High Affinity Binding of [3H]Propionyl-[Met(O2)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
- Sandrine Sagan1,
- Jean-Claude Beaujouan2,
- Yvette Torrens2,
- Monique Saffroy2,
- Gérard Chassaing1,
- Jacques Glowinski2 and
- Solange Lavielle1
- 1Laboratoire de Chimie Organique Biologique, URA CNRS 493, Université Pierre et Marie Curie, 75252 Paris Cedex 05, France (S.S., G.C., S.L.), and 2Chaire de Neuropharmacologie, Institut National de la Santé et de la Recherche Médicale U114, Collège de France, 75005 Paris, France (J.-C.B., Y.T., M.S., J.G.)
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 (Kd = 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.
Footnotes
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Send reprint requests to: Dr. Solange Lavielle, Laboratoire de Chimie Organique Biologique, URA CNRS 493, Université Paris 6, 4, place Jussieu, 75252 Paris Cedex 05, France. E-mail:lavielle{at}ccr.jussieu.fr
- Abbreviations:
- SP
- substance P
- NK
- neurokinin
- ALIE-124
- propionyl-[Met(O2)11]substance P(7–11)
- CHO
- Chinese hamster ovary
- PI
- phosphatidylinositol
- BSA
- bovine serum albumin
- PMSF
- phenylmethylsulfonyl fluoride
- DMSO
- dimethylsulfoxide
- PLC
- phospholipase C
- hNK
- human neurokinin
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
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- Received November 19, 1996.
- Accepted March 27, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
