Equilibrium and kinetic measurements of muscarinic receptor antagonism on living cells using bead injection spectroscopy

Bead injection spectroscopy (BIS) techniques are introduced for automated m easurement of pharmacological antagonism by functional assay. Chinese hamst er ovary cells that express the rat type 1 muscarinic receptor are cultured on microbeads and used as a renewable biological target for muscarinic rec eptor antagonist ligands. A now injection instrument is used to reproducibl y sample and capture the cells in a jet ring chamber. The effect of the ant agonist pirenzepine on the carbachol-induced intracellular calcium response of the cells is measured with a fluorescence microscope photometry system. The BTS functional assay is used to quantify both equilibrium and kinetic pharmacological values for pirenzepine. In addition, two muscarinic recepto r antagonists (pirenzepine and atropine) are assayed to compare their relat ive efficacy at diminishing the calcium response. Due to the precision of t he automated fluid/bead handling protocols, and reproducibility of the meas ured calcium response, the quantification of useful pharmacological informa tion from living cells by BIS techniques is demonstrated.