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 activity. In order to find new compounds modulating the activity of these receptors we have developed a cellular screening system that measures the biological activity of receptors coupled to the IP3/DAG signal transduction pathway via the transcriptional activation of a reporter gene. For the establishment of neurokinin test cell lines the reporter cell line A20, stably transformed with the luciferase gene under the control 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 genes for the human neurokinin receptors NK1, NK2 or NK3, respectively. In these cell lines, expression of luciferase was inducible by substance P, neurokinin A and neurokinin B, respectively. The order of potency of the three neurokinins substance P, neurokinin A and neurokinin B was consistent with published data and results from ligand binding studies performed with the NK1 and NK2 test cell lines. The agonistic effect 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.