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 glow-signal luciferase reporter gene assay to simultaneously evaluate drug activity at two different seven-transmembrane receptor subtypes. Stable cell lines (CHO) transfected with either human corticotrophin releasing hormone 1 (hCRH1) receptors and a firefly luciferase reporter gene or hCRH2 and a Renilla luciferase reporter gene were created to provide different luciferase readouts for CRH1 and CRH2 receptors, respectively. Cells were combined for stimulation and measurement of luciferase luminescence in a 96-well plate format assay. The nonselective CRH agonists rat/human CRH and sauvagine caused concentration-dependent increases in luminescence via activation of CRH1 (firefly luciferase; pEC50 = 8.40 +/- 0.06 and 8.39 +/- 0.08, respectively, n = 8) and CRH2 (Renilla luciferase; pEC50 = 8.89 +/- 0.14 and 8.92 +/- 0.13, respectively, n = 8) receptors. The nonselective CRH antagonist astressin blocked these agonist-induced increases in luciferase at both CRH1 and CRH2 receptors. The selective CRH1 antagonist CP154,526 blocked r/hCRH- and sauvagine-induced increases in luciferase at CRH1 receptors only. These data report the expected pharmacology for CRH1 and CRH2 receptors. This assay enabled two receptor subtypes to be studied simultaneously in the same 96-well plate and generated robust data with low variability. It has the potential advantage of significant time and cost savings, with application to both basic research and compound screening.

Rat renal cortical slices: Maintenance of viability and use in in vitro nephrotoxicity testing.

The majority of in vitro toxicology studies employing precision-cut rat renal slices are carried out using simple incubation systems over relatively short periods of time (usually up to 8 hr). We aimed to develop a system for longer-term (up to 24 hr) culture of rat renal slices. The viability of precision-cut rat renal cortical slices was therefore investigated under a variety of incubation conditions. Slices cultured using a dynamic organ culture system were found to be more viable than those cultured in shaken multiwell dishes. This improved viability was evident after only 2 hr of culture (as assessed by the measurement of LDH leakage). An oxygen enriched gaseous environment was found to be important for the maintenance of slice viability using the dynamic organ culture system and conditions were established which permitted the culture of viable slices for periods up to 24 hr. Using these optimized incubation conditions, less than 8% LDH leakage was observed at 24 hr and slices retained 84% of fresh ATP values. Histologically, few signs of slice degeneration were noted until 20 hr of culture. The renal slice system was shown to discriminate between the most toxic and least toxic of a series of cephalosporin antibiotics, based on measurements of slice ATP content.