Characterization of species-related differences in the pharmacology of tachykinin NK receptors 1, 2 and 3.

Tachykinin NK receptors (NKRs) differ to a large degree among species with respect to their affinities for small molecule antagonists. The aims of the present study were to clone NKRs from gerbil (NK2R and NK3R) and dog (NK1R, NK2R and NK3R) in which the sequence was previously unknown and to investigate the potency of several NKR antagonists at all known human, dog, gerbil and rat NKRs. The NKR protein coding sequences were cloned and expressed in CHO cells. The inhibitory concentrations of selective and non-selective NKR antagonists were determined by inhibition of agonist-induced mobilization of intracellular Ca2+. Receptor homology models were constructed based on the rhodopsin crystal structure to investigate and identify the antagonist binding sites and interaction points in the transmembrane (TM) regions of the NKRs. Data collected using the cloned dog NK1R confirmed that the dog NK1R displays similar pharmacology as the human and the gerbil NK1R, but differs greatly from the mouse and the rat NK1R. Despite species-related amino acid (AA) differences located close to the antagonist binding pocket of the NK2R, they did not affect the potency of the antagonists ZD6021 and saredutant. Two AA differences located close to the antagonist binding site of NK3R likely influence the NK3R antagonist potency, explaining the 3-10-fold decrease in potency observed for the rat NK3R. For the first time, detailed pharmacological experiments in vitro with cloned NKRs demonstrate that not only human, but also dog and gerbil NKR displays similar antagonist pharmacology while rat diverges significantly with respect to NK1R and NK3R.

Molecular cloning, mutations and effects of NK1 receptor antagonists reveal the human-like pharmacology of gerbil NK1 receptors.

The present study investigates the pharmacology of the cloned neurokinin 1 receptor from the gerbil (gNK(1)R), a species claimed to have human-like NK(1)R (hNK(1)R) pharmacology. The amino acid sequence of NK(1)R was cloned. The hNK(1)R, rat NK(1)R (rNK(1)R), gNK(1)R and mutants of the gNK(1)R were expressed in CHO cells. The affinity and potency of NKR agonists and the NK(1)R antagonists CP99994 and RP67580 (NK(1)R-selective) and ZD6021 (NK1/2R) were assessed in vitro by monitoring [(3)H]-SarMet SP binding and substance P-evoked mobilization of intracellular Ca(2+). The gerbil foot tap (GFT) method was used to assess the potency of the antagonists in vivo. The gNK(1)R coding sequence displayed an overall 95% and 97% homology with hNK(1)R and rNK(1)R, respectively. The affinity of the NK(1)R-selective agonist (3)H-SarMet SP for human and gerbil NK(1)R was similar (2.0 and 3.1 nM) but lower for rNK(1)R (12.4 nM). The rank order potency of the agonists for NK(1)R was SP > or = ASMSP > or = NKA >>> pro7NKB in all species. The NK(1)R antagonists, ZD6021 and CP99994, had comparable affinity and potency for gerbil and human NK(1)R, but were 1000-fold less potent for rNK(1)R. In contrast, RP67580 had comparable affinity and potency for all three species. Mutations in positions 116 and 290 did not affect agonist potency at the gNK(1)R while the potency of the antagonists ZD6021 and CP99994 were markedly decreased (10-20-fold). It is concluded that gNK(1)R has similar antagonist pharmacology as the human-like orthologue and that species differences in antagonist function depend on key residues in the coding sequence and antagonist structure.