Characterization of a thromboxane A2/prostaglandin H2 receptor in Guinea pig lung membranes using a radioiodinated thromboxane mimetic

Thromboxane A2 (TXA2) and prostaglandin H2 (PGH2) are potent constrictors of airway smooth muscle and may mediate some of the pulmonary effects of leukotrienes. To date, the TXA2/PGH2 receptor in lung has not been well characterized. In this report, we describe the evaluation of the TXA2/PGH2 receptor in guinea pig lung membranes using the new radiolabeled TXA2 mimetic (1S(1 alpha,2 beta(5Z),3 alpha(1E,3S*),4 alpha))-7-(3-(3-hydroxy-4-(4'- iodophenoxy)-1-butenyl)-7-oxabicyclo-(2.2.1)heptan-2-yl)-5-h eptenoic acid (IBOP). IBOP elicited a dose-dependent contraction of guinea pig lung parenchymal strips (EC50 = 3.03 +/- 0.97 nM, three experiments), which was blocked by the TXA2/PGH2 antagonists SQ29548 (pKB = 7.44 +/- 0.2, three experiments), BM13505 (pKB = 6.29 +/- 0.26, three experiments), and I-PTA-OH (pKB = 5.82 +/- 0.36, three experiments). In radioligand binding studies, the binding of (125I)IBOP to guinea pig lung membranes prepared from perfused lungs was saturable, displaceable, and dependent upon protein concentration. Binding was optimal at pH 6.5 and was enhanced by the addition of mono- and divalent cations. The standard assay buffer was 25 mM 3-(N-morpholino)propanesulfonic acid, pH 6.5, 100 mM NaCl, 5 mM MgCl2. Binding was inhibited by pretreatment with dithiothreitol, N-ethylmaleimide, or beta-mercaptoethanol. Binding was unaffected by the addition of guanine nucleotide analogs at concentrations upmore » to 300 microM. Analysis of the time course of binding of (125)IBOP at 30 degrees yielded k-1 = 0.0447 min-1, k1 = 2.49 x 10(8) M-1 min-1, and Kd = k-1/k1 = 180 pM. Computer analysis of equilibrium binding studies using nonlinear methods (LUNDON-1) revealed a single class of noninteracting binding sites with a Kd of 86.9 +/- 11.9 pM and a Bmax of 81.8 +/- 7.7 fmol/mg of protein (three experiments).« less