The expression of endothelin-1 and endothelin-converting enzyme-1 (ECE-1) are independently regulated in bovine aortic endothelial cells

The endopeptidase called endothelin-converting enzyme-1 (ECE-1) is thought to play a physiological role in endothelin-l (ET-1) biosynthesis. For human ECE-1, differential splicing of messenger RNA (mRNA) results in the synthesis of three isoforms, termed ECE-1a, ECE-1b, and ECE-1c. The isoform(s) responsible for the hydrolysis of the biosynthetic intermediate big ET-1 in endothelial cells have yet to be assigned. To investigate whether the expression of mRNAs for preproET-1 and ECE-1 are regulated in parallel, a variety of conditions were used to compare levels of ET-1 synthesis by bovine aortic endothelial cells (BAECs) with levels of mRNA for preproET-1, ECE-la, ECE-lc, and the combined ECE-1 isoforms (ECE-1a/b/c), Stimulation of BAECs with tumor necrosis factor-alpha or transforming growth factor-beta increased ET-1 synthesis, and treatment of BAECs with 2-chloroadenosine or staurosporine caused concentration-dependent reductions in ET-1 synthesis. Estimates of mRNA levels by reverse transcription-polymerase chain reaction (RT-PCR) with linear cycling conditions showed changes in preproET-1 expression to correlate well with ET-I secretion. In contrast, RT-PCR analysis of ECE-1 expression by using primer pairs to measure ECE-la, ECE-lc, or all the ECE-1 isoforms simultaneously showed no correlation between their mRNA levels and those of preproET-1. This indicates that under the conditions investigated, expression of ECE-1 is not coordinated with ET-1 synthesis in BAECs.