmonary smooth muscle cells, showing that stimulation of these cells with cytokines, to mimic pathological pulmonary conditions can induce COX-2 expression, and thereby increase production of prostanoids (Wort et al. 2002). Because prostanoids have been shown to improve the balance between ET clearance and production (Langleben et al. 1999), and because blockade of COX has been shown to affect pulmonary vascular tone only when ET receptors are unblocked (Gomez-Alamillo et al. 2003), this slight increase in pulmonary prostanoids may have acted to limit ET-induced pulmonary vasoconstriction (Wort et al. 2002) rather than to induce pulmonary vasodilatation itself. Indeed, the present study also shows that, in the presence of tezosentan, PVR is not different before and after inhibition of COX, suggesting that the main action of prostanoids in the pulmonary vasculature is to suppress the ET-system.

In contrast to the findings under resting conditions, there was no effect of COX inhibition on pulmonary vasomotor tone during exercise in the presence or absence of eNOS inhibition, suggesting that the vasodilator effect of prostanoids is lost during exercise. In accordance with these findings, the pulmonary vasodilator effect of tezosentan during exercise was similar in the presence and absence of COX inhibition.

NO and pulmonary hypertension

NO is a very potent vasodilator of the pulmonary vasculature, and endogenous NO contributes to the regulation of pulmonary vascular tone in normal swine (Albertini et al. 1996; Duncker et al. 2000). Physiologically, NO is produced by eNOS in response to increases in shear stress and/or in response to receptor activation, and produces vasodilatation through activation of soluble guanylyl cyclase in vascular smooth muscle, followed by reduction in intracellular calcium concentrations as well as reduction in myosin light chain phosphorylation, both of which are essential for smooth muscle cell contraction (Hampl & Herget, 2000). Besides its direct vasodilator effect on the pulmonary vasculature, NO can also cause vasodilatation through inhibition of the ET system. Thus, NO inhibits the production of ET through endothelin-converting enzyme (Lavallee et al. 2001) and modulates ETA receptor sensitivity (Wiley & Davenport, 2001). Indeed, the vasodilatation in response to ETA/ETB receptor blockade with tezosentan is larger after inhibition of NO synthesis (present study, Houweling et al. 2005), suggesting that part of the vasodilator effect of NO is exerted through inhibition of ET-mediated vasoconstriction.

The direct vasoconstrictor effect of inhibition of NO synthesis within the pulmonary vascular bed may be modified by the central effects of inhibition of eNOS. Thus, inhibition of central NO will result in an increase in sympathetic outflow (Sartori et al. 2005). Conversely, the systemic pressor response was accompanied by a decrease in heart rate, probably mediated via the baroreceptor reflex. In both normal and MI swine, NLA resulted in similar decrements in heart rate, indicating that in both groups the baroreceptor reflex outweighed any effects of central eNOS inhibition. The resultant attenuated sympathetic activity will result in blunted β-receptor-mediated pulmonary vasodilatation (Stubenitsky et al. 1998), thereby potentially enhancing the vasoconstriction produced by inhibition of endo-thelial NO production. However, the increase in pulmonary vascular resistance produced by NLA was much more pronounced than that previously observed following complete β-adrenergic receptor blockade (Stubenitsky et al. 1998), suggesting that the NLA-induced increase in pulmonary vascular resistance was principally the result of inhibition of local NO synthesis.

Studies on the role of NO in regulation of pulmonary vasomotor tone in pulmonary hypertension have yielded variable results in that the contribution of NO is either increased, decreased or unaltered, depending on the model of pulmonary hypertension as well as the severity and duration of the disease. In early stages of pulmonary hypertension, pulmonary vasoconstriction may increase shear stress, thereby increasing NO production and counteracting the development of pulmonary hypertension (Hampl & Herget, 2000). However, when pulmonary hypertension persists for longer periods of time, the chronically elevated pressure may contribute to endothelial dysfunction, thereby decreasing NO production and promoting the development of pulmonary hypertension (Ontkean et al. 1991; Cooper et al. 1998; Ben Driss et al. 2000). The present study shows that, in accordance with a previous study from our laboratory (Haitsma et al. 2002), the pulmonary vasoconstriction induced by inhibition of eNOS with NLA was similar in swine with MI as compared to normal swine, suggesting that basal NO production is maintained ∼2 weeks after induction of MI. Apparently, the MI-induced pulmonary hypertension is not severe enough and/or did not last long enough to provoke perturbations in the NO system. These data are in accordance with a study in humans, showing that basal NO activity is reduced in patients with congestive heart failure and severely elevated levels of pulmonary arterial pressure and pulmonary vascular resistance, but not in patients with mildly elevated levels of pulmonary arterial pressure and pulmonary vascular resistance (Cooper et al. 1998).

