Abstract 19347: The Mouse Mast Cell Protease 4 is Involved in the Generation of Endothelin-1 in vitro and in vivo

Abstract

The serine protease chymase converts intravenously (iv)- administered big-endothelin-1 to endothelin-1 (ET-1) in the mouse in vivo. The mouse mast cell protease 4 (mMCP-4) is among eight chymase-like murine isoforms and is involved in the generation of angiotensin-II (Ang-II) from its precursor Ang-I. It remains to be determined which chymase isoform is involved in the endothelin-converting enzyme -independent production of ET-1. In the present study, the contribution of mMCP-4 was determined in the conversion of exogenous Big-ET-1 in vitro and in vivo as well as in the tissue production of endogenous ET-1. In vitro analyses were performed from cardiac left ventricle, aorta, kidneys and lungs homogenates derived from wild type (WT) and mMCP-4 KO mice. The pressor response in anaesthetized mice to Big-ET-1, but not ET-1 (1-31) or ET-1 was significantly reduced in mMCP-4 KO mice compared to their WT congeners. Furthermore, a selective chymase inhibitor, TY-51469, significantly reduced the pressor response to Big-ET-1 in WT but not mMCP-4 KO mice. Radio-telemetry analysis in free-moving, conscious mice revealed no differences in basal blood pressure between WT and mMCP-4 KO mice. TY-51469 also inhibited the in vitro hydrolysis a chymotrypsin-sensitive fluorogenic peptide in soluble fractions derived from WT but not mMCP-4 KO tissues. HPLC-MALDI-MS analysis revealed that supernatants from WT but not mMCP-4 KO mice could yield ET-1 (1-31) from Big-ET-1 in vitro, and that this conversion is blocked by TY-51469 in WT samples. The in vivo conversion of iv-administered Big-ET-1 to plasmatic ET-1 (1-31) and ET-1 was also reduced in mMCP-4 KO mice compared to their WT congeners. Finally, tissue levels of immunoreactive ET-1 were reduced by 40% in lung homogenates from mMCP-4 KO mice when compared to those from WT congeners. We conclude that mMCP-4 is the predominantly involved chymase isoform in the production of endogenous ET-1 in the mouse model.