Interaction of the substance P receptor antagonist RP 67580 with the rat brain NK 1 receptor expressed in transfected CHO cells

Abstract

In the present study, we describe the effects of RP 67580, a substance P non-peptide antagonist, in binding and second messenger experiments performed using transfected Chinese hamster ovary cells expressing the rat NK 1 receptor. The cDNA sequence encoding the rat brain substance P receptor was transfected in Chinese hamster ovary cells, and cellular clones which stably express the corresponding protein were isolated. [ 3H]Substance P binding was performed in homogenates of these transfected cells and revealed the presence of NK 1 receptors in displacement experiments, using peptide analogs of three mammalian tachykinins (substance P, neurokinin A, neurokinin B). Scatchard analysis indicated a K d value of 0.33 ± 0.13 nM and a B max value of 5.83 ± 1.16 pmol/mg of protein. RP 67580, a selective NK 1-receptor antagonist was found to displace the specific binding of [ 3H]substance P. When [ 3H]RP 67580 was used as a ligand, it displayed a high affinity (K D value: 1.22 ± 0.27 nM) in transfected cell homogenates and only competed with NK 1 receptor ligands. Substance P stimulated the hydrolysis of phosphoinositide in a time- and concentration-dependent manner and this effect was mimicked by selective agonists of the NK 1 receptor ([Pro 9]SP and septide). RP 67580 did not induce any accumulation of inositol phosphates, but was found to inhibit the inositol phosphate increase mediated by substance P, without affecting the maximal response. From these results, one may conclude that the receptor expressed by the transfected Chinese hamster ovary cells revealed similar binding characteristics as the NK 1 receptor present in the rat brain and also confirmed the high affinity and the antagonist properties of RP 67580.