Characterization of the enzyme involved in the processing of big endothelin-1 in human lung epithelial cells

Pulm Pharmacol Ther. 1998 Apr-Jun;11(2-3):209-13. doi: 10.1006/pupt.1998.0140.

Abstract

Biosynthesis of active endothelin-1 (ET-1) implies an enzymatic processing of the inactive precursor Big ET-1 (1-39) into the mature, 21 amino acid peptide. The aim of this study was to characterize in airway and alveolar epithelial cells the enzymes responsible for this activation. BEAS-2B and A549 cells, which both produce ET-1, were studied in vitro as models for bronchiolar and alveolar cells, respectively. Both cell lines were able to convert exogenously added Big ET-1 (0.1 microM) into ET-1, suggesting a cell surface or an extracellular processing. The conversion was inhibited by phosphoramidon in both cell lines with an IC50 approximately 1 microM, but not by thiorphan, a specific inhibitor of neutral endopeptidase 24.11 (NEP). The endogenous production of serum-stimulated BEAS-2B and A549 cells was not inhibited by thiorphan, and phosphoramidon showed inhibition only at high concentration (>100 microM). Western blotting following electrophoresis in reducing conditions demonstrated a protein of MR 110 corresponding to the ECE-1 monomer in both BEAS-2B and A549 cells, as well as in whole lung extracts. By RT-PCR we revealed the mRNA encoding for the ECE-1b and/or -1c subtype, but not ECE-1a, in both cell lines. We conclude that BEAS-2B and A549 cells are able to process either endogenous or exogenous Big ET-1 by ECE-1 and that isoforms 1b and 1c could be involved in this processing with no significant role of NEP.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cell Line
  • Endothelin-1 / biosynthesis*
  • Endothelins / metabolism*
  • Epithelial Cells / enzymology
  • Glycopeptides / metabolism
  • Humans
  • Lung / enzymology*
  • Neprilysin / metabolism
  • Protease Inhibitors / metabolism
  • Protein Isoforms
  • Protein Precursors / metabolism*
  • RNA, Messenger / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Signal Transduction
  • Thiorphan / metabolism

Substances

  • Endothelin-1
  • Endothelins
  • Glycopeptides
  • Protease Inhibitors
  • Protein Isoforms
  • Protein Precursors
  • RNA, Messenger
  • Thiorphan
  • Neprilysin
  • phosphoramidon