title: Novel method for the study of receptor Ca2+ signalling exemplified by the NK1 receptor.
abstract: We have used a novel technology (NovoStar from BMG Labtechnologies) for the study of the Ca2+ signalling of the human tackykinin NK1 (hNK-I receptor). The NovoStar is a microplate reader based on fluorescence and luminescence. The instrument implements a robotic pipettor arm and two microplate carriers, typically one for samples and one for cells. The robotic pipettor arm can transfer sample (agonist or antagonist) from the sample plate or other liquid containers to the cell plate, facilitating the study of Ca2+ signalling to such a degree that the instrument can be used for Medium Throughput Screening (MTS). Using the NovoStar we have found the molecular pharmacology of the NK1 receptor to be comparable to that observed in classical signal transduction assays. Thus, we have observed an EC50 value of 3 nM for substance P induced Ca2+ response. This value corresponds well with previously published values for substance P induced IP and cAMP turnover. [1] Using the NovoStar technology we have studied the pharmacological profile of the well known non-peptide NKI receptor antagonists CP96,345 and SR140,333 [2,3] in respect of inhibition of the Ca2+ response induced by substance P. Interestingly, the antagonistic potency of the antagonists depended greatly on the experimental design, e.g., a dependency of timing in the addition of antagonists vs. agonist was noted. Also, metal-ion site engineered NK1 receptors [2] were tested for the ability of metal-ions to inhibit signalling. It is concluded that the NovoStar is a reliable tool for the study of receptor Ca2+ signalling, both as a research tool and as a MTS system.
authors.affiliation: 7TM Pharma A/S, Rønnegade 2, DK-2100 Copenhagen, Denmark. ahed@7tm.com
authors.forename: A
authors.initials: A
authors.lastname: Heding
authors.forename: C E
authors.initials: CE
authors.lastname: Elling
authors.forename: T W
authors.initials: TW
authors.lastname: Schwartz
mesh_terms: D000818:Animals; D001713:Biphenyl Compounds / Q000494:pharmacology; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010716:Phosphatidylinositols / Q000378:metabolism; D010880:Piperidines / Q000494:pharmacology; D011812:Quinuclidines / Q000494:pharmacology; D018040:Receptors, Neurokinin-1 / Q000378:metabolism*; D013237:Stereoisomerism; D013373:Substance P / Q000037:antagonists & inhibitors / Q000494:pharmacology; D015032:Zinc / Q000494:pharmacology
chemical_list: D001713:Biphenyl Compounds; D010716:Phosphatidylinositols; D010880:Piperidines; D011812:Quinuclidines; D018040:Receptors, Neurokinin-1; C085215:SR 140333; D013373:Substance P; D000242:Cyclic AMP; D015032:Zinc; D002118:Calcium; C066892:CP 96345