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 Chine se hamster ovary (CHO) cells expressing human tachykinin neurokinin (N K)-1 receptors, ALIE-124 displaced [H-3][Pro(9)]substance P (SP) from its binding site at micromolar concentrations. However, ALIE-124 stimu lated 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 compet itors, with 10-fold greater potency for sites labeled with [H-3]ALIE-1 24 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][P ros]SP-specific binding sites. NKB and [Lys(5),methyl-Leu(9),Nle(10)]N KA(4-10) displaced [H-3]ALIE-124 binding but with lower potency, where as senktide had no affinity. The existence of [H-3]ALIE-124 specific b inding sites was also demonstrated in rat submandibular gland. In this tissue, [H-3]ALIE-124 specific maximal binding was higher, reaching 4 0-50% of that achieved with [H-3][Pro(9)]SP.