Characterization of human peripheral blood monocyte thromboxane A2 receptors

Thromboxane A2 (TxA2) is the predominant eicosanoid metabolite produced by activated human blood-borne monocytes. This study was designed to characterize TxA2 receptors on human peripheral blood monocytes via identification of radioligand binding characteristics using the stable TxA2 mimetic [125I]-BOP ([1S,(1 alpha,2 beta(5Z),3 alpha(1E,3S*),4 alpha]-7-[3-hydroxy-4-(4'- iodophenoxy-1-butenyl)-7-oxabicyclo-[2.2.1]heptan-2-yl]-5-he ptenoic acid) and via analysis of second messenger signal transduction pathways. Scatchard analysis of the binding of [125I]-BOP in human monocyte membranes revealed a single class of binding sites, Kd = 1.49 +/- 0.14 nM and Bmax = 696 +/- 113 fmol/mg membrane protein (n = 8). [125I]-BOP interacted specifically with a TxA2 receptor, as shown by competition binding studies with a range of TxA2 receptor agonists and antagonists that gave a rank order of potency of (-)-9-chlorobenzyl-6-fluoro-1,2,3,4-tetrahydrocarbazol-1-yl acetic acid > I-BOP = I-SAP > ONO11113 > SQ29548 > U46619 > (+)-9-chlorobenzyl-6-fluoro-1,2,3,4- tetrahydrocarbazol-1-yl acetic acid. I-BOP caused a dose-dependent increase in intracellular free calcium (EC50 = 7.14 +/- 1.1 nM, n = 4), which was attenuated by preincubation with the TxA2 receptor antagonists SQ29548 (1 microM) and (-)-9-chlorobenzyl-6-fluoro-1,2,3,4-tetrahydrocarbazol-1-yl acetic acid (100 nM). This study provides further evidence for a TxA2 receptor in monocytes and supports the potential for future characterization of TxA2 receptor subtypes using molecular techniques.