High affinity binding of [H-3]propionyl-[Met(O-2)(11)]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(O-2)(11)]substance P(7-11) [ALIE-124 or propionyl-[Met(O-2)(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 [H-3][Pro(9)]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-3]ALlE-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. [H-3]ALIE-124 specific maximal binding represented only 15-20% of that observed with [H-3][Pro(9)]SP in CHO cells. Septide-like peptides, including septide and NKA, were potent competitors (in the nanomolar range) of [H-3]ALIE-124 specific binding site. Interestingly, SP and [Pro(9)]SP were also potent competitors, with 10-fold greater potency for sites labeled with [H-3]ALIE-124 than for sites labeled with [H-3][Pro(9)]SP. The NK-1 antagonist RP 67580 also showed a higher potency for [H-3]ALIE-124 than for [H-3][Pros]SP-specific binding sites. NKB and [Lys(5),methyl-Leu(9),Nle(10)]NKA(4-10) displaced [H-3]ALIE-124 binding but with lower potency, whereas senktide had no affinity. The existence of [H-3]ALIE-124 specific binding sites was also demonstrated in rat submandibular gland. In this tissue, [H-3]ALIE-124 specific maximal binding was higher, reaching 40-50% of that achieved with [H-3][Pro(9)]SP.