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

• Published in: Molecular pharmacology 2001-02, Vol.59 (2), p.163-169
• Main Authors: Barker, S, Khan, N Q, Wood, E G, Corder, R
• Format: Article
• Language: English
• Subjects: Animals; Aspartic Acid Endopeptidases - biosynthesis; Aspartic Acid Endopeptidases - drug effects; Aspartic Acid Endopeptidases - genetics; Cattle; Cells, Cultured; Endothelin-1 - biosynthesis; Endothelin-Converting Enzymes; Gene Expression - drug effects; Metalloendopeptidases; Muscle, Smooth, Vascular - drug effects; Muscle, Smooth, Vascular - metabolism; Muscle, Smooth, Vascular - physiology; Oligodeoxyribonucleotides, Antisense - pharmacology; Protein Isoforms - biosynthesis; Protein Isoforms - genetics; Pulmonary Artery - cytology; Pulmonary Artery - drug effects; Pulmonary Artery - metabolism; RNA, Messenger - biosynthesis; RNA, Messenger - drug effects; Tumor Necrosis Factor-alpha - pharmacology
• ISSN: 0026-895X; 1521-0111
• DOI: 10.1124/mol.59.2.163

Endothelin-1 (ET-1) is secreted from endothelial and vascular smooth muscle cells (VSMC) after intracellular hydrolysis of big ET-1 by endothelin converting 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 VSMC 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. Reverse 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 ECE-1b and ECE-1c were expressed in BPASMC, with ECE-1c being the predominant isoform. Basal release of ET-1 from BPASMC was low. Treatment for 24 h with tumor necrosis factor-α (TNFα) 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 TNFα, whereas amounts of ECE-1b mRNA were decreased significantly. Treatment of BPASMC with a phosphorothioate antisense ODN to ECE-1c caused a marked reduction in ECE-1c mRNA levels and ECE-1 protein levels. However, basal and TNFα-stimulated ET-1 release were largely unaffected by the ECE-1c antisense ODN despite the inhibition of ECE-1c synthesis. Hence, an endopeptidase distinct from ECE-1 is mainly responsible big ET-1 processing in BPASMC.