Endothelin (ET), a 21 amino acid peptide, containing two disulfide bridges, is the most potent vasoconstrictor known to date. Two G-protein-coupled ET receptors with seven transmembrane spanning domains have been described, cloned from human tissue and defined as ETA and ETB receptors. Whereas ETA receptors are selective for ET-1 and ET-2 over ET-3, ET receptors bind all three isopeptides with similar potency. Ro 46-2005 is a new synthetic non-peptide endothelin (ET) receptor antagonist with IC50 220 nM for ETA. It binds to natural ETA receptors containing tissue, i.e. human smooth muscle cells and rat mesangial cells, and also to recombinant human ETA receptors with similar potencies[3]. In a binding assay study, human smooth muscle cells, rat endothelial cells on membranes were performed in 250 μl 50 mM Tris buffer (pH 7.4, 25 mM MnC12, 1 mM EDTA, 0.5% (w/v) BSA) containing 5 - 35 μg protein, 32 pM 125I-labelled ET and increasing amounts of unlabelled ligands. In binding assays with [125I]ET-3 on ETA receptor a tracer concentration of 213 pM was used. Ro 46-2005 competed for the binding of ET-1 on cells carrying ETA receptor, i.e. human smooth muscle cells and rat endothelial cells, with IC50 values of 220 ± 60 nM and 430 ± 140 nM, respectively. Its potency for binding to ETB receptors was similar on membranes of human placenta (IC50 160 ± 77 nM) and porcine cerebellum (IC50 227 ± 92 nM)[4].
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