Figure 3
From: Low affinity glucocorticoid binding site ligands as potential anti-fibrogenics
Radiolabelled dexamethasone interacts in a specific and saturable manner with rat liver microsomes and binding is competed by selected compounds. Male rat liver microsomes were incubated in duplicate with increasing concentrations of radiolabelled dexamethasone (ligand) with or without excess unlabelled dexamethasone and allowed to reach equilibrium on ice. A small volume of each incubation was removed to determine the total ligand concentration ([L0]) prior to removal of free unbound ligand by dextran/charcoal adsorption. Specifically bound ligand at equilibrium ([LRe]) was calculated by subtracting radioactive counts present in samples which also contained excess unlabelled dexamethasone after dextran-charcoal adsorption (and was typically < 5%). Free ligand concentration at equilibrium was calculated by subtracting specifically bound ligand at equilibrium from the total ligand concentration (i.e. [L0] - [LRe]) and assumes receptor-ligand stoichiometry of 1:1. Results typical of six separate preparations (a). Male rat liver microsomes were incubated with 50 nM [3H] dexamethasone as outlined in methods section with or without excess unlabelled dexamethasone (to determine non-specific binding) or a range of unlabelled compounds (added with ethanol vehicle such that final ethanol concentration was 1%, also present in controls). After overnight incubation on ice, free ligand was removed by dextran-charcoal adsorption and specifically bound radiolabelled dexamethasone determined (b).