CHARACTERIZATION OF THE THROMBOXANE RECEPTOR MEDIATING PROSTACYCLIN RELEASE FROM CULTURED ENDOTHELIAL-CELLS

The thromboxane A2 (TXA2) mimetic, 9,11-dideoxy-11,9-epoxymethano-prostaglandin F2-alpha (U46619), mobilized calcium in the bovine aortic endothelial cell line AG4762 and stimulated release of prostacyclin from these cells. The U46619-stimulated release of prostacyclin could be inhibited by TXA2 antagonists with the order of potency [ls-[1 < a, 2 < b(5z), 3<b, 4 < a]]-7-[3-[[2-[(phenyl-amino) carbonyl]hydrazinolmethyl]-7-oxabicyclo-[2.2.1]hept-2-yl]-5-heptenoic acid (SQ29548) > 4-[2-(4-chlorobenzene-sulphonamido) ethyl]phenylacetic acid (BM13505) > 4-[2-(phenylsulphonamido)ethyl] phenoxyacetic acid (BM13177), which was consistent with release being mediated by a TXA2 (TP) receptor. The TP receptor ligands, [H-3]SQ29548 and 9,11-dimethylmethano-16(3-[I-125]iodo-4-hydroxyphenyl)-13, 14-dihydro-13-aza-l5-omega-o-tetranor-thromboxane ([I-125]-PTA-OH), both appeared to bind to a homogenous population of sites in AG4762 cell membranes. The affinities of [H-3]SQ29548 and [I-125]PTA-OH were almost-equal-to 10 nM and almost-equal-to 0.3 nM, respectively, and the density of sites labelled by either ligand was almost-equal-to 25 fmol/mg protein. Under conditions where equilibrium was approached, the specific binding of [H-3]SQ29548 or [I-125]PTA-OH was displaced by SQ29548, BM13505 and BM13177 with the same order of potency and similar apparent affinities as in the functional assay, suggesting that these binding sites represent bona fide TP receptors.