Inactivation of endothelin-1 by an enzyme of the vascular endothelial cells

Abstract

We previous Investigated the inactivation of endothelln-1 by deamidase (lysosomal protective protein), present in many ceils, including vascular smooth muscle cells. This enzyme, which we originally purified from human platelets, preferentially hydrolyzes peptides at the C-terminus with hydrophobic amino acids in the Pi or P,' position or both and thereby inactivates endothelin-1, which has a C-terminal sequence oflie'Mle^-Trp^-OH. We tested for the presence of deamidase in cultured bovine aortic endothelial cells. The final supernatant or the homogenized cells (S3) cleaved the deamidase substrate dansyi-Phe-Leu-Arg at a rate of 1J nmol/m in per 10 cells at pH 5.5 at 37°C. Endothelin-1 was completely inactivated by the S3 fraction as determined on rat thoracic aorta strips. The major site of inactivation was the IleM-Trp31 bond, established by high performance liquid chromatography and by amino acid analysis where the main product was des-TrpJI-endothelin-l. The hydrolysis of endothelin-1 (5.9 nmol/min per milligram of protein at pH 5.5 at 23°C) by S3 was blocked mainly by inhibitors of deamidase, including diisopropyl fluorophosphate, but not by inhibitors of some other peptidases. This is the first report of a novel pathway of endothelin-1 metabolism in endothelial cells. Thus, endothelial cells, besides being the source of endothelin-1, contain an enzyme that inactivates it. (Hypertension 1993;21:925-928) © 1993 American Heart Association, Inc.