title: The two-step conversion of big endothelin 1 to endothelin 1 and degradation of endothelin 1 by subcellular fractions from human polymorphonuclear leukocytes.
abstract: The metabolism of big endothelin 1 (bET) and endothelin 1 (ET-1) by subcellular fractions from human polymorphonuclear leukocytes (PMNs) was investigated by bioassay and reversed-phase high-performance liquid chromatography. More than 80% of endothelin-converting activity was recovered from the cytosolic fraction, which in addition to ET-1 generated other peptides from bET. The processing of bET to all its metabolites including ET-1 was prevented by the serine protease inhibitor 3,4-dichloroisocoumarin (DCI; 50 microM) or the elastase inhibitor ONO-5046 (100 microM) but not by phenylmethylsulfonyl fluoride (PMSF; 143 microM), another serine protease inhibitor. Paradoxically, human leukocyte elastase, despite generating a bET fragmentation pattern similar to that of PMN cytosol, produced very little ET-1. However, subsequent treatment of the elastase-derived metabolites of bET with PMN cytosol in the presence of ONO-5046 dramatically increased the amount of ET-1 formed. The generation of ET-1 following this intervention was inhibited by DCI. The PMN membrane preparation degraded ET-1 to a major metabolite, similar to that produced from ET-1 by elastase, and several minor products, paralleled by a loss of its smooth muscle contracting activity. The degradation of ET-1 by PMN microsomes was prevented by DCI, PMSF, or ONO-5046. Our results suggest that an elastase-initiated serine protease cascade is responsible for the sequential conversion of bET to ET-1 by the PMN cytosol. Elastase also partly accounts for the ET-metabolizing properties of PMN microsomes.
authors.affiliation: William Harvey Research Institute, St. Bartholomew's Hospital Medical College, United Kingdom.
authors.forename: S
authors.initials: S
authors.lastname: Kaw
authors.forename: M
authors.initials: M
authors.lastname: Hecker
authors.forename: J R
authors.initials: JR
authors.lastname: Vane
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000378:metabolism; D019332:Endothelin-1; D016232:Endothelins / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007601:Jugular Veins / Q000187:drug effects / Q000502:physiology; D008297:Male; D008861:Microsomes / Q000378:metabolism; D009119:Muscle Contraction / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology*; D011480:Protease Inhibitors / Q000494:pharmacology; D011498:Protein Precursors / Q000378:metabolism*; D011817:Rabbits; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D019332:Endothelin-1; D016232:Endothelins; D011480:Protease Inhibitors; D011498:Protein Precursors