Effect of WIN 64338, a B2 bradykinin receptor antagonist on guinea-pig tracheal smooth muscle cells in culture.

This study investigated the effect of the non-peptidic B2 bradykinin (BK) receptor antagonist WIN 64338 on BK binding and BK-induced inositol phosphate formation on guinea-pig tracheal smooth muscle cells in culture. The presence of specific and saturable binding sites for BK was demonstrated using [3H]BK. Scatchard analysis indicates a single population of binding sites for [3H]BK with a maximal density (Bmax) of 245.4 +/- 71 fmol/mg of protein and an equilibrium dissociation constant (Kd) of 87.7 +/- 0.12 pM. The order of potency of] B2 BK receptor ligands was Hoe 140 = NPC 17731 > BK > WIN 64338 > D- Arg0[Hyp3, D-Phe7]-BK > > des-Arg9-BK, while B1 BK receptor antagonist, des-Arg9-[Leu8]-BK, was without effect on [3H]BK binding. These results demonstrate the presence of B2 Bk receptors on cultured tracheal smooth muscle cells. The cells were stimulated by BK, and inositol phosphate formation was determined by anion exchange chromatography. The stimulating effect of BK on inositol phosphate formation was concentration dependent (1 nM to 10 microM). The B1 BK agonist des-Arg9-BK did not induce inositol phosphate formation. The order of potency of B2 antagonists to inhibit BK-induced inositol phosphate formation was Hoe 140 = NPC 17731 > WIN 64338 > D-Arg0[Hyp3, D-Phe7]-BK. This study demonstrates that WIN 64338 is able to displace [3H]BK binding and to inhibit B2-BK-induced inositol phosphate formation on cultured guinea-pig tracheal smooth muscle cells.