Use of a dual firefly and Renilla luciferase reporter gene assay to simultaneously determine drug selectivity at human corticotrophin releasing hormone 1 and 2 receptors

The aim of this study was to investigate and validate the use of a dual glo w-signal luciferase reporter gene assay to simultaneously evaluate drug act ivity at two different seven-transmembrane receptor subtypes. Stable cell l ines (CHO) transfected with either human corticotrophin releasing hormone 1 (hCRH(1)) receptors and a firefly luciferase reporter gene or hCRH(2) and a Renilla luciferase reporter gene were created to provide different lucife rase readouts for CRH, and CRH, receptors, respectively. Cells were combine d for stimulation and measurement of luciferase luminescence in a 96-well p late format assay. The nonselective CRH agonists rat/human CRH and sauvagin e caused concentration-dependent increases in luminescence via activation o f CRH1 (fire By luciferase; pEC(50) = 8.40 +/- 0.06 and 8.39 +/- 0.08, resp ectively, n = 8) and CRH2 (Renilla luciferase; pEC(50) = 8.89 +/- 0.14 and 8.92 +/- 0.13, respectively, n = 8) receptors. The nonselective CRH antagon ist astressin blocked these agonist-induced increases in luciferase at both CRH, and CRH, receptors. The selective CRH, antagonist CP154,526 blocked r /hCRH- and sauvagine-induced increases in luciferase at CRH, receptors only . These data report the expected pharmacology for CRH, and CRH, receptors. This assay enabled two receptor subtypes to be studied simultaneously in th e same 96-well plate and generated robust data with low variability. It has the potential advantage of significant time and cost savings, with applica tion to both basic research and compound screening. (C) 2000 Academic Press .