Human endothelin-converting enzyme (ECE-1) has been shown to exist as three isoforms (ECE-1a, ECE-1b and ECE-1c) diverging in their N-terminal sequence and displaying different patterns of subcellular localization. We report here the cloning of ECE-1d, a novel isoform of 767 amino acids, which is generated from the same gene via the existence of an additional promoter located upstream from the third exon of the ECE-1 gene. ECE-1d converting activity is comparable to that of the other three isoenzymes. In contrast to ECE-1b, ECE-1d is expressed at the cell surface, although less strongly than ECE-1a. We have also shown, by identifying ECE-1b and ECE-1d in rat, that the ECE-1 diversity is conserved between human and rodent, suggesting its physiological relevance. The mRNA levels of the four isoforms were assessed in the two species in various cell types, revealing some differences. In particular, the ECE-1a isoform, strongly expressed at the plasma membrane, was found to be highly expressed in primary cultures of endothelial cells but absent from primary cultures of smooth muscle cells.

Taken together, these data suggest that overexpression of ET-1 and down-regulation of ET(B) receptors in the lung are determinants of circulating endothelin in CHF. As a corollary, increased plasma endothelin may provide evidence of pulmonary endothelial dysfunction in CHF.

Thrombin has been shown to stimulate endothelin release by endothelial cells, but the ability of thrombin to induce endothelin in nonendothelial cells is less well-known. Incubation of rat aortic smooth muscle cells with thrombin resulted in a stimulation of preproendothelin-1 (preproET-1) mRNA expression. This induction of preproET-1 mRNA expression by thrombin was accompanied by the release of immunoreactive peptide ET-1 into the extracellular medium. The synthetic thrombin receptor activator peptide (TRAP) confirmed ligand-specific receptor action to induce preproET-1 mRNA. Nuclear run-on analysis revealed that the transcriptional rate of preproET-1 mRNA increases twofold after 1 h of incubation with thrombin. In cells treated with thrombin, the half-life of preproET-1 mRNA was identical to that in untreated control cells. These results demonstrated that thrombin regulates endothelin synthesis at a transcriptional level but does not influence mRNA stability. Inhibition of protein kinase C (PKC) with selective inhibitors (chelerythrine and bisindolylmaleimide I) before thrombin stimulation failed to significantly inhibit preproET-1 gene expression. Inhibition of mitogen-activated protein (MAP) kinase kinase and protein tyrosine kinase decreased preproET-1 mRNA expression in thrombin-stimulated smooth muscle cells. Furthermore, addition of an activator of peroxisome proliferator-activated receptors-alpha (PPARalpha), fenofibrate, prevented the preproET-1 gene induction in response to thrombin. These results demonstrated that thrombin-induced endothelin gene transcription involved MAP kinase kinase rather than the PKC cascade in smooth muscle cells, which was repressed by PPARalpha stimulation.

Tissue production of ET- 1 is up-regulated in experimental critical hind limb ischemia. Inhibition of the endothelin system by a mixed ET(A/B) receptor antagonist may protect, at least in part, against muscle injury.