High Affinity Binding of [H]Propionyl-[Met(O2) ]Substance P(7–11), a Tritiated Septide-Like Peptide, in Chinese Hamster Ovary Cells Expressing Human Neurokinin-1 Receptors and in Rat Submandibular Glands

Propionyl-[Met(O2) ]substance P(7–11) [ALIE-124 or propionyl-[Met(O2) ]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 [H][Pro]substance P (SP) from its binding site at micromolar concentrations. However, ALIE-124 stimulated phosphatidylinositol hydrolysis, as previously shown for septide-like peptides. With [H]ALIE-124 (95 Ci/mmol), we have been able to reveal a high affinity binding site in CHO cells (Kd 5 6.6 6 1.0 nM), with a low maximal binding capacity. [H]ALIE-124 specific maximal binding represented only 15–20% of that observed with [H][Pro]SP in CHO cells. Septide-like peptides, including septide and NKA, were potent competitors (in the nanomolar range) of [H]ALIE-124 specific binding site. Interestingly, SP and [Pro]SP were also potent competitors, with 10-fold greater potency for sites labeled with [H]ALIE-124 than for sites labeled with [H][Pro]SP. The NK-1 antagonist RP 67580 also showed a higher potency for [H]ALIE-124 than for [H][Pro]SP-specific binding sites. NKB and [Lys,methylLeu,Nle]NKA(4–10) displaced [H]ALIE-124 binding but with lower potency, whereas senktide had no affinity. The existence of [H]ALIE-124 specific binding sites was also demonstrated in rat submandibular gland. In this tissue, [H]ALIE-124 specific maximal binding was higher, reaching 40–50% of that achieved with [H][Pro]SP.