Radioligand binding assays and their analysis.

Radioligand binding is widely used to characterize receptors and determine their anatomical distribution, particularly the superfamily of seven transmembrane-spanning G protein-coupled receptors for both established transmitters such as endothelin-1 and an increasing number of orphan receptors recently paired with their cognate ligands. Three types of assay are described. In saturation experiments, tissue sections, cultured cells, or homogenates are incubated with an increasing concentration of a radiolabeled ligand, which can be a labeled analog of a naturally occurring transmitter, hormone, or synthetic drug. Analysis using iterative nonlinear curve-fitting programs, such as KELL, measures the affinity of the labeled ligand for a receptor (equilibrium dissociation constant, K ( D )), receptor density (B (max)), and Hill slope (nH). The affinity and selectivity of an unlabeled ligand to compete for the binding of a fixed concentration of a radiolabeled ligand to a receptor are determined using a competition binding assay. Kinetic assays measure the rate of association to or dissociation from a receptor from which a kinetic K ( D ) may be derived. Quantitative autoradiography and image analysis is a sensitive technique to detect low levels of radiolabeled ligands and determine the anatomical distribution of receptors in sections that retain the morphology of the tissue. The measurement of bound radioligand within discrete regions of autoradiographical images using -computer-assisted image analysis is described.