In accordance with the maintained basal NO production in swine with MI (Haitsma et al. 2002), the present study shows that the vasodilatation induced by ETA/ETB receptor blockade in MI swine was increased after inhibition of NO synthesis to a similar extent as in normal swine. These findings indicate that NO acts to limit the ET-induced pulmonary vasoconstriction to a similar degree in MI and normal swine. Thus, the increased sensitivity of the pulmonary vasculature to ET as observed in our swine model (Houweling et al. 2006) cannot be explained by a loss of NO-mediated inhibition of ET-induced vasoconstriction.

Endothelial control of the systemic resistance vessels after MI

NO and prostanoids exert a vasodilator influence on the systemic vasculature of normal swine, while ET exerts a vasoconstrictor influence. The effects of endo-genous NO, prostanoids and ET on systemic vasomotor tone are similar in normal swine and swine with MI. Moreover, in both normal swine and swine with MI, the vasodilatation induced by endogenous NO and prostanoids is in part mediated through inhibition of ET-mediated vasoconstriction (Langleben et al. 1999; Lavallee et al. 2001; Wort et al. 2002; Kelly et al. 2004) as evidenced by a larger effect of ETA/ETB receptor blockade after blocking NO or prostanoid production, and in part a direct effect, as SVR does not return to baseline levels after ETA/ETB receptor blockade.

In normal swine, the vasoconstrictor effect of combined inhibition of NO and prostanoids is larger than the sum of the individual effects, suggesting that inhibition of one endogenous vasodilator is compensated by increased production of the other dilator (Merkus et al. 2004). These data are in accordance with findings in isolated endothelial cells as well as in vivo (Puybasset et al. 1996; Osanai et al. 2000; Marcelin-Jimenez & Escalante, 2001). Additionally, the effect of ETA/ETB receptor blockade is significantly enhanced when production of both NO and prostanoids is inhibited, suggesting that NO and prostanoids act in concert to limit ET-induced vasoconstriction. In contrast, in swine with MI, the effects of inhibition of NO and prostanoid production on systemic vasomotor tone are additive. Moreover, the effect of ETA/ETB receptor blockade is not further enhanced after combined inhibition of NO and prostanoids. These findings could be interpreted to suggest that after MI the effect of loss of NO or prostanoids on vasomotor tone and on ET-mediated vasoconstriction is not compensated by increased production of the other endothelial vasodilator.

Conclusion

The present study demonstrates that ∼2 weeks after MI, there is an increased pulmonary vasoconstrictor influence of endogenous ET. We have previously shown that this increased pulmonary vasoconstrictor influence is probably due to increased plasma ET levels in combination with an increased sensitivity of the pulmonary vasculature to ET (Houweling et al. 2006). In contrast, the pulmonary vasodilator influence of NO was well maintained after MI, while the inhibition of the ET-mediated vasoconstrictor influence by NO was also unmitigated. Moreover, increased production of prostanoids after MI acted to limit the increase in pulmonary vascular resistance under resting conditions. This effect of prostanoids was mediated by a reduction of the ET-induced pulmonary vasoconstriction. Taken together these findings indicate that the increased vasoconstrictor influence of ET in the pulmonary circulation after MI is not the result of a loss of vasodilator influence of NO or prostanoids. Moreover, our findings provide a rationale for the therapeutic use of endothelin receptor blockers to alleviate pulmonary hypertension.

Acknowledgments

This study was supported by The Netherlands Heart Foundation Grants 2000T038 (to D.J.D.) and 2000T042 (to D.M.). Technical assistance of Revelino Vieira and Mario Brandwijk is gratefully acknowledged.

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