Tezosentan reduces the microvascular filtration coefficient in isolated lungs from rats subjected to cecum ligation and puncture

Abstract

Introduction: We recently demonstrated that the non-selective endothelin-1 (ET-1) receptor blocker tezosentan antagonizes ovine acute lung injury (ALI) following infusion of endotoxin or ET-1 by reducing the enhanced lung microvascular pressure, although we could not exclude the possibility of a simultaneous decline in microvascular permeability. In the present study, our aim was to find out if tezosentan reverses the rise in microvascular filtration coefficient (Kfc) in rat lungs that have been isolated and perfused 12 h after cecum ligation and puncture (CLP) or infusion of ET-1.

Methods: Wistar rats (n = 42) were subjected to CLP. Postoperatively, rats were randomized to a CLP group (n = 7) and a CLP + tezosentan group (n = 7); the latter received tezosentan 30 mg/kg. A sham-operated group (n = 5) underwent laparotomy without CLP. Twelve hours postoperatively, the lungs were isolated and perfused with blood from similarly treated rats that also were used to assess plasma concentration of ET-1 and protein kinase Calpha (PKCalpha) in lung tissue. Additionally, isolated blood perfused lungs from healthy rats were randomized to a control group (n = 8), an ET-1 group (n = 7) subjected to pulmonary arterial injection of ET-1 10 nM, and an ET-1 + tezosentan group (n = 7) that received tezosentan 30 mg/kg. All lung preparations received papaverine 0.1 microg/kg added to the perfusate for vasoplegia. Pulmonary hemodynamic variables, Kfc and lung compliance (CL) were assessed.

Results: After CLP, the plasma concentration of ET-1 increased. Papaverine abolished the vasoconstrictor response to ET-1 and the pulmonary vascular pressures remained close to baseline throughout the experiments. Both CLP and injection of ET-1 caused significant changes in Kfc and CL that were prevented in tezosentan-treated rats. Compared to sham-operated animals, CLP increased the content of PKCalpha by 50% and 70% in the cytosolic and the membrane fractions of lung tissue homogenates, respectively. Tezosentan prevented the upregulation of PKCalpha in the membrane fraction.

Conclusion: In rat lungs isolated and perfused after CLP, tezosentan precludes both the increase in Kfc and the upregulation of PKCalpha in the membrane fraction of lung tissue.

Figure 1

Pulmonary arterial pressure responses (ΔP_PA) to endothelin-1 (ET-1) before and after papaverine administration in isolated lungs. Data are presented as mean ± SEM. ^†p < 0.05 from baseline.

Figure 2

Plasma concentrations of endothelin-1 (ET-1) in rats determined 12 h after surgical interventions. Data are presented as mean ± SEM. Sham, sham-operated group (n = 5); CLP, cecum ligation and puncture group (n = 7); CLP+Tezo, cecum ligation and puncture + tezosentan group (n = 7). *p < 0.05 between CLP and CLP+Tezo groups; ^†p < 0.05 between Sham and CLP groups.

Figure 3

Microvascular filtration coefficient (Kfc) and compliance (C_L) in lungs isolated 12 h after surgical interventions. Data are presented as mean ± SEM. Sham, sham-operated group (n = 5); CLP, cecum ligation and puncture group (n = 7); CLP+Tezo, cecum ligation and punction + tezosentan group (n = 7). *p < 0.05 between CLP and CLP+Tezo groups; ^† p < 0.05 between Sham and CLP groups; ^‡ p < 0.05 from t = 0 minutes in Sham group; ^&p < 0.05 from t = 0 minutes in the CLP group; ^§p < 0.05 from t = 0 minutes in the CLP+Tezo group.

Figure 4

Microvascular filtration coefficient (Kfc) and compliance (C_L) after endothelin-1 (ET-1) administration in isolated lungs from healthy rats. Data are presented as mean ± SEM. Control, control group (n = 8); ET-1, endothelin-1 group (n = 7); ET-1+Tezo, endothelin-1+tezosentan group (n = 7). *p < 0.05 between ET-1 and ET-1+Tezo groups; ^†p < 0.05 between control and ET-1 groups; ^‡p < 0.05 from t = 0 minutes in the control group; ^&p < 0.05 from t = 0 minutes in the ET-1 group; ^§p < 0.05 from t = 0 minutes in the ET-1+Tezo group.

Figure 5

Protein kinase Cα in lungs after surgical interventions. (a) Cytosolic and (b) membrane fractions. Data are presented as mean ± SEM. Sham, sham-operated group (n = 5); CLP, cecum ligation and puncture group (n = 6); CLP+Tezo, cecum ligation and puncture + tezosentan group (n = 6). ^†p < 0.05 between Sham and CLP groups; ^‡p < 0.05 between Sham and CLP+Tezo groups; *p > 0.05 between CLP and CLP+Tezo groups.

Figure 6

Protein kinase Cα in lungs after endothelin-1 (ET-1) administration. (a) Cytosolic and (b) membrane fractions. Data are presented as mean ± SEM. Control, control group (n = 4); ET-1, endothelin-1 group (n = 4); ET-1+Tezo, endothelin-1+tezosentan group (n = 4). ^†p < 0.05 between control and ET-1 groups; *p > 0.05 between ET-1 and ET-1+Tezo groups.