EFFECTS OF VASOACTIVE AND INFLAMMATORY MEDIATORS ON ENDOTHELIN-1 EXPRESSION IN PULMONARY ENDOTHELIAL-CELLS

Endothelin-1 (ET-1) is known to be involved in a variety of pathophysiologic conditions, especially of the pulmonary vasculature. The aim of this study was to investigate physiologic mediators potentially involved in the pathogenesis of pulmonary hypertension, for their effects on ET-1 gene expression at both the transcriptional and translational level. Rat microvascular and pulmonary artery endothelial cells grown in culture were exposed to vasoactive mediators (thrombin or an anoxic gas mixture) and inflammatory mediators (lipopolysaccharide, interleukin 1 alpha, interleukin 1 beta, or tumor necrosis factor alpha) for various time periods. The change in prepro-ET-1 (ppET-1) mRNA levels in these cells in response to stimuli was a time-dependent phenomenon. The inflammatory mediators caused an acute rise in ppET-1 mRNA levels whereby peak induction occurred after 1 h with a rapid decline to control levels by 4 h. The vasoactive mediators elicited a more sustained response whereby a significant elevation in ppET-1 mRNA expression occurred quickly and remained elevated through 4 h. The pattern of induction was more rapid for thrombin than for anoxic gas exposure. Radioimmunoassay analysis demonstrated a similar response for thrombin and the inflammatory mediators in ET-1 mature peptide release, whereas the effect of anoxic gas exposure was divergent. Significant elevations were noted after 6 h for thrombin as well as each of the inflammatory mediators except IL-1 alpha. In response to the anoxic gas exposure, however, a significant rise in ET-1 peptide release was not evident until after 24 h. To determine the level at which ppET-1 mRNA induction is regulated, cells were cotreated with each of the stimuli and actinomycin D or cycloheximide. Results indicate that the induction of ppET-1 mRNA levels is likely due to de novo transcription, as well as mRNA stabilization. In summary, inflammatory and vasoactive agents are important regulators of ET-1 gene expression in rat pulmonary endothelial cells; most important, we observed a differential response at the mRNA or peptide level depending on the mediator involved.