Effect of an antisense oligodeoxynucleotide to endothelin-converting enzyme-1c (ECE-1c) on ECE-1c mRNA, ECE-1 protein and endothelin-1 synthesis in bovine pulmonary artery smooth muscle cells

Endothelin-1 (ET-1) is secreted from endothelial and vascular smooth muscle cells (VSMC) after intracellular hydrolysis of big ET-1 by endothelin conv erting enzyme (ECE). The metallopeptidase called ECE-1 is widely thought to be the physiological ECE, but unequivocal evidence of this role has yet to be provided. Endothelial cells express four isoforms of ECE-1 (ECE-1a, ECE -1b, ECE-1c, and ECE-1d), but the identity of ECE-1 isoforms expressed in V SMC is less clear. Here, we describe the characterization of ECE-1 isoforms in bovine pulmonary artery smooth muscle cells (BPASMC) and the effect on ET-1 synthesis of an antisense oligodeoxynucleotide (ODN) to ECE-1c. Revers e transcriptase-polymerase chain reaction (RT-PCR) evaluation of total RNA from BPASMC showed that ECE-1a and ECE-1d were not expressed. Sequencing of cloned ECE-1 cDNA products and semiquantitative RT-PCR demonstrated that E CE-1b and ECE-1c were expressed in BPASMC, with ECE-1c being the predominan t isoform. Basal release of ET-1 from BPASMC was low. Treatment for 24 h wi th tumor necrosis factor-alpha (TNF alpha) stimulated ET-1 production by up to 10-fold with parallel increases in levels of preproET-1 mRNA. Levels of ECE-1c mRNA were also raised after TNF alpha, whereas amounts of ECE-1b mR NA were decreased significantly. Treatment of BPASMC with a phosphorothioat e antisense ODN to ECE-1c caused a marked reduction in ECE-1c mRNA levels a nd ECE-1 protein levels. However, basal and TNF alpha -stimulated ET-1 rele ase were largely unaffected by the ECE-1c antisense ODN despite the inhibit ion of ECE-1c synthesis. Hence, an endopeptidase distinct from ECE-1 is mai nly responsible big ET-1 processing in BPASMC.