Tezosentan-induced attenuation of lung injury in endotoxemic sheep is associated with reduced activation of protein kinase C

INTRODUCTION Studies in vitro reveal that endothelin-1 (ET-1) activates the alpha isoform of protein kinase C (PKC-alpha) in cultures of endothelial cells, thereby deranging cellular integrity. Sepsis and endotoxemia are associated with increased plasma concentrations of ET-1 that induce acute lung injury (ALI). We recently reported that non-selective ET-1 receptor blockade attenuates ALI in sheep by reducing the endotoxin-induced increase in extravascular lung water index (EVLWI). The aim of this study was to find out whether this attenuation is associated with reduced translocation of PKC-alpha from the cytosolic to the membrane fraction of lung tissue homogenate. METHODS Seventeen awake, instrumented sheep were randomly assigned to a sham-operated group (n = 3), a lipopolysaccharide (LPS) group (n = 7) receiving an intravenous infusion of Escherichia coli 15 ng/kg per min for 24 hours, and a tezosentan group (n = 7) subjected to LPS and, from 4 hours, an intravenous injection of tezosentan 3 mg/kg followed by infusion at 1 mg/kg per hour for the reminder of the experiment. Pulmonary micro-occlusion pressure (Pmo), EVLWI, plasma concentrations of ET-1, tumor necrosis factor-a (TNF-a), and interleukin-8 (IL-8) were determined every 4 hours. Western blotting was used to assess PKC-alpha. RESULTS In non-treated sheep a positive correlation was found between the plasma concentration of ET-1 and Pmo in the late phase of endotoxemia (12 to 24 hours). A positive correlation was also noticed between Pmo and EVLWI in the LPS and the LPS plus tezosentan groups, although the latter was significantly reduced in comparison with LPS alone. In both endotoxemic groups, plasma concentrations of ET-1, TNF-alpha, and IL-8 increased. In the LPS group, the cytosolic fraction of PKC-alpha decreased by 75% whereas the membrane fraction increased by 40% in comparison with the sham-operated animals. Tezosentan completely prevented the changes in PKC-alpha in both the cytosolic and the membrane fractions, concomitantly causing a further increase in the plasma concentrations of ET-1, TNF-alpha, and IL-8. CONCLUSION In endotoxemic sheep, ET-1 receptor blockade alleviates lung injury as assessed by a decrease in EVLWI paralleled by a reduction in Pmo and the prevention of activation of PKC-alpha.