FUNCTIONAL-CHARACTERIZATION OF THE HUMAN NEUROKININ RECEPTORS NK1, NK2, AND NK3 BASED ON A CELLULAR-ASSAY SYSTEM

The neurokinin receptor family is known to modulate phospholipase C ac tivity. In order to find new compounds modulating the activity of thes e receptors we have developed a cellular screening system that measure s the biological activity of receptors coupled to the IP3/DAG signal t ransduction pathway via the transcriptional activation of a reporter g ene. For the establishment of neurokinin test cell lines the reporter cell line A20, stably transformed with the luciferase gene under the c ontrol of a promoter containing TPA response elements (TRE), which did not respond to neurokinin agonists, was used. Stable test cell lines were developed by transfecting the reporter cell line A20 with the gen es for the human neurokinin receptors NK1, NK2 or NK3, respectively. I n these cell lines, expression of luciferase was inducible by substanc e P, neurokinin A and neurokinin B, respectively. The order of potency of the three neurokinins substance P, neurokinin A and neurokinin B w as consistent with published data and results from ligand binding stud ies performed with the NK1 and NK2 test cell lines. The agonistic effe ct of the neurokinins could be inhibited in a dose-dependent manner by simultaneous addition of neurokininspecific antagonists like the non- peptide antagonists CP-99,994 and SR 48968.