Establishment of a cell-based screening system for NK-1 antagonists using SP-conjugated fluorescence; effects of plant extracts on the NK-1 receptor binding in U-373MG cultures

Substance P (SP) is a peptide neurotransmitter binding to neurokinin-1 (NK-1) receptors which are common in the central nervous systems. The interaction between SP and NK-1 receptor has been associated with a number of diseases, including inflammatory bowel disease, liver diseases, asthma, diabetes, migraine, emesis, depression and pain [2]. There is no such study for the discovery of a new agent from plant products even though their action could present value as a target in the treatment of many diseases. In this study, we have established a cell-based system for screening NK-1 receptor antagonists from plant extracts using a fluorescence probe, SP conjugated Oregon Green®488 (Molecular Probes, Eugene, OR), and U-373MG human malignant glioma cells, which dominantly expresses NK receptors [3–5]. The treatment of 10 nM SP-Oregon Green®488 to the -373MG culture resulted in the significant increase of the fluorescence intensity, which was selectively inhibited by L-733,060, a selective synthetic NK-1 antagonist having high affinity on human NK-1 receptor (6). L-733,060 blocked the binding of the SP fluorescence probe in a dose-dependent manner at the concentrations ranging from 0.1 to 100 nM (IC₅₀=1.85 nM). We were able to investigate some prospective plant extracts with the NK-1 receptor antagonist activity using the assay system.

Acknowledgement: This study was supported by a grant from the Ministry of Commerce, Industry and Energy (MOCIE) and the Korea Institute of Industrial Technology Evaluation & Planning (ITEP) through the Biohealth Products Research Center of Inje University.

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