Inhibition of Radiolabeled Leukotriene-Binding by AS-35 in Guinea Pig Lung Membrane Fraction.

The inhibitory effects of 9-[(4-acetyl-3-hydroxy-2-n-propylphenoxy) methyl]-3-(1H-tetrazol-5-yl)-4H-pyrido[1,2-a]pyrimidin-4-one (AS-35), a peptide leukotriene (LT) antagonist, on specific bindings of radiolabeled LTC4 and LTD4 in guinea pig lung membrane were investigated to clarify the mechanism by which this agent inhibited LT-induced physiological responses. Binding assays were performed at 20 degrees C in 50 mM Tris-HCl buffer (pH 7.4) containing 10 mM CaCl2, 10 mM MgCl2 and 10 mM cysteine in the absence (LTD4 binding assay) or presence (LTC4 binding assay) of 80 mM L-serine borate. Scatchard analysis of each LT specific binding indicated a single and high affinity binding site with a Kd of 0.21 +/- 0.05 nM and Bmax of 808 +/- 71 fmol/mg protein for [3H]-LTD4, and with a Kd of 21.6 +/- 3.8 nM and Bmax of 74.9 +/- 2.6 pmol/mg protein for [3H]-LTC4. Competition binding studies showed that AS-35 antagonized [3H]-LTD4 specific binding with a Ki value of 92.7 nM. In contrast, AS-35 was 100 times less effective in inhibiting [3H]-LTC4 specific binding, compared with [3H]-LTD4 specific binding. These results indicate that AS-35 interacts directly with peptide LTs receptors, especially the LTD4 specific binding site to produce its pharmacological effects.