Pivotal role of mouse mast cell protease 4 in the conversion and pressor properties of Big-endothelin-1

Abstract

The serine protease chymase has been reported to generate intracardiac angiotensin-II (Ang-II) from Ang-I as well as an intermediate precursor of endothelin-1 (ET-1), ET-1 (1-31) from Big-ET-1. Although humans possess only one chymase, several murine isoforms are documented, each with its own specific catalytic activity. Among these, mouse mast cell protease 4 (mMCP-4) is the isoform most similar to the human chymase for its activity. The aim of this study was to characterize the capacity of mMCP-4 to convert Big-ET-1 into its bioactive metabolite, ET-1, in vitro and in vivo in the mouse model. Basal mean arterial pressure did not differ between wild-type (WT) and mMCP-4(-/-) mice. Systemic administration of Big-ET-1 triggered pressor responses and increased blood levels of immunoreactive (IR) ET-1 (1-31) and ET-1 that were reduced by more than 50% in mMCP-4 knockout (-/-) mice compared with WT controls. Residual responses to Big-ET-1 in mMCP-4(-/-) mice were insensitive to the enkephalinase/neutral endopeptidase inhibitor thiorphan and the specific chymase inhibitor TY-51469 {2-[4-(5-fluoro-3-methylbenzo[b]thiophen-2-yl)sulfonamido-3-methanesulfonylphenyl]thiazole-4-carboxylic acid}. Soluble fractions from the lungs, left cardiac ventricle, aorta, and kidneys of WT but not mMCP-4(-/-) mice generated ET-1 (1-31) from exogenous Big-ET-1 in a TY-51469-sensitive fashion as detected by high-performance liquid chromatography/ matrix-assisted laser desorption/ionization-mass spectrometry. Finally, pulmonary endogenous levels of IR-ET-1 were reduced by more than 40% in tissues derived from mMCP-4(-/-) mice compared with WT mice. Our results show that mMCP-4 plays a pivotal role in the dynamic conversion of systemic Big-ET-1 to ET-1 in the mouse model.

Fig. 1.

Chymase-like enzymatic activity in the soluble fraction of lung (A), left heart ventricle (B), aorta (C) and kidney (D) homogenates from WT or mMCP-4(−/−) mice, treated with either vehicle (PBS) or a specific chymase inhibitor (TY-51469). ***P < 0.001; n = 6.

Fig. 2.

In vitro conversion of Big-ET-1 to ET-1 (1–31) in soluble fractions from lung homogenates, with HPLC-MS identification of conversion results from WT (A), mMCP-4(−/−) (B), and WT tissues treated with TY-51469 (C).

Fig. 3.

Quantification of the in vitro conversion of Big ET-1 to ET-1 (1–31) in homogenates from the lungs (A), left heart ventricle (B), aorta (C), and kidneys (D) from WT (treated with vehicle or TY-51469) or mMCP-4(−/−) mice using HPLC area under the curve arbitrary units (AU); n = 5.

Fig. 4.

Maximal variation in mean arterial pressure (▵MAP) following the intravenous administration of Big-ET-1 (A), ET-1 (1–31) (B), and ET-1 (C) in WT and mMCP-4(−/−) mice. *P < 0.05; **P < 0.01; n = 6.

Fig. 5.

Time course of the variation of the mean arterial pressure (▵MAP) in response to i.v. administration of Big ET-1 (1 nmol/kg) [(A) WT mice; (B) mMCP-4(−/−) mice] or ET-1 (1-31) (1 nmol/kg) [(C) WT mice; (D) mMCP-4(−/−) mice] in mice pre-treated with either a chymase (TY-51469), a NEP (thiorphan) or an ECE inhibitor (CGS 35066). *P < 0.05 vs. PBS; #P < 0.05 vs. thiorphan; §P < 0.05 vs. CGS 35066; n = 5–9.

Fig. 6.

In vivo conversion of Big-ET-1 into ET-1 (1–31) and ET-1 in WT and mMCP-4(−/−) mice. Quantification of the plasma levels of immunoreactive endothelin-1 (1–31) (A) and IR-ET-1 (C) after the intravenous administration of Big-ET-1 1.0 nmol/kg. In (B), the plasma levels of IR-ET-1 (1–31) were determined following the intravenous administration of ET-1 (1–31) (0.1 nmol/kg). **P < 0.01; n = 5–6.

Fig. 7.

Endogenous tissue levels of IR-ET-1 in the lungs, left cardiac ventricle, the aorta, and the kidneys. **P < 0.05; n = 6–7.