Received for publication July 9, 2008.
Revised November 3, 2008.
Accepted for publication November 4, 2008.

CHYMASE-DEPENDENT CONVERSION OF BIG ENDOTHELIN-1 IN THE MOUSE IN VIVO

Abstract

The aim of this study was to identify the role of chymase in the conversion of exogenously administered big endothelin-1 in the mouse in vivo. Real time PCR analysis detected mRNA of mucosal mast cell chymase 4 and 5 as well as endothelin converting enzyme 1a and neutral endopeptidase 24.11 in pulmonary, cardiac and aorta homogenates derived from C57BL/6J mice with the later tissue expressing the highest levels of both chymase isoforms. Furthermore, hydrolysis of a fluorogenic peptide substrate, Suc-Leu-Leu-Val-Tyr-MCA, was sensitive to the chymase inhibitors Suc-Val-Pro-Phe^P (OPh)₂ (200 µM) and chymostatin (100 µM) in supernatants extracted from the same tissue homogenates. In anesthetized mice, big endothelin-1, endothelin-1 (1-31) and endothelin-1 triggered pressor responses (ED50s; 0.67, 0.89 and 0.16 nmol/kg) which were all reduced or potentiated by selective ET_A or ET_B receptor antagonists, respectively (BQ-123 or BQ-788; each at 1 mg/kg). The pressor responses to big endothelin-1 were significantly reduced by the neutral endopeptidase inhibitor thiorphan (1 mg/kg) or the endothelin converting enzyme inhibitor CGS 35066 (0.1 mg/kg). In contrast, the responses to endothelin-1 (1-31) were abolished by thiorphan, but unaffected by CGS 35066. In addition, Suc-Val-Pro-Phe^P (OPh)₂ (20-40 mg/kg) reduced by more than 60% the hemodynamic response to big endothelin-1 but not to endothelin-1 (1-31) and endothelin-1. Finally intravenous administration of big endothelin-1 induced Suc-Val-Pro-Phe^P (OPh)₂-sensitive increases in plasma immunoreactive levels of endothelin-1 (1-31) and endothelin-1. The present study suggests that chymase plays a pivotal role in the conversion and cardiovascular properties of big endothelin-1 in vivo.

Key words: Chymase, Endothelin-1 (1-31), Mouse, Real Time PCR, Suc-Val-Pro-PheP (OPh)2, blood pressure