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 polymerase chain reaction analysis detected mRNA of mucosal mast cell chymases 4 and 5, endothelin-converting enzyme 1a, and neutral endopeptidase 24.11 in pulmonary, cardiac, and aorta homogenates derived from C57BL/6J mice, with the latter tissue expressing the highest levels of both chymase isoforms. Furthermore, hydrolysis of a fluorogenic peptide substrate, Suc-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin, was sensitive to the chymase inhibitors Suc-Val-Pro-PheP(OPh)2 (200 μM) and chymostatin [(S)-1-carboxy-2-phenylethyl]-carbamoyl-α-[2-iminohexahydro-4(S)-pyrimidyl]-(S)-Gly-X-Phe-al, where X can be the amino acid Leu, Val, or Ile) (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) that were all reduced or potentiated by selective endothelin ETA or ETB receptor antagonists, respectively, BQ-123 (cyclo[d-Asp-Pro-d-Val-Leu-d-Trp]) or BQ-788 (N-[N-[N-[(2,6-dimethyl-1-piperidinyl)carbonyl]-4-methyl-l-leucyl]-1-(methoxycarbonyl)-d-tryptophyl]-d-norleucine sodium salt), each at 1 mg/kg. The pressor responses to big endothelin-1 were significantly reduced by the neutral endopeptidase inhibitor thiorphan (dl-3-mercapto-2-benzylpropanoylglycine) (1 mg/kg) or the endothelin-converting enzyme inhibitor CGS 35066 [α-[(S)-(phosphonomethyl)amino]-3-dibenzofuranopropanoic acid] (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-PheP(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-PheP-(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.
Footnotes
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This work was supported by the Canadian Institutes for Health Research and by the Etienne Lebel Clinical Research Center.
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The authors of the present manuscript declare that there are no financial links, including consultancies with manufacturers of material or devices described in the article, and no links to the pharmaceutical industry or regulatory agencies or any other potential conflicts of interest.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.108.142992.
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ABBREVIATIONS: ECE, endothelin-converting enzyme; ET, endothelin; phosphoramidon, N-(α-rhamno-pyranosyl-oxy-hydroxy-phosphinyl)-Leu-Trp disodium salt; NEP, neutral endopeptidase; Big ET-1 (1–38), big endothelin-1 (1–38); chymostatin, [(S)-1-carboxy-2-phenylethyl]-carbamoyl-α-[2-iminohexahydro-4(S)-pyrimidyl]-(S)-Gly-X-Phe-al, where X can be the amino acid Leu, Val, or Ile; mMCP, murine mast cell protease; PCR, polymerase chain reaction; F, forward; R, reverse; MCA, 7-amino-4-methylcoumarin; MAP, mean arterial pressure; HR, heart rate; BQ-123, cyclo(d-Asp-Pro-d-Val-Leu-d-Trp; BQ-788, N-[N-[N-[(2,6-dimethyl-1-piperidinyl)carbonyl]-4methyl-l-leucyl]-1-(methoxycarbonyl)-d-tryptophyl]-d-norleucine sodium salt; thiorphan, dl-3-mercapto-2-benzylpropanoylglycine; CGS 35066, α-[(S)-(phosphonomethyl)amino]-3-dibenzofuranopropanoic acid; HPLC, high-pressure liquid chromatography; RIA, radioimmunoassay; EIA, enzymatic immunoassay; IR, immunoreactive; PBS, phosphate-buffered saline; NK-2301, 2-(5-formylamino-6-oxo-2-phenyl-1,6-dihydropyrimidine-1-yl)-N-[[3,4-dioxo-1-phenyl-7-(2-pyridyloxy)]-2-heptyl]acetamide; SUN-C8257, 3-[(3-amino-4-carboxy)phenylsulfonyl]-7-chloroquinazoline-2,4(1H,3H)-dione; TY-51184, 2-[4-(5-fluoro-3-methylbenzo-[b]thiophen-2-yl)sulfonamide-3-methanesulfonylphenyl] oxazole-4-carboxylic acid; IRL 1620, Suc-[Glu9,Ala11,15]-endothelin-1(8-21).
- Received July 5, 2008.
- Accepted November 4, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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