Abstract

Propionyl-[Met(O₂)¹¹]substance P(7–11) [ALIE-124 or propionyl-[Met(O₂)¹¹]SP(7–11)] has been designed as a septide-like ligand adequate for tritiation and, therefore, adequate for binding studies. In Chinese hamster ovary (CHO) cells expressing human tachykinin neurokinin (NK)-1 receptors, ALIE-124 displaced [³H][Pro⁹]substance P (SP) from its binding site at micromolar concentrations. However, ALIE-124 stimulated phosphatidylinositol hydrolysis, as previously shown for septide-like peptides. With [³H]ALIE-124 (95 Ci/mmol), we have been able to reveal a high affinity binding site in CHO cells (K_d = 6.6 ± 1.0 nm), with a low maximal binding capacity. [³H]ALIE-124 specific maximal binding represented only 15–20% of that observed with [³H][Pro⁹]SP in CHO cells. Septide-like peptides, including septide and NKA, were potent competitors (in the nanomolar range) of [³H]ALIE-124 specific binding site. Interestingly, SP and [Pro⁹]SP were also potent competitors, with 10-fold greater potency for sites labeled with [³H]ALIE-124 than for sites labeled with [³H][Pro⁹]SP. The NK-1 antagonist RP 67580 also showed a higher potency for [³H]ALIE-124 than for [³H][Pro⁹]SP-specific binding sites. NKB and [Lys⁵,methyl-Leu⁹,Nle¹⁰]NKA(4–10) displaced [³H]ALIE-124 binding but with lower potency, whereas senktide had no affinity. The existence of [³H]ALIE-124 specific binding sites was also demonstrated in rat submandibular gland. In this tissue, [³H]ALIE-124 specific maximal binding was higher, reaching 40–50% of that achieved with [³H][Pro⁹]SP.